Norbert Frey

Cardiac hypertrophy is the heart's response to a variety of extrinsic and intrinsic stimuli that impose increased biomechanical stress. While hypertrophy can eventually normalize wall tension, it is associated with an unfavorable outcome and threatens affected patients with sudden death or progression to overt heart failure. Accumulating evidence from studies in human patients and animal models suggests that in most instances hypertrophy is not a compensatory response to the change in mechanical load, but rather is a maladaptive process. Accordingly, modulation of myocardial growth without adversely affecting contractile function is increasingly recognized as a potentially auspicious approach in the prevention and treatment of heart failure. In this review, we summarize recent insights into hypertrophic signaling and consider several novel antihypertrophic strategies.

Document Reviewers: Rudolf A. de Boer (CPG Review Coordinator) (Netherlands), P. Christian Schulze (CPG Review Coordinator) (Germany), Magdy Abdelhamid (Egypt), Victor Aboyans (France), Stamatis Adamopoulos (Greece), Stefan D. Anker (Germany), Elena Arbelo (Spain), Riccardo Asteggiano (Italy), Johann Bauersachs (Germany), Antoni Bayes-Genis (Spain), Michael A. Borger (Germany), Werner Budts (Belgium), Maja Cikes (Croatia), Kevin Damman (Netherlands), Victoria Delgado (Netherlands), Paul Dendale (Belgium), Polychronis Dilaveris (Greece), Heinz Drexel (Austria), Justin Ezekowitz (Canada), Volkmar Falk (Germany), Laurent Fauchier (France), Gerasimos Filippatos (Greece), Alan Fraser (United Kingdom), Norbert Frey (Germany), Chris P. Gale (United Kingdom), Finn Gustafsson (Denmark), Julie Harris (United Kingdom), Bernard Iung (France), Stefan Janssens (Belgium), Mariell Jessup (United States of America), Aleksandra Konradi (Russia), Dipak Kotecha (United Kingdom), Ekaterini Lambrinou (Cyprus), Patrizio Lancellotti (Belgium), Ulf Landmesser (Germany), Christophe Leclercq (France), Basil S. Lewis (Israel), Francisco Leyva (United Kingdom), AleVs Linhart (Czech Republic), Maja-Lisa Løchen (Norway), Lars H. Lund (Sweden), Donna Mancini (United States of America), Josep Masip (Spain), Davor Milicic (Croatia), Christian Mueller (Switzerland), Holger Nef (Germany), Jens-Cosedis Nielsen (Denmark), Lis Neubeck (United Kingdom), Michel Noutsias (Germany), Steffen E. Petersen (United Kingdom), Anna Sonia Petronio (Italy), Piotr Ponikowski (Poland), Eva Prescott (Denmark), Amina Rakisheva (Kazakhstan), Dimitrios J. Richter (Greece), Evgeny Schlyakhto (Russia), Petar Seferovic (Serbia), Michele Senni (Italy), Marta Sitges (Spain), Miguel Sousa-Uva (Portugal), Carlo G. Tocchetti (Italy), Rhian M. Touyz (United Kingdom), Carsten Tschoepe (Germany), Johannes Waltenberger (Germany/Switzerland) All experts involved in the development of these guidelines have submitted declarations of interest. These have been compiled in a report and published in a supplementary document simultaneously to the guidelines. The report is also available on the ESC website www.escardio.org/guidelines For the Supplementary Data which include background information and detailed discussion of the data that have provided the basis for the guidelines see European Heart Journal online.

Recent studies call into question the necessity of hypertrophic growth of the heart as a "compensatory" response to hemodynamic stress. These findings, coupled with recent progress in dissecting the molecular bases of hypertrophy, raise the prospect of suppressing hypertrophy without provoking circulatory insufficiency. In this article, we focus on signaling pathways that hold promise as potential targets for therapeutic intervention. We also summarize observations from animal models and clinical trials that suggest benefit from an antihypertrophic strategy.

Background Cardiac muscle hypercontractility is a key pathophysiological abnormality in hypertrophic cardiomyopathy, and a major determinant of dynamic left ventricular outflow tract (LVOT) obstruction. Available pharmacological options for hypertrophic cardiomyopathy are inadequate or poorly tolerated and are not disease-specific. We aimed to assess the efficacy and safety of mavacamten, a first-in-class cardiac myosin inhibitor, in symptomatic obstructive hypertrophic cardiomyopathy. Methods In this phase 3, randomised, double-blind, placebo-controlled trial (EXPLORER-HCM) in 68 clinical cardiovascular centres in 13 countries, patients with hypertrophic cardiomyopathy with an LVOT gradient of 50 mm Hg or greater and New York Heart Association (NYHA) class II–III symptoms were assigned (1:1) to receive mavacamten (starting at 5 mg) or placebo for 30 weeks. Visits for assessment of patient status occurred every 2–4 weeks. Serial evaluations included echocardiogram, electrocardiogram, and blood collection for laboratory tests and mavacamten plasma concentration. The primary endpoint was a 1·5 mL/kg per min or greater increase in peak oxygen consumption (pVO2) and at least one NYHA class reduction or a 3·0 mL/kg per min or greater pVO2 increase without NYHA class worsening. Secondary endpoints assessed changes in post-exercise LVOT gradient, pVO2, NYHA class, Kansas City Cardiomyopathy Questionnaire-Clinical Summary Score (KCCQ-CSS), and Hypertrophic Cardiomyopathy Symptom Questionnaire Shortness-of-Breath subscore (HCMSQ-SoB). This study is registered with ClinicalTrials.gov, NCT03470545. Findings Between May 30, 2018, and July 12, 2019, 429 adults were assessed for eligibility, of whom 251 (59%) were enrolled and randomly assigned to mavacamten (n=123 [49%]) or placebo (n=128 [51%]). 45 (37%) of 123 patients on mavacamten versus 22 (17%) of 128 on placebo met the primary endpoint (difference +19·4%, 95% CI 8·7 to 30·1; p=0·0005). Patients on mavacamten had greater reductions than those on placebo in post-exercise LVOT gradient (−36 mm Hg, 95% CI −43·2 to −28·1; p<0·0001), greater increase in pVO2 (+1·4 mL/kg per min, 0·6 to 2·1; p=0·0006), and improved symptom scores (KCCQ-CSS +9·1, 5·5 to 12·7; HCMSQ-SoB −1·8, −2·4 to −1·2; p<0·0001). 34% more patients in the mavacamten group improved by at least one NYHA class (80 of 123 patients in the mavacamten group vs 40 of 128 patients in the placebo group; 95% CI 22·2 to 45·4; p<0·0001). Safety and tolerability were similar to placebo. Treatment-emergent adverse events were generally mild. One patient died by sudden death in the placebo group. Interpretation Treatment with mavacamten improved exercise capacity, LVOT obstruction, NYHA functional class, and health status in patients with obstructive hypertrophic cardiomyopathy. The results of this pivotal trial highlight the benefits of disease-specific treatment for this condition. Funding MyoKardia.

Hypertrophic growth is an adaptive response of the heart to diverse pathological stimuli and is characterized by cardiomyocyte enlargement, sarcomere assembly, and activation of a fetal program of cardiac gene expression. A variety of Ca(2+)-dependent signal transduction pathways have been implicated in cardiac hypertrophy, but whether these pathways are independent or interdependent and whether there is specificity among them are unclear. Previously, we showed that activation of the Ca(2+)/calmodulin-dependent protein phosphatase calcineurin or its target transcription factor NFAT3 was sufficient to evoke myocardial hypertrophy in vivo. Here, we show that activated Ca(2+)/calmodulin-dependent protein kinases-I and -IV (CaMKI and CaMKIV) also induce hypertrophic responses in cardiomyocytes in vitro and that CaMKIV overexpressing mice develop cardiac hypertrophy with increased left ventricular end-diastolic diameter and decreased fractional shortening. Crossing this transgenic line with mice expressing a constitutively activated form of NFAT3 revealed synergy between these signaling pathways. We further show that CaMKIV activates the transcription factor MEF2 through a posttranslational mechanism in the hypertrophic heart in vivo. Activated calcineurin is a less efficient activator of MEF2-dependent transcription, suggesting that the calcineurin/NFAT and CaMK/MEF2 pathways act in parallel. These findings identify MEF2 as a downstream target for CaMK signaling in the hypertrophic heart and suggest that the CaMK and calcineurin pathways preferentially target different transcription factors to induce cardiac hypertrophy.

The adult myocardium responds to a variety of pathologic stimuli by hypertrophic growth that frequently progresses to heart failure. The calcium/calmodulin-dependent protein phosphatase calcineurin is a potent transducer of hypertrophic stimuli. Calcineurin dephosphorylates members of the nuclear factor of activated T cell (NFAT) family of transcription factors, which results in their translocation to the nucleus and activation of calcium-dependent genes. Glycogen synthase kinase-3 (GSK-3) phosphorylates NFAT proteins and antagonizes the actions of calcineurin by stimulating NFAT nuclear export. To determine whether activated GSK-3 can act as an antagonist of hypertrophic signaling in the adult heart in vivo, we generated transgenic mice that express a constitutively active form of GSK-3 beta under control of a cardiac-specific promoter. These mice were physiologically normal under nonstressed conditions, but their ability to mount a hypertrophic response to calcineurin activation was severely impaired. Similarly, cardiac-specific expression of activated GSK-3 beta diminished hypertrophy in response to chronic beta-adrenergic stimulation and pressure overload. These findings reveal a role for GSK-3 beta as an inhibitor of hypertrophic signaling in the intact myocardium and suggest that elevation of cardiac GSK-3 beta activity may provide clinical benefit in the treatment of pathologic hypertrophy and heart failure.

Chronic kidney disease (CKD) is a worldwide public health threat that increases risk of death due to cardiovascular complications, including left ventricular hypertrophy (LVH). Novel therapeutic targets are needed to design treatments to alleviate the cardiovascular burden of CKD. Previously, we demonstrated that circulating concentrations of fibroblast growth factor (FGF) 23 rise progressively in CKD and induce LVH through an unknown FGF receptor (FGFR)-dependent mechanism. Here, we report that FGF23 exclusively activates FGFR4 on cardiac myocytes to stimulate phospholipase Cγ/calcineurin/nuclear factor of activated T cell signaling. A specific FGFR4-blocking antibody inhibits FGF23-induced hypertrophy of isolated cardiac myocytes and attenuates LVH in rats with CKD. Mice lacking FGFR4 do not develop LVH in response to elevated FGF23, whereas knockin mice carrying an FGFR4 gain-of-function mutation spontaneously develop LVH. Thus, FGF23 promotes LVH by activating FGFR4, thereby establishing FGFR4 as a pharmacological target for reducing cardiovascular risk in CKD.

Temporal resolution of cellular features associated with a severe COVID-19 disease trajectory is needed for understanding skewed immune responses and defining predictors of outcome. Here, we performed a longitudinal multi-omics study using a two-center cohort of 14 patients. We analyzed the bulk transcriptome, bulk DNA methylome, and single-cell transcriptome (>358,000 cells, including BCR profiles) of peripheral blood samples harvested from up to 5 time points. Validation was performed in two independent cohorts of COVID-19 patients. Severe COVID-19 was characterized by an increase of proliferating, metabolically hyperactive plasmablasts. Coinciding with critical illness, we also identified an expansion of interferon-activated circulating megakaryocytes and increased erythropoiesis with features of hypoxic signaling. Megakaryocyte- and erythroid-cell-derived co-expression modules were predictive of fatal disease outcome. The study demonstrates broad cellular effects of SARS-CoV-2 infection beyond adaptive immune cells and provides an entry point toward developing biomarkers and targeted treatments of patients with COVID-19.

Venoarterial extracorporeal membrane oxygenation (VA-ECMO) is increasingly used to treat cardiogenic shock. However, VA-ECMO might hamper myocardial recovery. The Impella unloads the left ventricle. This study aimed to evaluate whether left ventricular unloading in patients with cardiogenic shock treated with VA-ECMO was associated with lower mortality.Data from 686 consecutive patients with cardiogenic shock treated with VA-ECMO with or without left ventricular unloading using an Impella at 16 tertiary care centers in 4 countries were collected. The association between left ventricular unloading and 30-day mortality was assessed by Cox regression models in a 1:1 propensity score-matched cohort.Left ventricular unloading was used in 337 of the 686 patients (49%). After matching, 255 patients with left ventricular unloading were compared with 255 patients without left ventricular unloading. In the matched cohort, left ventricular unloading was associated with lower 30-day mortality (hazard ratio, 0.79 [95% CI, 0.63-0.98]; P=0.03) without differences in various subgroups. Complications occurred more frequently in patients with left ventricular unloading: severe bleeding in 98 (38.4%) versus 45 (17.9%), access site-related ischemia in 55 (21.6%) versus 31 (12.3%), abdominal compartment in 23 (9.4%) versus 9 (3.7%), and renal replacement therapy in 148 (58.5%) versus 99 (39.1%).In this international, multicenter cohort study, left ventricular unloading was associated with lower mortality in patients with cardiogenic shock treated with VA-ECMO, despite higher complication rates. These findings support use of left ventricular unloading in patients with cardiogenic shock treated with VA-ECMO and call for further validation, ideally in a randomized, controlled trial.

Programmed necrosis (necroptosis) represents a newly identified mechanism of cell death combining features of both apoptosis and necrosis. Like apoptosis, necroptosis is tightly regulated by distinct signalling pathways. A key regulatory role in programmed necrosis has been attributed to interactions of the receptor-interacting protein kinases, RIP1 and RIP3. However, the specific functional role of RIP3-dependent signalling and necroptosis in the heart is unknown. The aims of this study were thus to assess the significance of necroptosis and RIP3 in the context of myocardial ischaemia. Immunoblots revealed strong expression of RIP3 in murine hearts, indicating potential functional significance of this protein in the myocardium. Consistent with a role in promoting necroptosis, adenoviral overexpression of RIP3 in neonatal rat cardiomyocytes and stimulation with TNF-α induced the formation of a complex of RIP1 and RIP3. Moreover, RIP3 overexpression was sufficient to induce necroptosis of cardiomyocytes. In vivo, cardiac expression of RIP3 was up-regulated upon myocardial infarction (MI). Conversely, mice deficient for RIP3 (RIP3−/−) showed a significantly better ejection fraction (45 ± 3.6 vs. 32 ± 4.4%, P < 0.05) and less hypertrophy in magnetic resonance imaging studies 30 days after experimental infarction due to left anterior descending coronary artery ligation. This was accompanied by a diminished inflammatory response of infarcted hearts and decreased generation of reactive oxygen species. Here, we show that RIP3-dependent necroptosis modulates post-ischaemic adverse remodelling in a mouse model of MI. This novel signalling pathway may thus be an attractive target for future therapies that aim to limit the adverse consequences of myocardial ischaemia.

Abstract Aims In spite of current medical treatment approaches, mortality of chronic heart failure (HF) remains high and novel treatment modalities are thus urgently needed. A recent theory proposes a possible impact of the intestinal microbiome on the incidence and clinical course of heart failure. This study sought to systematically investigate, if there are specific changes of the intestinal microbiome in heart failure patients. Methods and results The intestinal microbiome of 20 patients with heart failure with reduced ejection fraction due to ischemic or dilated cardiomyopathy was investigated by applying high‐throughput sequencing of the bacterial 16S rRNA gene. Microbial profiles were compared to those of matched controls in which heart failure was ruled out by clinical assessment and NT‐proBNP serum levels ( n = 20) . According to the Shannon diversity index (which measures the intra‐individual alpha‐diversity) based on the distribution of operational taxonomic units (OTUs), HF cases showed a nominally significantly lower diversity index compared to controls ( P nom. = 0.01), and testing for genera abundance showed a tendency towards a decreased alpha diversity of HF patients. Beta‐diversity measures (inter‐individual diversity) revealed a highly significant separation of HF cases and controls, (e.g. P weighted UniFracv = 0.004). Assessing the individual abundance of core measurable microbiota (CMM), a significant decrease of Coriobacteriaceae , Erysipelotrichaceae and Ruminococcaceae was observed on the family level. In line with that, Blautia , Collinsella , uncl. Erysipelotrichaceae and uncl. Ruminococcaceae showed a significant decrease in HF cases compared to controls on the genus level. Conclusions Heart failure patients showed a significantly decreased diversity of the intestinal microbiome as well as a downregulation of key intestinal bacterial groups. Our data point to an altered intestinal microbiome as a potential player in the pathogenesis and progression of heart failure.

Abstract Aims Transcatheter aortic valve implantation (TAVI) has emerged as established treatment option in patients with symptomatic aortic stenosis. Technical developments in valve design have addressed previous limitations such as suboptimal deployment, conduction disturbances, and paravalvular leakage. However, there are only limited data available for the comparison of newer generation self-expandable valve (SEV) and balloon-expandable valve (BEV). Methods and results SOLVE-TAVI is a multicentre, open-label, 2 × 2 factorial, randomized trial of 447 patients with aortic stenosis undergoing transfemoral TAVI comparing SEV (Evolut R, Medtronic Inc., Minneapolis, MN, USA) with BEV (Sapien 3, Edwards Lifesciences, Irvine, CA, USA). The primary efficacy composite endpoint of all-cause mortality, stroke, moderate/severe prosthetic valve regurgitation, and permanent pacemaker implantation at 30 days was powered for equivalence (equivalence margin 10% with significance level 0.05). The primary composite endpoint occurred in 28.4% of SEV patients and 26.1% of BEV patients meeting the prespecified criteria of equivalence [rate difference −2.39 (90% confidence interval, CI −9.45 to 4.66); P equivalence = 0.04]. Event rates for the individual components were as follows: all-cause mortality 3.2% vs. 2.3% [rate difference −0.93 (90% CI −4.78 to 2.92); P equivalence &amp;lt; 0.001], stroke 0.5% vs. 4.7% [rate difference 4.20 (90% CI 0.12 to 8.27); P equivalence = 0.003], moderate/severe paravalvular leak 3.4% vs. 1.5% [rate difference −1.89 (90% CI −5.86 to 2.08); P equivalence = 0.0001], and permanent pacemaker implantation 23.0% vs. 19.2% [rate difference −3.85 (90% CI −10.41 to 2.72) in SEV vs. BEV patients; P equivalence = 0.06]. Conclusion In patients with aortic stenosis undergoing transfemoral TAVI, newer generation SEV and BEV are equivalent for the primary valve-related efficacy endpoint. These findings support the safe application of these newer generation percutaneous valves in the majority of patients with some specific preferences based on individual valve anatomy.

Hypomorphic mutations in the DNA repair enzyme RNase H2 cause the neuroinflammatory autoimmune disorder Aicardi-Goutières syndrome (AGS). Endogenous nucleic acids are believed to accumulate in patient cells and instigate pathogenic type I interferon expression. However, the underlying nucleic acid species amassing in the absence of RNase H2 has not been established yet. Here, we report that murine RNase H2 knockout cells accumulated cytosolic DNA aggregates virtually indistinguishable from micronuclei. RNase H2-dependent micronuclei were surrounded by nuclear lamina and most of them contained damaged DNA. Importantly, they induced expression of interferon-stimulated genes (ISGs) and co-localized with the nucleic acid sensor cGAS. Moreover, micronuclei associated with RNase H2 deficiency were cleared by autophagy. Consequently, induction of autophagy by pharmacological mTOR inhibition resulted in a significant reduction of cytosolic DNA and the accompanied interferon signature. Autophagy induction might therefore represent a viable therapeutic option for RNase H2-dependent disease. Endogenous retroelements have previously been proposed as a source of self-nucleic acids triggering inappropriate activation of the immune system in AGS. We used human RNase H2-knockout cells generated by CRISPR/Cas9 to investigate the impact of RNase H2 on retroelement propagation. Surprisingly, replication of LINE-1 and Alu elements was blunted in cells lacking RNase H2, establishing RNase H2 as essential host factor for the mobilisation of endogenous retrotransposons.

Adeno-associated virus (AAV) forms the basis for several commercial gene therapy products and for countless gene transfer vectors derived from natural or synthetic viral isolates that are under intense preclinical evaluation. Here, we report a versatile pipeline that enables the direct side-by-side comparison of pre-selected AAV capsids in high-throughput and in the same animal, by combining DNA/RNA barcoding with multiplexed next-generation sequencing. For validation, we create three independent libraries comprising 183 different AAV variants including widely used benchmarks and screened them in all major tissues in adult mice. Thereby, we discover a peptide-displaying AAV9 mutant called AAVMYO that exhibits superior efficiency and specificity in the musculature including skeletal muscle, heart and diaphragm following peripheral delivery, and that holds great potential for muscle gene therapy. Our comprehensive methodology is compatible with any capsids, targets and species, and will thus facilitate and accelerate the stratification of optimal AAV vectors for human gene therapy.

It is currently unclear if active left ventricular (LV) unloading should be used as a primary treatment strategy or as a bailout in patients with cardiogenic shock (CS) treated with venoarterial extracorporeal membrane oxygenation (VA-ECMO).This study sought to evaluate the association between timing of active LV unloading and implantation of VA-ECMO with outcomes of patients with CS.Data from 421 patients with CS treated with VA-ECMO and active LV unloading at 18 tertiary care centers in 4 countries were analyzed. Patients were stratified by timing of device implantation in early vs delayed active LV unloading (defined by implantation before up to 2 hours after VA-ECMO). Adjusted Cox and logistic regression models were fitted to evaluate the association between early active LV unloading and 30-day mortality as well as successful weaning from ventilation.Overall, 310 (73.6%) patients with CS were treated with early active LV unloading. Early active LV unloading was associated with a lower 30-day mortality risk (HR: 0.64; 95% CI: 0.46-0.88) and a higher likelihood of successful weaning from ventilation (OR: 2.17; 95% CI: 1.19-3.93) but not with more complications. Importantly, the relative mortality risk increased and the likelihood of successful weaning from ventilation decreased almost proportionally with the time interval between VA-ECMO implantation and (delayed) initiation of active LV unloading.This exploratory study lends support to the use of early active LV unloading in CS patients on VA-ECMO, although the findings need to be validated in a randomized controlled trial.

Immune checkpoint inhibitors (ICIs) are approved for multiple cancers but can result in ICI-associated myocarditis, an infrequent but life-threatening condition. Elevations in cardiac biomarkers, specifically troponin-I (cTnI), troponin-T (cTnT), and creatine kinase (CK), are used for diagnosis. However, the association between temporal elevations of these biomarkers with disease trajectory and outcomes has not been established.We analyzed the diagnostic accuracy and prognostic performances of cTnI, cTnT, and CK in patients with ICI myocarditis (n=60) through 1-year follow-up in 2 cardio-oncology units (APHP Sorbonne, Paris, France and Heidelberg, Germany). A total of 1751 (1 cTnT assay type), 920 (4 cTnI assay types), and 1191 CK sampling time points were available. Major adverse cardiomyotoxic events (MACE) were defined as heart failure, ventricular arrhythmia, atrioventricular or sinus block requiring pacemaker, respiratory muscle failure requiring mechanical ventilation, and sudden cardiac death. Diagnostic performance of cTnI and cTnT was also assessed in an international ICI myocarditis registry.Within 72 hours of admission, cTnT, cTnI, and CK were increased compared with upper reference limits (URLs) in 56 of 57 (98%), 37 of 42 ([88%] P=0.03 versus cTnT), and 43 of 57 ([75%] P<0.001 versus cTnT), respectively. This increased rate of positivity for cTnT (93%) versus cTnI ([64%] P<0.001) on admission was confirmed in 87 independent cases from an international registry. In the Franco-German cohort, 24 of 60 (40%) patients developed ≥1 MACE (total, 52; median time to first MACE, 5 [interquartile range, 2-16] days). The highest value of cTnT:URL within the first 72 hours of admission performed best in terms of association with MACE within 90 days (area under the curve, 0.84) than CK:URL (area under the curve, 0.70). A cTnT:URL ≥32 within 72 hours of admission was the best cut-off associated with MACE within 90 days (hazard ratio, 11.1 [95% CI, 3.2-38.0]; P<0.001), after adjustment for age and sex. cTnT was increased in all patients within 72 hours of the first MACE (23 of 23 [100%]), whereas cTnI and CK values were less than the URL in 2 of 19 (11%) and 6 of 22 (27%) of patients (P<0.001), respectively.cTnT is associated with MACE and is sensitive for diagnosis and surveillance in patients with ICI myocarditis. A cTnT:URL ratio <32 within 72 hours of diagnosis is associated with a subgroup at low risk for MACE. Potential differences in diagnostic and prognostic performances between cTnT and cTnI as a function of the assays used deserve further evaluation in ICI myocarditis.

Calcineurin, a calcium/calmodulin-regulated protein phosphatase, modulates gene expression in cardiac and skeletal muscles during development and in remodeling responses such as cardiac hypertrophy that are evoked by environmental stresses or disease. Recently, we identified two genes encoding proteins (MCIP1 and MCIP2) that are enriched in striated muscles and that interact with calcineurin to inhibit its enzymatic activity. In the present study, we show that expression of MCIP1 is regulated by calcineurin activity in hearts of mice with cardiac hypertrophy, as well as in cultured skeletal myotubes. In contrast, expression of MCIP2 in the heart is not altered by activated calcineurin but responds to thyroid hormone, which has no effect on MCIP1. A approximately 900-bp intragenic segment located between exons 3 and 4 of the MCIP1 gene functions as an alternative promoter that responds to calcineurin. This region includes a dense cluster of 15 consensus binding sites for NF-AT transcription factors. Because MCIP proteins can inhibit calcineurin, these results suggest that MCIP1 participates in a negative feedback circuit to diminish potentially deleterious effects of unrestrained calcineurin activity in cardiac and skeletal myocytes. Inhibitory effects of MCIP2 on calcineurin activity may be pertinent to gene switching events driven by thyroid hormone in striated muscles. The full text of this article is available at http://www. circresaha.org.

The calcium- and calmodulin-dependent protein phosphatase calcineurin has been implicated in the transduction of signals that control the hypertrophy of cardiac muscle and slow fiber gene expression in skeletal muscle. To identify proteins that mediate the effects of calcineurin on striated muscles, we used the calcineurin catalytic subunit in a two-hybrid screen for cardiac calcineurin-interacting proteins. From this screen, we discovered a member of a novel family of calcineurin-interacting proteins, termed calsarcins, which tether calcineurin to alpha-actinin at the z-line of the sarcomere of cardiac and skeletal muscle cells. Calsarcin-1 and calsarcin-2 are expressed in developing cardiac and skeletal muscle during embryogenesis, but calsarcin-1 is expressed specifically in adult cardiac and slow-twitch skeletal muscle, whereas calsarcin-2 is restricted to fast skeletal muscle. Calsarcins represent a novel family of sarcomeric proteins that link calcineurin with the contractile apparatus, thereby potentially coupling muscle activity to calcineurin activation.

beta-Adrenergic receptor (betaAR) signaling, which elevates intracellular cAMP and enhances cardiac contractility, is severely impaired in the failing heart. Protein kinase A (PKA) is activated by cAMP, but the long-term physiological effect of PKA activation on cardiac function is unclear. To investigate the consequences of chronic cardiac PKA activation in the absence of upstream events associated with betaAR signaling, we generated transgenic mice that expressed the catalytic subunit of PKA in the heart. These mice developed dilated cardiomyopathy with reduced cardiac contractility, arrhythmias, and susceptibility to sudden death. As seen in human heart failure, these abnormalities correlated with PKA-mediated hyperphosphorylation of the cardiac ryanodine receptor/Ca(2+)-release channel, which enhances Ca(2+) release from the sarcoplasmic reticulum, and phospholamban, which regulates the sarcoplasmic reticulum Ca(2+)-ATPase. These findings demonstrate a specific role for PKA in the pathogenesis of heart failure, independent of more proximal events in betaAR signaling, and support the notion that PKA activity is involved in the adverse effects of chronic betaAR signaling.

The Z-disc is a highly specialized multiprotein complex of striated muscles that serves as the interface of the sarcomere and the cytoskeleton.In addition to its role in muscle contraction, its juxtaposition to the plasma membrane suggests additional functions of the Z-disc in sensing and transmitting external and internal signals.Recently, we described two novel striated muscle-specific proteins, calsarcin-1 and calsarcin-2, that bind ␣-actinin on the Z-disc and serve as intracellular binding proteins for calcineurin, a calcium/calmodulindependent phosphatase shown to be integral in cardiac hypertrophy as well as skeletal muscle differentiation and fiber-type specification.Here, we describe an additional member of the calsarcin family, calsarcin-3, which is expressed specifically in skeletal muscle and is enriched in fast-twitch muscle fibers.Like calsarcin-1 and calsarcin-2, calsarcin-3 interacts with calcineurin, and the Z-disc proteins ␣-actinin, ␥-filamin, and telethonin.In addition, we show that calsarcins interact with the PDZ-LIM domain protein ZASP/Cypher/Oracle, which also localizes to the Z-disc.Calsarcins represent a novel family of sarcomeric proteins that serve as focal points for the interactions of an array of proteins involved in Z-disc structure and signal transduction in striated muscle.

Biomechanical stress ie, attributable to pressure overload, leads to cardiac hypertrophy and may ultimately cause heart failure. Yet, it is still unclear how mechanical stress is sensed and transduced on the molecular level. To systematically elucidate the underlying signal transduction pathways, we analyzed the gene expression profile of stretched cardiomyocytes on a genome-wide scale in comparison with other inducers of hypertrophy such as pharmacological stimulation. Neonatal rat ventricular cardiomyocytes were either stretched biaxially or stimulated with phenylephrine (PE), both resulting in a similar degree of hypertrophy. Microarray analyses revealed 164 genes >2.0-fold up- and 21 genes <0.5-fold downregulated (P<0.01). Differential expression was confirmed by real-time polymerase chain reaction. Genes of the "fetal gene program" such as BNP were induced by both stretch (4.2x) and PE (2.9x). We also verified upregulation of known stretch-responsive genes, including HSP70 (20.9x) and c-myc (3.0x). Moreover, several genes were found to be preferentially induced by stretch, such as the cardioprotective cytokine GDF15 (24.8x) and heme oxygenase 1 (Hmox1, 10.8x; both confirmed on protein level). Neither PE nor endothelin-1 upregulated GDF15 and Hmox1, whereas angiotensin II significantly induced both genes. Conversely, the AT(1) receptor blocker irbesartan markedly blunted stretch-mediated GDF15 and Hmox1 upregulation, suggesting that the angiotensin receptor transduces the biomechanical induction of these genes. In conclusion, we report a comprehensive gene expression profile of cardiomyocytes subjected to biomechanical stress in comparison with pharmacologically induced hypertrophy. Our data imply that a stretch-specific gene program exists, which is mediated, at least in part, by angiotensin II-dependent signaling.

During the last 15 years, the perception of the cardiac z-disc has undergone substantial changes. Initially viewed as a structural component at the lateral boundaries of the sarcomere, the cardiac z-disc has increasingly become recognized as a nodal point in cardiomyocyte signal transduction and disease. This minireview thus focuses on novel components and recent developments in z-disc biology and their role in cardiac signaling and disease.

Dilated cardiomyopathy (DCM) is one of the leading causes for cardiac transplantations and accounts for up to one-third of all heart failure cases. Since extrinsic and monogenic causes explain only a fraction of all cases, common genetic variants are suspected to contribute to the pathogenesis of DCM, its age of onset, and clinical progression. By a large-scale case-control genome-wide association study we aimed here to identify novel genetic risk loci for DCM.Applying a three-staged study design, we analysed more than 4100 DCM cases and 7600 controls. We identified and successfully replicated multiple single nucleotide polymorphism on chromosome 6p21. In the combined analysis, the most significant association signal was obtained for rs9262636 (P = 4.90 × 10(-9)) located in HCG22, which could again be replicated in an independent cohort. Taking advantage of expression quantitative trait loci (eQTL) as molecular phenotypes, we identified rs9262636 as an eQTL for several closely located genes encoding class I and class II major histocompatibility complex heavy chain receptors.The present study reveals a novel genetic susceptibility locus that clearly underlines the role of genetically driven, inflammatory processes in the pathogenesis of idiopathic DCM.

Cardiac hypertrophy is an independent risk for heart failure (HF) and sudden death. Deciphering signalling pathways dependent on extracellular calcium (Ca(2+)) influx that control normal and pathological cardiac growth may enable identification of novel therapeutic targets. The objective of the present study is to determine the role of the Ca(2+) release-activated Ca(2+) (CRAC) channel Orai1 and stromal interaction molecule 1 (Stim1) in postnatal cardiomyocyte store operated Ca(2+) entry (SOCE) and impact on normal and hypertrophic postnatal cardiomyocyte growth. Employing a combination of siRNA-mediated gene silencing, cultured neonatal rat ventricular cardiomyocytes together with indirect immunofluorescence, epifluorescent Ca(2+) imaging and site-specific protein phosphorylation and real-time mRNA expression analysis, we show for the first time that both Orai1 and Stim1 are present in cardiomyocytes and required for SOCE due to intracellular Ca(2+) store depletion by thapsigargin. Stim1-KD but not Orai1-KD significantly decreased diastolic Ca(2+) levels and caffeine-releasable Ca(2+) from the sarcoplasmic reticulum (SR). Conversely, Orai1-KD but not Stim1-KD significantly diminished basal NRCM cell size, anp and bnp mRNA levels and activity of the calcineurin (CnA) signalling pathway although diminishing both Orai1 and Stim1 proteins similarly attenuated calmodulin kinase II (CamKII) and ERK1/2 activity under basal conditions. Both Orai1- and Stim1-KD completely abrogated phenylephrine (PE) mediated hypertrophic NRCM growth and enhanced natriuretic factor expression by inhibiting G(q)-protein conveyed activation of the CamKII and ERK1/2 signalling pathway. Interestingly, only Orai1-KD but not Stim1-KD prevented Gq-mediated CaN-dependent prohypertrophic signalling. This study shows for the first time that both Orai1 and Stim1 have a key role in cardiomyocyte SOCE regulating both normal and hypertrophic postnatal cardiac growth in vitro.

MicroRNA (miRNA) levels in serum have recently emerged as potential novel biomarkers for various diseases. miRNAs are routinely measured by standard quantitative real-time PCR (qPCR); however, the high sensitivity of qPCR demands appropriate normalization to correct for nonbiological variation. Presently, RNU6B (U6) is used for data normalization of circulating miRNAs in many studies. However, it was suggested that serum levels of U6 themselves might differ between individuals. Therefore, no consensus has been reached on the best normalization strategy in 'circulating miRNA'. We analyzed U6 levels as well as levels of spiked-in SV40-RNA in sera of 44 healthy volunteers, 203 intensive care unit patients and 64 patients with liver fibrosis. Levels of U6 demonstrated a high variability in sera of healthy donors, patients with critical illness and liver fibrosis. This high variability could also be confirmed in sera of mice after the cecal ligation and puncture procedure. Most importantly, levels of circulating U6 were significantly upregulated in sera of patients with critical illness and sepsis compared with controls and correlated with established markers of inflammation. In patients with liver fibrosis, U6 levels were significantly downregulated. In contrast, levels of spiked-in SV40 displayed a significantly higher stability both in human cohorts (healthy, critical illness, liver fibrosis) and in mice. Thus, we conclude that U6 levels in the serum are dysregulated in a disease-specific manner. Therefore, U6 should not be used for data normalization of circulating miRNAs in inflammatory diseases and previous studies using this approach should be interpreted with caution. Further studies are warranted to identify specific regulatory processes of U6 levels in sepsis and liver fibrosis.

Down-regulation of miR-150 was recently linked to inflammation and bacterial infection. Furthermore, reduced serum levels of miR-150 were reported from a small cohort of patients with sepsis. We thus aimed at evaluating the diagnostic and prognostic value of miR-150 serum levels in patients with critically illness and sepsis.miR-150 serum levels were analyzed in a cohort of 223 critically ill patients of which 138 fulfilled sepsis criteria and compared to 76 healthy controls. Results were correlated with clinical data and extensive sets of routine and experimental biomarkers.Measurements of miR-150 serum concentrations revealed only slightly reduced miR-150 serum levels in critically ill patients compared to healthy controls. Furthermore miR-150 levels did not significantly differ in critically ill patients with our without sepsis, indicating that miR-150 serum levels are not suitable for diagnostic establishment of sepsis. However, serum levels of miR-150 correlated with hepatic or renal dysfunction. Low miR-150 serum levels were associated with an unfavorable prognosis of patients, since low miR-150 serum levels predicted mortality with high diagnostic accuracy compared with established clinical scores and biomarkers.Reduced miR-150 serum concentrations are associated with an unfavorable outcome in patients with critical illness, independent of the presence of sepsis. Besides a possible pathogenic role of miR-150 in critical illness, our study indicates a potential use of circulating miRNAs as a prognostic rather than diagnostic marker in critically ill patients.

Osteopontin, a glycoprotein that can be detected in plasma, was found to be upregulated in several animal models of cardiac failure and may thus represent a new biomarker that facilitates risk stratification in patients with heart failure. We therefore tested whether osteopontin plasma levels are elevated in patients with chronic heart failure and whether they provide independent prognostic information.We analyzed osteopontin plasma levels in 420 patients with chronic heart failure due to significantly impaired left ventricular systolic function and correlated the results with disease stage and prognostic information (median follow-up of 43 months). We found that osteopontin plasma levels were significantly elevated in patients with heart failure as compared with healthy control subjects (532 versus 382 ng/mL, P=0.008), irrespective of heart failure origin (ischemic versus dilated cardiomyopathy). Furthermore, osteopontin levels were higher in patients with moderate to severe heart failure than in patients with no or mild symptoms (672 ng/mL for New York Heart Association class III/IV versus 479 ng/mL for class I/II, P<0.0001). Estimated 4-year death rates in patients with osteopontin levels above or below a cutoff value derived from receiver operating characteristic analyses were 56.5% and 28.4%, respectively (hazard ratio 3.4, 95% confidence interval 2.2 to 5.3, P<0.0001). In a multivariable model that included demographic, clinical, and biochemical parameters such as N-terminal prohormone brain natriuretic peptide, osteopontin emerged as an independent predictor of death (hazard ratio 2.3, 95% confidence interval 1.4 to 3.5, P<0.001).Our findings suggest that osteopontin might be useful as a novel prognostic biomarker in patients with chronic heart failure.

The composition of skeletal muscle, in terms of the relative number of slow- and fast-twitch fibers, is tightly regulated to enable an organism to respond and adapt to changing physical demands. The phosphatase calcineurin and its downstream targets, transcription factors of the nuclear factor of activated T cells (NFAT) family, play a critical role in this process by promoting the formation of slow-twitch, oxidative fibers. Calcineurin binds to calsarcins, a family of striated muscle-specific proteins of the sarcomeric Z-disc. We show here that mice deficient in calsarcin-2, which is expressed exclusively by fast-twitch muscle and encoded by the myozenin 1 (Myoz1) gene, have substantially reduced body weight and fast-twitch muscle mass in the absence of an overt myopathic phenotype. Additionally, Myoz1 KO mice displayed markedly improved performance and enhanced running distances in exercise studies. Analysis of fiber type composition of calsarcin-2-deficient skeletal muscles showed a switch toward slow-twitch, oxidative fibers. Reporter assays in cultured myoblasts indicated an inhibitory role for calsarcin-2 on calcineurin, and Myoz1 KO mice exhibited both an excess of NFAT activity and an increase in expression of regulator of calcineurin 1-4 (RCAN1-4), indicating enhanced calcineurin signaling in vivo. Taken together, these results suggest that calsarcin-2 modulates exercise performance in vivo through regulation of calcineurin/NFAT activity and subsequent alteration of the fiber type composition of skeletal muscle.

For years, the term "apoptosis" was used synonymously with programmed cell death. However, it was recently discovered that receptor interacting protein 3 (RIP3)-dependent "necroptosis" represents an alternative programmed cell death pathway activated in many inflamed tissues. Here, we show in a genetic model of chronic hepatic inflammation that activation of RIP3 limits immune responses and compensatory proliferation of liver parenchymal cells (LPC) by inhibiting Caspase-8-dependent activation of Jun-(N)-terminal kinase in LPC and nonparenchymal liver cells. In this way, RIP3 inhibits intrahepatic tumor growth and impedes the Caspase-8-dependent establishment of specific chromosomal aberrations that mediate resistance to tumor-necrosis-factor-induced apoptosis and underlie hepatocarcinogenesis. Moreover, RIP3 promotes the development of jaundice and cholestasis, because its activation suppresses compensatory proliferation of cholangiocytes and hepatic stem cells. These findings demonstrate a function of RIP3 in regulating carcinogenesis and cholestasis. Controlling RIP3 or Caspase-8 might represent a chemopreventive or therapeutic strategy against hepatocellular carcinoma and biliary disease.

We aimed to test, for the first time, the feasibility of intracoronary delivery of an innovative, injectable bioabsorbable scaffold (IK-5001), to prevent or reverse adverse left ventricular remodeling and dysfunction in patients after ST-segment-elevation myocardial infarction.Patients (n=27) with moderate-to-large ST-segment-elevation myocardial infarctions, after successful revascularization, were enrolled. Two milliliters of IK-5001, a solution of 1% sodium alginate plus 0.3% calcium gluconate, was administered by selective injection through the infarct-related coronary artery within 7 days after myocardial infarction. IK-5001 is assumed to permeate the infarcted tissue, cross-linking into a hydrogel and forming a bioabsorbable cardiac scaffold. Coronary angiography, 3 minutes after injection, confirmed that the injection did not impair coronary flow and myocardial perfusion. Furthermore, IK-5001 deployment was not associated with additional myocardial injury or re-elevation of cardiac biomarkers. Clinical assessments, echocardiographic studies, 12-lead electrocardiograms, 24-hour Holter monitoring, blood tests, and completion of Minnesota Living with Heart Failure Questionnaires were repeated during follow-up visits at 30, 90, and 180 days after treatment. During a 6-month follow-up, these tests confirmed favorable tolerability of the procedure, without device-related adverse events, serious arrhythmias, blood test abnormalities, or death. Serial echocardiographic studies showed preservation of left ventricular indices and left ventricular ejection fraction.This first-in-man pilot study shows that intracoronary deployment of an IK-5001 scaffold is feasible and well tolerated. Our results have promoted the initiation of a multicenter, randomized controlled trial to confirm the safety and efficacy of this new approach in high-risk patients after ST-segment-elevation myocardial infarction.http://www.clinicaltrials.gov. Unique identifier: NCT01226563.

Objective: Serum levels of microRNA have been proposed as biomarkers in various inflammatory diseases. However, up to now, their clinical relevance in critical illness and sepsis is unclear. Design: Single-center clinical study. Setting: Fourteen-bed medical ICU of the University Hospital Aachen, university laboratory research unit. Subjects and Patients: Experimental sepsis model in C57Bl/6 mice; 223 critically ill patients in comparison with 76 healthy volunteers. Interventions: We used the model of cecal pole ligation and puncture for induction of polymicrobial sepsis in mice and measured alterations in serum levels of six different microRNAs with a known function in inflammatory diseases upon induction of septic disease. These results from mice were translated into a large and well-characterized cohort of critically ill patients admitted to the medical ICU. Measurements and Main Results: Serum miR-133a was then measured in 223 critically ill patients (138 with sepsis and 85 without sepsis) and 76 controls and associated with disease severity, organ failure, and prognosis. Significant alterations of miR-133a, miR-150, miR-155, and miR-193b* were found in mice after cecal pole ligation and puncture–induced sepsis. Among all regulated microRNAs, miR-133a displayed the most prominent and concordant up-regulation in sepsis, and this microRNA was therefore chosen for further investigation in the human. Here, significantly elevated miR-133a levels were found in critically ill patients at ICU admission, when compared with healthy controls, especially in patients with sepsis. Correlation analyses revealed significant correlations of miR-133a with disease severity, classical markers of inflammation and bacterial infection, and organ failure. Strikingly, high miR-133a levels were predictive for an unfavorable prognosis and represented a strong independent predictor for both ICU and long-term mortality in critically ill patients. Conclusions: miR-133a serum levels were significantly elevated in critical illness and sepsis. High miR-133a levels were associated with the severity of disease and predicted an unfavorable outcome of critically ill patients.

miRNAs are novel regulators of organ fibrosis. miR-133a plays a role in cardiac and muscle remodeling, but its function in the liver is unclear. We therefore aimed at evaluating a possible function of miR-133a in hepatofibrogenesis.miR-133a levels were measured in whole liver samples from different murine hepatic fibrosis models and human liver tissue from patients with liver cirrhosis. The cell-specific regulation of miR-133a was assessed in FACS-sorted hepatic cell subpopulations. Murine and human primary hepatic stellate cells (HSC) were isolated and treated with different cytokines to evaluate upstream regulators of miR-133a. Moreover, GRX cells were transfected with synthetic miR-133a and the effect on extracellular matrix (ECM) gene regulation was assessed. Finally, miR-133a serum levels were measured in a cohort of patients with chronic liver diseases and correlated with disease progression.Overall miR-133a expression levels were unchanged in whole RNA extracts from fibrotic murine and human livers. However, miR-133a was specifically downregulated in HSC during fibrogenesis. Treatment of primary murine and human HSC with transforming growth factor (TGF)-β resulted in a significant downregulation of miR-133a in these cells. In turn, overexpression of miR-133a in primary murine HSC led to decreased expression of collagens. In addition, miR-133a serum levels were increased in patients with chronic liver disease and indicated the presence and progression of liver cirrhosis.Evidence is presented for a novel antifibrotic functional role of miR-133a in hepatofibrogenesis. miR-133a may thus represent a target for diagnostic and therapeutic strategies in liver fibrosis.

Background: In clinical practice, local anesthesia with conscious sedation (CS) is performed in roughly 50% of patients undergoing transcatheter aortic valve replacement. However, no randomized data assessing the safety and efficacy of CS versus general anesthesia (GA) are available. Methods: The SOLVE-TAVI (Comparison of Second-Generation Self-Expandable Versus Balloon-Expandable Valves and General Versus Local Anesthesia in Transcatheter Aortic Valve Implantation) trial is a multicenter, open-label, 2×2 factorial, randomized trial of 447 patients with aortic stenosis undergoing transfemoral transcatheter aortic valve replacement comparing CS versus GA. The primary efficacy end point was powered for equivalence (equivalence margin 10% with significance level 0.05) and consisted of the composite of all-cause mortality, stroke, myocardial infarction, infection requiring antibiotic treatment, and acute kidney injury at 30 days. Results: The primary composite end point occurred in 27.2% of CS and 26.4% of GA patients (rate difference, 0.8 [90% CI, −6.2 to 7.8]; P equivalence =0.015). Event rates for the individual components were as follows: all-cause mortality, 3.2% versus 2.3% (rate difference, 1.0 [90% CI, −2.9 to 4.8]; P equivalence &lt;0.001); stroke, 2.4% versus 2.8% (rate difference, −0.4 [90% CI, −3.8 to 3.8]; P equivalence &lt;0.001); myocardial infarction, 0.5% versus 0.0% (rate difference, 0.5 [90% CI, −3.0 to 3.9]; P equivalence &lt;0.001), infection requiring antibiotics 21.1% versus 22.0% (rate difference, −0.9 [90% CI, −7.5 to 5.7]; P equivalence =0.011); acute kidney injury, 9.0% versus 9.2% (rate difference, −0.2 [90% CI, −5.2 to 4.8]; P equivalence =0.0005). There was a lower need for inotropes or vasopressors with CS (62.8%) versus GA (97.3%; rate difference, −34.4 [90% CI, −41.0 to −27.8]). Conclusions: Among patients with aortic stenosis undergoing transfemoral transcatheter aortic valve replacement, use of CS compared with GA resulted in similar outcomes for the primary efficacy end point. These findings suggest that CS can be safely applied for transcatheter aortic valve replacement. Registration: URL: https://www.clinicaltrials.gov ; Unique identifier: NCT02737150.

Bioabsorbable cardiac matrix (BCM) is a novel device that attenuates adverse left ventricular (LV) remodeling after large myocardial infarctions in experimental models.This study aimed to analyze whether BCM, compared with saline control, would result in less LV dilation and fewer adverse clinical events between baseline and 6 months.In an international, randomized, double-blind, controlled trial, 303 subjects with large areas of infarction despite successful primary percutaneous coronary intervention (PCI) of ST-segment elevation myocardial infarction (STEMI) were randomized 2:1 to BCM or saline injected into the infarct-related artery 2 to 5 days after primary PCI. The primary outcome was mean change from baseline in LV end-diastolic volume index (LVEDVI) at 6 months. Secondary outcomes included change in Kansas City Cardiomyopathy Questionnaire score, 6-minute walk time, and New York Heart Association functional class at 6 months. The primary safety endpoint was a composite of cardiovascular death, recurrent MI, target-vessel revascularization, stent thrombosis, significant arrhythmia requiring therapy, or myocardial rupture through 6 months.In total, 201 subjects were assigned to BCM and 102 to saline control. There was no significant difference in change in LVEDVI from baseline to 6 months between the groups (mean change ± SD: BCM 14.1 ± 28.9 ml/m(2) vs. saline 11.7 ± 26.9 ml/m(2); p = 0.49). There was also no significant difference in the secondary endpoints. The rates of the primary safety outcome were similar between the 2 groups (BCM 11.6% vs. saline 9.1%; p = 0.37).Intracoronary deployment of BCM 2 to 5 days after successful reperfusion in subjects with large myocardial infarction did not reduce adverse LV remodeling or cardiac clinical events at 6 months. (IK-5001 for the Prevention of Remodeling of the Ventricle and Congestive Heart Failure After Acute Myocardial Infarction [PRESERVATION I]; NCT01226563).

Abstract Systemic forms of amyloidosis affecting the heart are mostly light-chain (AL) and transthyretin (ATTR) amyloidoses. The latter is caused by deposition of misfolded transthyretin, either in wild-type (ATTRwt) or mutant (ATTRv) conformation. For diagnostics, specific serum biomarkers and modern non-invasive imaging techniques, such as cardiovascular magnetic resonance imaging (CMR) and scintigraphic methods, are available today. These imaging techniques do not only complement conventional echocardiography, but also allow for accurate assessment of the extent of cardiac involvement, in addition to diagnosing cardiac amyloidosis. Endomyocardial biopsy still plays a major role in the histopathological diagnosis and subtyping of cardiac amyloidosis. The main objective of the diagnostic algorithm outlined in this position statement is to detect cardiac amyloidosis as reliably and early as possible, to accurately determine its extent, and to reliably identify the underlying subtype of amyloidosis, thereby enabling subsequent targeted treatment.

Coronavirus diseases 2019 (COVID-19) has become a worldwide pandemic affecting people at high risk and particularly at advanced age, cardiovascular and pulmonary disease. As cardiovascular patients are at high risk but also have dyspnea and fatigue as leading symptoms, prevention, diagnostics and treatment in these patients are important to provide adequate care for those with or without COVID-19 but most importantly when comorbid cardiovascular conditions are present. Severe COVID-19 with acute respiratory distress (ARDS) is challenging as patients with elevated myocardial markers such as troponin are at enhanced high risk for fatal outcomes. As angiotensin-converting enzyme 2 (ACE2) is regarded as the viral receptor for cell entry and as the Coronavirus is downregulating this enzyme, which provides cardiovascular and pulmonary protection, there is ongoing discussions on whether treatment with cardiovascular drugs, which upregulate the viral receptor ACE2 should be modified. As most of the COVID-19 patients have cardiovascular comorbidities like hypertension, diabetes, coronary artery disease and heart failure, which imposes a high risk on these patients, cardiovascular therapy should not be modified or even withdrawn. As cardiac injury is a common feature of COVID-19 associated ARDS and is linked with poor outcomes, swift diagnostic management and specialist care of cardiovascular patients in the area of COVID-19 is of particular importance and deserves special attention.

The inflammatory response after myocardial infarction (MI) is a precisely regulated process that greatly affects subsequent remodeling. Here, we show that basophil granulocytes infiltrated infarcted murine hearts, with a peak occurring between days 3 and 7. Antibody-mediated and genetic depletion of basophils deteriorated cardiac function and resulted in enhanced scar thinning after MI. Mechanistically, we found that basophil depletion was associated with a shift from reparative Ly6Clo macrophages toward increased numbers of inflammatory Ly6Chi monocytes in the infarcted myocardium. Restoration of basophils in basophil-deficient mice by adoptive transfer reversed this proinflammatory phenotype. Cellular alterations in the absence of basophils were accompanied by lower cardiac levels of IL-4 and IL-13, two major cytokines secreted by basophils. Mice with basophil-specific IL-4/IL-13 deficiency exhibited a similarly altered myeloid response with an increased fraction of Ly6Chi monocytes and aggravated cardiac function after MI. In contrast, IL-4 induction in basophils via administration of the glycoprotein IPSE/α-1 led to improved post-MI healing. These results in mice were corroborated by the finding that initially low counts of blood basophils in patients with acute MI were associated with a worse cardiac outcome after 1 year, characterized by a larger scar size. In conclusion, we show that basophils promoted tissue repair after MI by increasing cardiac IL-4 and IL-13 levels.

In principle, electrode-based bioelectrical signal acquisition can be complemented by biomagnetic sensing and therefore requires a more detailed assessment, especially because of the availability of novel noncryogenic sensor technologies. The current development of thin-film magnetoelectric (ME) sensors ensures that ME technology is becoming a prospective candidate for biomagnetometry. The main obstacle for large-scale usage is the lack of extremely low noise floors at the final sensor system output. This article highlights the current state of ME sensor development based on a magnetocardiography (MCG) pilot study involving a healthy volunteer in a magnetically shielded chamber. For assessment, an ME prototype (converse ME thin-film sensors) will be applied for the first time. This sensor type ensures a noise amplitude spectral density below 20 pT / <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"> <tex-math notation="LaTeX">$\sqrt {\text {Hz}}$ </tex-math></inline-formula> at 10 Hz by using a sophisticated magnetic layer system. The main aim of this pilot study is to evaluate the applicability of this promising sensor for the detection of a human heart signal and to evaluate the sensor output with competitive optical magnetometry technology. A magnetic equivalent of a human R wave could be successfully detected within a 1-min measurement period with the sensor presented here. Finally, the article will provide an outlook on future ME perspectives and challenges, especially for cardiovascular applications.

Abstract In recent years, SGLT2 inhibitors have become an integral part of heart failure therapy, and several mechanisms contributing to cardiorenal protection have been identified. In this study, we place special emphasis on the atria and investigate acute electrophysiological effects of dapagliflozin to assess the antiarrhythmic potential of SGLT2 inhibitors. Direct electrophysiological effects of dapagliflozin were investigated in patch clamp experiments on isolated atrial cardiomyocytes. Acute treatment with elevated-dose dapagliflozin caused a significant reduction of the action potential inducibility, the amplitude and maximum upstroke velocity. The inhibitory effects were reproduced in human induced pluripotent stem cell-derived cardiomyocytes, and were more pronounced in atrial compared to ventricular cells. Hypothesizing that dapagliflozin directly affects the depolarization phase of atrial action potentials, we examined fast inward sodium currents in human atrial cardiomyocytes and found a significant decrease of peak sodium current densities by dapagliflozin, accompanied by a moderate inhibition of the transient outward potassium current. Translating these findings into a porcine large animal model, acute elevated-dose dapagliflozin treatment caused an atrial-dominant reduction of myocardial conduction velocity in vivo. This could be utilized for both, acute cardioversion of paroxysmal atrial fibrillation episodes and rhythm control of persistent atrial fibrillation. In this study, we show that dapagliflozin alters the excitability of atrial cardiomyocytes by direct inhibition of peak sodium currents. In vivo , dapagliflozin exerts antiarrhythmic effects, revealing a potential new additional role of SGLT2 inhibitors in the treatment of atrial arrhythmias.

<h2>Abstract</h2><h3>Background</h3> The clinical chemistry score (CCS) comprising high-sensitivity cardiac troponins (hs-cTn), glucose and estimated glomerular filtration rate has been previously validated with superior accuracy for detection and risk stratification of acute myocardial infarction (AMI) compared to hs-cTn alone. <h3>Methods</h3> The CCS was compared to other biomarker-based algorithms for rapid rule-out and prognostication of AMI including the hs-cTnT limit-of-blank (LOB, <3 ng/L) or limit-of-detection (LOD, <5 ng/L) and a dual marker strategy (DMS) (copeptin <10 pmol/L and hs-cTnT ≤14 ng/L) in 1506 emergency department (ED) patients with symptoms suggestive of acute coronary syndrome. Negative predictive values (NPV) and sensitivities for AMI rule-out, and 12-month combined endpoint rates encompassing mortality, myocardial re-infarction, as well as stroke were assessed. <h3>Results</h3> NPVs of 100% (95% CI: 98.3–100%) were observed for CCS = 0, hs-cTnT LoB and hs-cTnT LoD with rule-out efficacies of 11.1%, 7.6% and 18.3% as well as specificities of 13.0% (95% CI: 9.9–16.6%), 8.8% (95% CI: 7.3–10.5%) and 21.4% (95% CI: 19.2–23.8%), respectively. A CCS ≤ 1 achieved a rule-out in 32.2% of all patients with a NPV of 99.6% (95% CI: 98.4–99.9%) and specificity of 37.4% (95% CI: 34.2–40.5%) compared to a rule-out efficacy of 51.2%, NPV of 99.0 (95% CI: 98.0–99.5) and specificity of 59.7% (95% CI: 57.0–62.4%) for the DMS. Rates of the combined end-point of death/AMI within 30 days ranged between 0.0% and 0.7% for all fast-rule-out protocols. <h3>Conclusions</h3> The CCS ensures reliable AMI rule-out with low short and long-term outcome rates for a specific ED patient subset. However, compared to a single or dual biomarker strategy, the CCS displays reduced efficacy and specificity, limiting its clinical utility.

Rupture or dissection of thoracic aortic aneurysms is still the leading cause of death for patients diagnosed with Marfan syndrome. Inflammation and matrix digestion regulated by matrix metalloproteases (MMPs) play a major role in the pathological remodeling of the aortic media. Regnase-1 is an endoribonuclease shown to cleave the mRNA of proinflammatory cytokines, such as interleukin-6. Considering the major anti-inflammatory effects of regnase-1, here, we aimed to determine whether adeno-associated virus (AAV)–mediated vascular overexpression of the protein could provide protection from the development and progression of aortic aneurysms in Marfan syndrome. The overexpression of regnase-1 resulted in a marked decrease in inflammatory parameters and elastin degradation in aortic smooth muscle cells <i>in vitro</i>. Intravenous injection of a vascular-targeted AAV vector resulted in the efficient transduction of the aortic wall and overexpression of regnase-1 in a murine model of Marfan syndrome, associated with lower circulating levels of proinflammatory cytokines and decreased MMP expression and activity. Regnase-1 overexpression strongly improved elastin architecture in the media and reduced aortic diameter at distinct locations. Therefore, AAV-mediated regnase-1 overexpression may represent a novel gene therapy approach for inhibiting aortic aneurysms in Marfan syndrome.

Human dilated cardiomyopathy (DCM), a disorder of the cardiac muscle, causes considerable morbidity and mortality and is one of the major causes of sudden cardiac death. Genetic factors play a role in the etiology and pathogenesis of DCM. Disease-associated genetic variations identified to date have been identified in single families or single sporadic patients and explain a minority of the etiology of DCM. We show that a 600-kb region of linkage disequilibrium (LD) on 5q31.2-3, harboring multiple genes, is associated with cardiomyopathy in three independent Caucasian populations (combined P-value = 0.00087). Functional assessment in zebrafish demonstrates that at least three genes, orthologous to loci in this LD block, HBEGF, IK, and SRA1, result independently in a phenotype of myocardial contractile dysfunction when their expression is reduced with morpholino antisense reagents. Evolutionary analysis across multiple vertebrate genomes suggests that this heart failure-associated LD block emerged by a series of genomic rearrangements across amphibian, avian, and mammalian genomes and is maintained as a cluster in mammals. Taken together, these observations challenge the simple notion that disease phenotypes can be traced to altered function of a single locus within a haplotype and suggest that a more detailed assessment of causality can be necessary.

Receptor-interacting protein kinase 3 (RIPK3) mediates necroptosis, a form of programmed cell death that promotes inflammation in various pathological conditions, suggesting that it might be a privileged pharmacological target. However, its function in glucose homeostasis and obesity has been unknown. Here we show that RIPK3 is over expressed in the white adipose tissue (WAT) of obese mice fed with a choline-deficient high-fat diet. Genetic inactivation of Ripk3 promotes increased Caspase-8-dependent adipocyte apoptosis and WAT inflammation, associated with impaired insulin signalling in WAT as the basis for glucose intolerance. Similarly to mice, in visceral WAT of obese humans, RIPK3 is overexpressed and correlates with the body mass index and metabolic serum markers. Together, these findings provide evidence that RIPK3 in WAT maintains tissue homeostasis and suppresses inflammation and adipocyte apoptosis, suggesting that systemic targeting of necroptosis might be associated with the risk of promoting insulin resistance in obese patients.

The optimal treatment of patients with acute and severe decompensation of aortic stenosis is unclear due to recent advances in transcatheter interventions and supportive therapies. Our aim was to assess the early outcome of emergency transcatheter aortic valve implantation (eTAVI) versus emergency balloon aortic valvuloplasty (eBAV) followed by TAVI under elective circumstances.Emergency conditions were defined as: cardiogenic shock with requirement of catecholamine therapy, severe acute dyspnoea (NYHA IV), cardiac resuscitation or mechanic respiratory support. The data were collected according to the Valve Academic Research Consortium 2 (VARC-2) criteria.In five German centres, 23 patients (logistic Euroscore 37.7%±18.1) underwent eTAVI and 118 patients underwent eBAV (logistic Euroscore 35.3%±20.8). In the eTAVI group, immediate procedural mortality was 8.7%, compared with 20.3% for the eBAV group (p=0.19). After 30 days, cardiovascular mortality for the eTAVI group was 23.8% and for the eBAV group 33.0% (p=0.40). Analyses adjusting for potential confounders did not provide evidence of a difference between groups. Of note, the elective TAVI performed after eBAV (n=32, logistic Euroscore 25.9%±13.9) displayed an immediate procedural mortality of 9.4% and a cardiovascular mortality after 30 days of 15.6%. Major vascular complications were significantly more likely to occur after eTAVI (p=0.01) as well as stroke (p=0.01).In this multicentre cohort, immediate procedural and 30-day mortality of eTAVI and eBAV were high, and mortality of secondary TAVI subsequent to eBAV was higher than expected. Randomised study data are required to define the role of emergency TAVI in tertiary care centres with current device generations.

Adequate protein turnover is essential for cardiac homeostasis. Different protein quality controls are involved in the maintenance of protein homeostasis, including molecular chaperones and co-chaperones, the autophagy-lysosomal pathway, and the ubiquitin-proteasome system (UPS). In the last decade, a series of evidence has underlined a major function of the UPS in cardiac physiology and disease. Particularly, recent studies have shown that dysfunctional proteasomal function leads to cardiac disorders. Hypertrophic and dilated cardiomyopathies are the two most prevalent inherited cardiomyopathies. Both are primarily transmitted as an autosomal-dominant trait and mainly caused by mutations in genes encoding components of the cardiac sarcomere, including a relevant striated muscle-specific E3 ubiquitin ligase. A growing body of evidence indicates impairment of the UPS in inherited cardiomyopathies as determined by measurement of the level of ubiquitinated proteins, the activities of the proteasome and/or the use of fluorescent UPS reporter substrates. The present review will propose mechanisms of UPS impairment in inherited cardiomyopathies, summarize the potential consequences of UPS impairment, including activation of the unfolded protein response, and underline some therapeutic options available to restore proteasome function and therefore cardiac homeostasis and function. This article is part of a Special Issue entitled "Protein Quality Control, the Ubiquitin Proteasome System, and Autophagy".

Cardiac type 2 ryanodine receptors (RyR2s) play a pivotal role in cellular electrophysiology and contractility. Increased RyR2-mediated diastolic sarcoplasmic reticulum (SR) Ca2+ release is linked to heart failure (HF) and arrhythmias. Dantrolene, a drug used for the treatment of malignant hyperthermia, is known to stabilize RyRs in skeletal muscle.The purpose of this study was to investigate the effects of dantrolene on arrhythmogenic triggers and contractile function in human atrial fibrillation (AF) and HF cardiomyocytes (CM).Human CM were isolated from either patients with HF (ventricular) or patients with AF (atrial), and Ca2+ imaging, patch-clamp, or muscle strip experiments were performed.After exposure to dantrolene, human atrial AF and left ventricular HF CM showed significant reductions in proarrhythmic SR Ca2+ spark frequency and diastolic SR Ca2+ leak. Moreover, dantrolene decreased the frequency of Ca2+ waves and spontaneous Ca2+ transients in HF CM. Patch-clamp experiments revealed that dantrolene significantly suppressed delayed afterdepolarizations in HF and AF CM. Importantly, dantrolene had no effect on action potential duration in AF or in HF CM. In addition, dantrolene had neutral effects on contractile force of human isometrically twitching ventricular HF trabeculae.Our study showed that dantrolene beneficially influenced disrupted SR Ca2+ homeostasis in human HF and AF CM. Cellular arrhythmogenic triggers were potently suppressed by dantrolene, whereas action potential duration and contractility were not affected. As a clinically approved drug for the treatment of malignant hyperthermia, dantrolene may be a potential antiarrhythmic drug for patients with rhythm disorders and merits further clinical investigation.

Thin-film magnetoelectric sensors are able to measure very low magnetic fields. As a consequence the hypothesis that magnetoelectric sensors could be used for biomagnetic measurements was often mentioned but never proven. In this contribution the first proof of this hypothesis will be given by the measurement of the (well-known) R-wave of the human heart. This will be achieved by closing the gap between the sensor sensitivity and the signal level by averaging. In order to guarantee a fast convergence of the averaging process even in very noisy (realistic) measurement environments, different adaptive averaging techniques in the time- and frequency-domain are pointed out. The evaluation by synthetic measurements shows an improvement of the averaging process by up to 20 dB in terms of signal-to-noise ratio for an instationary measurement scenario in comparison to the conventional averaging after 750 average periods. Finally, measurements of the R-wave of a human heart are performed.

Secreted and membrane-bound proteins, which account for 1/3 of all proteins, play critical roles in heart health and disease. The endoplasmic reticulum (ER) is the site for synthesis, folding, and quality control of these proteins. Loss of ER homeostasis and function underlies the pathogenesis of many forms of heart disease.To investigate mechanisms responsible for regulating cardiac ER function, and to explore therapeutic potentials of strengthening ER function to treat heart disease.Screening a range of signaling molecules led to the discovery that Pak (p21-activated kinase)2 is a stress-responsive kinase localized in close proximity to the ER membrane in cardiomyocytes. We found that Pak2 cardiac deleted mice (Pak2-CKO) under tunicamycin stress or pressure overload manifested a defective ER response, cardiac dysfunction, and profound cell death. Small chemical chaperone tauroursodeoxycholic acid treatment of Pak2-CKO mice substantiated that Pak2 loss-induced cardiac damage is an ER-dependent pathology. Gene array analysis prompted a detailed mechanistic study, which revealed that Pak2 regulation of protective ER function was via the IRE (inositol-requiring enzyme)-1/XBP (X-box-binding protein)-1-dependent pathway. We further discovered that this regulation was conferred by Pak2 inhibition of PP2A (protein phosphatase 2A) activity. Moreover, IRE-1 activator, Quercetin, and adeno-associated virus serotype-9-delivered XBP-1s were able to relieve ER dysfunction in Pak2-CKO hearts. This provides functional evidence, which supports the mechanism underlying Pak2 regulation of IRE-1/XBP-1s signaling. Therapeutically, inducing Pak2 activation by genetic overexpression or adeno-associated virus serotype-9-based gene delivery was capable of strengthening ER function, improving cardiac performance, and diminishing apoptosis, thus protecting the heart from failure.Our findings uncover a new cardioprotective mechanism, which promotes a protective ER stress response via the modulation of Pak2. This novel therapeutic strategy may present as a promising option for treating cardiac disease and heart failure.

Abstract Aims Uromodulin is produced exclusively in the kidney and secreted into both urine and blood. Serum levels of uromodulin are correlated with kidney function and reduced in chronic kidney disease (CKD) patients, but physiological functions of serum uromodulin are still elusive. This study investigated the role of uromodulin in medial vascular calcification, a key factor associated with cardiovascular events and mortality in CKD patients. Methods and results Experiments were performed in primary human (HAoSMCs) and mouse (MOVAS) aortic smooth muscle cells, cholecalciferol overload and subtotal nephrectomy mouse models and serum from CKD patients. In three independent cohorts of CKD patients, serum uromodulin concentrations were inversely correlated with serum calcification propensity. Uromodulin supplementation reduced phosphate-induced osteo-/chondrogenic transdifferentiation and calcification of HAoSMCs. In human serum, pro-inflammatory cytokines tumour necrosis factor α (TNFα) and interleukin-1β (IL-1β) co-immunoprecipitated with uromodulin. Uromodulin inhibited TNFα and IL-1β-induced osteo-/chondrogenic signalling and activation of the transcription factor nuclear factor kappa-light-chain-enhancer of activated β cells (NF-kB) as well as phosphate-induced NF-kB-dependent transcriptional activity in HAoSMCs. In vivo, adeno-associated virus (AAV)-mediated overexpression of uromodulin ameliorated vascular calcification in mice with cholecalciferol overload. Conversely, cholecalciferol overload-induced vascular calcification was aggravated in uromodulin-deficient mice. In contrast, uromodulin overexpression failed to reduce vascular calcification during renal failure in mice. Carbamylated uromodulin was detected in serum of CKD patients and uromodulin carbamylation inhibited its anti-calcific properties in vitro. Conclusions Uromodulin counteracts vascular osteo-/chondrogenic transdifferentiation and calcification, at least in part, through interference with cytokine-dependent pro-calcific signalling. In CKD, reduction and carbamylation of uromodulin may contribute to vascular pathology.

Abstract Dysregulation of extracellular signal-regulated kinases (ERK1/2) is linked to several diseases including heart failure, genetic syndromes and cancer. Inhibition of ERK1/2, however, can cause severe cardiac side-effects, precluding its wide therapeutic application. ERK T188 -autophosphorylation was identified to cause pathological cardiac hypertrophy. Here we report that interference with ERK-dimerization, a prerequisite for ERK T188 -phosphorylation, minimizes cardiac hypertrophy without inducing cardiac adverse effects: an ERK-dimerization inhibitory peptide (EDI) prevents ERK T188 -phosphorylation, nuclear ERK1/2-signaling and cardiomyocyte hypertrophy, protecting from pressure-overload-induced heart failure in mice whilst preserving ERK1/2-activity and cytosolic survival signaling. We also examine this alternative ERK1/2-targeting strategy in cancer: indeed, ERK T188 -phosphorylation is strongly upregulated in cancer and EDI efficiently suppresses cancer cell proliferation without causing cardiotoxicity. This powerful cardio-safe strategy of interfering with ERK-dimerization thus combats pathological ERK1/2-signaling in heart and cancer, and may potentially expand therapeutic options for ERK1/2-related diseases, such as heart failure and genetic syndromes.

Increased shedding of extracellular vesicles (EVs)-small, lipid bilayer-delimited particles with a role in paracrine signalling-has been associated with human pathologies, e.g. atherosclerosis, but whether this is true for cardiac diseases is unknown.Here, we used the surface antigen CD172a as a specific marker of cardiomyocyte (CM)-derived EVs; the CM origin of CD172a+ EVs was supported by their content of cardiac-specific proteins and heart-enriched microRNAs. We found that patients with aortic stenosis, ischaemic heart disease, or cardiomyopathy had higher circulating CD172a+ cardiac EV counts than did healthy subjects. Cellular stress was a major determinant of EV release from CMs, with hypoxia increasing shedding in in vitro and in vivo experiments. At the functional level, EVs isolated from the supernatant of CMs derived from human-induced pluripotent stem cells and cultured in a hypoxic atmosphere elicited a positive inotropic response in unstressed CMs, an effect we found to be dependent on an increase in the number of EVs expressing ceramide on their surface. Of potential clinical relevance, aortic stenosis patients with the highest counts of circulating cardiac CD172a+ EVs had a more favourable prognosis for transcatheter aortic valve replacement than those with lower counts.We identified circulating CD172a+ EVs as cardiac derived, showing their release and function and providing evidence for their prognostic potential in aortic stenosis patients.

The randomized SOLVE-TAVI (compariSon of secOnd-generation seLf-expandable vs. balloon-expandable Valves and gEneral vs. local anesthesia in Transcatheter Aortic Valve Implantation) trial compared newer-generation self-expanding valves (SEV) and balloon-expandable valves (BEV) as well as local anesthesia with conscious sedation (CS) and general anesthesia (GA) in patients undergoing transfemoral transcatheter aortic valve replacement (TAVR). Both strategies showed similar outcomes at 30 days.The purpose of this study was to compare clinical outcomes during 1-year follow-up in the randomized SOLVE-TAVI trial.Using a 2 × 2 factorial design 447 intermediate- to high-risk patients with severe, symptomatic aortic stenosis were randomly assigned to transfemoral TAVR using either the SEV (Evolut R, Medtronic Inc., Minneapolis, Minnesota) or the BEV (Sapien 3, Edwards Lifesciences, Irvine, California) as well as CS or GA at 7 sites.In the valve-comparison strategy, rates of the combined endpoint of all-cause mortality, stroke, moderate or severe paravalvular leakage, and permanent pacemaker implantation were similar between the BEV and SEV group (n = 84, 38.3% vs. n = 87, 40.4%; hazard ratio: 0.94; 95% confidence interval: 0.70 to 1.26; p = 0.66) at 1 year. Regarding the anesthesia comparison, the combined endpoint of all-cause mortality, stroke, myocardial infarction, and acute kidney injury occurred with similar rates in the GA and CS groups (n = 61, 25.7% vs. n = 54, 23.8%; hazard ratio: 1.09; 95% confidence interval: 0.76 to 1.57; p = 0.63).In intermediate- to high-risk patients undergoing transfemoral TAVR, newer-generation SEV and BEV as well as CS and GA showed similar clinical outcomes at 1 year using a combined clinical endpoint. (SecOnd-generation seLf-expandable Versus Balloon-expandable Valves and gEneral Versus Local Anesthesia in TAVI [SOLVE-TAVI]; NCT02737150).

Patients undergoing surgery for severe aortic stenosis (SAS) can be treated with either transcatheter aortic valve implantation (TAVI) or surgical aortic valve replacement (SAVR). The choice of procedure depends on several factors, including the clinical judgement of the heart team and patient preferences, which are captured by actively informing and involving patients in a process of shared decision making (SDM). We synthesised the most up-to-date and accessible evidence on the benefits and risks that may be associated with TAVI versus SAVR to support SDM in this highly personalised decision-making process.Systematic review and meta-analysis.MEDLINE (Ovid), Embase (Ovid) and the Cochrane Central Register of Controlled Trials (CENTRAL; Wiley) were searched from January 2000 to August 2020 with no language restrictions. Reference lists of included studies were searched to identify additional studies.Randomised controlled trials (RCTs) that compared TAVI versus SAVR in patients with SAS and reported on all-cause or cardiovascular mortality, length of stay in intensive care unit or hospital, valve durability, rehospitalisation/reintervention, stroke (any stroke or major/disabling stroke), myocardial infarction, major vascular complications, major bleeding, permanent pacemaker (PPM) implantation, new-onset or worsening atrial fibrillation (NOW-AF), endocarditis, acute kidney injury (AKI), recovery time or pain were included.Two independent reviewers were involved in data extraction and risk of bias (ROB) assessment using the Cochrane tool (one reviewer extracted/assessed the data, and the second reviewer checked it). Dichotomous data were pooled using the Mantel-Haenszel method with random-effects to generate a risk ratio (RR) with 95% CI. Continuous data were pooled using the inverse-variance method with random-effects and expressed as a mean difference (MD) with 95% CI. Heterogeneity was assessed using the I2 statistic.8969 records were retrieved and nine RCTs (61 records) were ultimately included (n=8818 participants). Two RCTs recruited high-risk patients, two RCTs recruited intermediate-risk patients, two RCTs recruited low-risk patients, one RCT recruited high-risk (≥70 years) or any-risk (≥80 years) patients; and two RCTs recruited all-risk or 'operable' patients. While there was no overall change in the risk of dying from any cause (30 day: RR 0.89, 95% CI 0.65 to 1.22; ≤1 year: RR 0.90, 95% CI 0.79 to 1.03; 5 years: RR 1.09, 95% CI 0.98 to 1.22), cardiovascular mortality (30 day: RR 1.03, 95% CI 0.77 to 1.39; ≤1 year: RR 0.90, 95% CI 0.76 to 1.06; 2 years: RR 0.96, 95% CI 0.83 to 1.12), or any type of stroke (30 day: RR 0.83, 95% CI 0.61 to 1.14;≤1 year: RR 0.94, 95% CI 0.72 to 1.23; 5 years: RR 1.07, 95% CI 0.88 to 1.30), the risk of several clinical outcomes was significantly decreased (major bleeding, AKI, NOW-AF) or significantly increased (major vascular complications, PPM implantation) for TAVI vs SAVR. TAVI was associated with a significantly shorter hospital stay vs SAVR (MD -3.08 days, 95% CI -4.86 to -1.29; 4 RCTs, n=2758 participants). Subgroup analysis generally favoured TAVI patients receiving implantation via the transfemoral (TF) route (vs non-TF); receiving a balloon-expandable (vs self-expanding) valve; and those at low-intermediate risk (vs high risk). All RCTs were rated at high ROB, predominantly due to lack of blinding and selective reporting.No overall change in the risk of death from any cause or cardiovascular mortality was identified but 95% CIs were often wide, indicating uncertainty. TAVI may reduce the risk of certain side effects while SAVR may reduce the risk of others. Most long-term (5-year) results are limited to older patients at high surgical risk (ie, early trials), therefore more data are required for low risk populations. Ultimately, neither surgical technique was considered dominant, and these results suggest that every patient with SAS should be individually engaged in SDM to make evidence-based, personalised decisions around their care based on the various benefits and risks associated with each treatment.CRD42019138171.

Mitral valve transcatheter edge-to-edge repair (TEER) has been established as a suitable alternative to mitral valve surgery in patients with severe mitral regurgitation (MR) and high surgical risk. The PASCAL system represents a novel device, potentially augmenting the toolkit for TEER. The aim of this study was to assess and compare short and 1 year safety and efficacy of the PASCAL and MitraClip systems for TEER.Procedural, short, and 1 year outcomes of a 1:2 propensity-matched cohort including 41 PASCAL and 82 MitraClip cases were investigated. Matching was based on clinical, laboratory, echocardiographic, and functional characteristics. The primary endpoints assessed were procedural success [as defined by the Mitral Valve Academy Research Consortium (MVARC)], residual MR, functional class, and a composite endpoint comprising death, heart failure hospitalization, and mitral valve re-intervention. We found for the PASCAL and the matched MitraClip cohort no significant differences in MVARC defined technical (90.2% vs. 95.1%, P = 0.44), device (90.2% vs. 89.0%, P = 1.0), or procedural (87.8% vs. 80.5%, P = 0.45) success rates. Accordingly, the overall MR reduction and improvement in New York Heart Association (NYHA) class were comparable (1 year follow-up: MR ≤ 2 95% vs. 93.6%, P = 1.0; NYHA ≤ 2 57.1% vs. 66.7%, P = 0.59). The composite outcome revealed no statistically significant difference between both devices (1 year follow-up: 31.7% vs. 37.8%, P = 0.55). Interestingly, we found at both short and 1 year follow-up a significantly higher rate of patients with none or trace MR in the PASCAL-treated cohort (short follow-up: 17.9% vs. 0%, P = 0.0081; 1 year follow-up: 25% vs. 0%, P = 0.0016). Conversely, the rate of aborted device implantations due to an elevated transmitral gradient was higher in PASCAL interventions (9.8% vs. 1.2%, P = 0.04).Transcatheter edge-to-edge repair using the PASCAL or MitraClip device results in favourable and comparable outcomes regarding safety, efficacy, and clinical improvement after 1 year.

Der Einsatz von digitalen Technologien in der Medizin entwickelt sich weitgehend parallel zur IT-Branche. Heute nutzt jeder Kardiologe/in in Deutschland IT-Systeme, um Termine zu vereinbaren, Patientendaten zu speichern und Arztbriefe zu verfassen. Warum ist es also notwendig, innerhalb der Deutschen Gesellschaft für Kardiologie (DGK) ein eigenes eCardiology-Programm einzuführen? Bisher hat die Digitalisierung ihr Versprechen, den Alltag von Ärzten und Patienten zu erleichtern, in vielerlei Hinsicht nicht erfüllt. Klinische EDV-Lösungen stammen vorwiegend von buchhalterischen Systemen ab und führen nur begrenzt zur Verbesserung der Patientenversorgung. Es konnten sogar negative Auswirkungen auf die Burn-out-Raten und die Arbeitszufriedenheit von medizinischem Personal beobachtet werden. Zusätzlich hat seit Mai 2018 die Einführung der europäischen Datenschutz-Grundverordnung mit ihren Grundsätzen der Zweckbindung und Datenminimierung die Speicherung und Nutzung medizinischer Daten erschwert. Dies hat auch grundlegende Implikationen für die Entwicklungen der künstlichen Intelligenz (KI), die auf der Verfügbarkeit und Qualität von Big Data (= große, komplexe Datenbestände) beruhen und das Gesundheitssystem nachhaltig verändern könnten. Der technologiegetriebene Fortschritt der Medizin endet dabei nicht an der Türschwelle eines Krankenhauses, sondern bewirkt einen maßgeblichen kulturellen Wandel in unserer Gesellschaft. Der Einsatz von sog. „Wearables“, Suchmaschinen im Internet und „Social Media“ ermöglicht den „e-Patienten“ immer öfter, selbst Diagnosen zu stellen und eigenständige Entscheidungen in Bezug auf ihre Gesundheit zu treffen. Ärzte sehen dadurch oftmals gut, aber auch falsch informierte Patienten, die von ihren eigenen Smart Devices zu gesundheitsrelevanten Daten beraten wurden. Die DGK erkennt die enormen Herausforderungen und das Potenzial der digitalen Medizin, die Prognose kardiovaskulärer Erkrankungen zu verbessern. Im Rahmen des eCardiology-Programms hat die Gesellschaft 5 Ausschüsse gegründet, die wichtige Aspekte von Digital Health fördern und vermitteln: transsektorale Zusammenarbeit, Mobile Health, Precision Digital Health, Gesellschaft und Politik sowie Ausbildung und Medien. Wir berichten hier über die Elemente jedes Ausschusses und seiner Arbeitsgruppen.

Left atrial appendage closure (LAAC) has emerged as an alternative to oral anticoagulation (OAC) for stroke prevention in patients with atrial fibrillation (AF). OAC treatment has been proven feasible in mild-to-moderate chronic kidney disease (CKD). In contrast, the optimal antithrombotic management of AF patients with end-stage renal disease (ESRD) is unknown and LAAC has not been proven in these patients in prospective randomized clinical trials.The objective of this study is to evaluate safety and efficacy of LAAC in patients with ESRD.Patients undergoing LAAC were collected in a German multicenter real-world observational registry. A composite endpoint consisting of the occurrence of ischemic stroke/transient ischemic attack, systemic embolism, and/or major clinical bleeding was assessed. Patients with ESRD were compared with propensity score-matched patients without severe CKD. ESRD was defined as a glomerular filtration rate < 15 ml/min/1.73 m2 or chronic hemodialysis treatment.A total of 604 patients were analyzed, including 57 with ESRD and 57 propensity-matched patients. Overall, 596 endocardial and 8 epicardial LAAC procedures were performed. Frequency of major complications was 7.0% (42/604 patients) in the overall cohort, 8.8% (5/57 patients) in patients with ESRD, and 10.5% (6/57 patients) in matched controls (p = 0.75). The estimated event-free survival of the combined endpoint after 500 days was 90.7 ± 4.5% in patients with ESRD and 90.2 ± 5.5% in matched controls (p = 0.33).LAAC had comparable procedural safety and clinical efficacy in patients with ESRD and patients without severe CKD.

The German Centre for Cardiovascular Research (DZHK) is one of the German Centres for Health Research and aims to conduct early and guideline-relevant studies to develop new therapies and diagnostics that impact the lives of people with cardiovascular disease. Therefore, DZHK members designed a collaboratively organised and integrated research platform connecting all sites and partners. The overarching objectives of the research platform are the standardisation of prospective data and biological sample collections among all studies and the development of a sustainable centrally standardised storage in compliance with general legal regulations and the FAIR principles. The main elements of the DZHK infrastructure are web-based and central units for data management, LIMS, IDMS, and transfer office, embedded in a framework consisting of the DZHK Use and Access Policy, and the Ethics and Data Protection Concept. This framework is characterised by a modular design allowing a high standardisation across all studies. For studies that require even tighter criteria additional quality levels are defined. In addition, the Public Open Data strategy is an important focus of DZHK. The DZHK operates as one legal entity holding all rights of data and biological sample usage, according to the DZHK Use and Access Policy. All DZHK studies collect a basic set of data and biosamples, accompanied by specific clinical and imaging data and biobanking. The DZHK infrastructure was constructed by scientists with the focus on the needs of scientists conducting clinical studies. Through this, the DZHK enables the interdisciplinary and multiple use of data and biological samples by scientists inside and outside the DZHK. So far, 27 DZHK studies recruited well over 11,200 participants suffering from major cardiovascular disorders such as myocardial infarction or heart failure. Currently, data and samples of five DZHK studies of the DZHK Heart Bank can be applied for.

Among low-risk patients with severe, symptomatic aortic stenosis who are eligible for both transcatheter aortic-valve implantation (TAVI) and surgical aortic-valve replacement (SAVR), data are lacking on the appropriate treatment strategy in routine clinical practice.In this randomized noninferiority trial conducted at 38 sites in Germany, we assigned patients with severe aortic stenosis who were at low or intermediate surgical risk to undergo either TAVI or SAVR. Percutaneous- and surgical-valve prostheses were selected according to operator discretion. The primary outcome was a composite of death from any cause or fatal or nonfatal stroke at 1 year.A total of 1414 patients underwent randomization (701 to the TAVI group and 713 to the SAVR group). The mean (±SD) age of the patients was 74±4 years; 57% were men, and the median Society of Thoracic Surgeons risk score was 1.8% (low surgical risk). The Kaplan-Meier estimate of the primary outcome at 1 year was 5.4% in the TAVI group and 10.0% in the SAVR group (hazard ratio for death or stroke, 0.53; 95% confidence interval [CI], 0.35 to 0.79; P<0.001 for noninferiority). The incidence of death from any cause was 2.6% in the TAVI group and 6.2% in the SAVR group (hazard ratio, 0.43; 95% CI, 0.24 to 0.73); the incidence of stroke was 2.9% and 4.7%, respectively (hazard ratio, 0.61; 95% CI, 0.35 to 1.06). Procedural complications occurred in 1.5% and 1.0% of patients in the TAVI and SAVR groups, respectively.Among patients with severe aortic stenosis at low or intermediate surgical risk, TAVI was noninferior to SAVR with respect to death from any cause or stroke at 1 year. (Funded by the German Center for Cardiovascular Research and the German Heart Foundation; DEDICATE-DZHK6 ClinicalTrials.gov number, NCT03112980.).

In addition to regulating cell motility, contractility, and cytokinesis, the actin cytoskeleton plays a critical role in the regulation of transcription and gene expression. We have previously identified a novel muscle-specific actin-binding protein, STARS (striated muscle activator of Rho signaling), which directly binds actin and stimulates serum-response factor (SRF)-dependent transcription. To further dissect the STARS/SRF pathway, we performed a yeast two-hybrid screen of a skeletal muscle cDNA library using STARS as bait, and we identified two novel members of the ABLIM protein family, ABLIM-2 and -3, as STARS-interacting proteins. ABLIM-1, which is expressed in retina, brain, and muscle tissue, has been postulated to function as a tumor suppressor. ABLIM-2 and -3 display distinct tissue-specific expression patterns with the highest expression levels in muscle and neuronal tissue. Moreover, these novel ABLIM proteins strongly bind F-actin, are localized to actin stress fibers, and synergistically enhance STARS-dependent activation of SRF. Conversely, knockdown of endogenous ABLIM expression utilizing small interfering RNA significantly blunted SRF-dependent transcription in C2C12 skeletal muscle cells. These findings suggest that the members of the novel ABLIM protein family may serve as a scaffold for signaling modules of the actin cytoskeleton and thereby modulate transcription. In addition to regulating cell motility, contractility, and cytokinesis, the actin cytoskeleton plays a critical role in the regulation of transcription and gene expression. We have previously identified a novel muscle-specific actin-binding protein, STARS (striated muscle activator of Rho signaling), which directly binds actin and stimulates serum-response factor (SRF)-dependent transcription. To further dissect the STARS/SRF pathway, we performed a yeast two-hybrid screen of a skeletal muscle cDNA library using STARS as bait, and we identified two novel members of the ABLIM protein family, ABLIM-2 and -3, as STARS-interacting proteins. ABLIM-1, which is expressed in retina, brain, and muscle tissue, has been postulated to function as a tumor suppressor. ABLIM-2 and -3 display distinct tissue-specific expression patterns with the highest expression levels in muscle and neuronal tissue. Moreover, these novel ABLIM proteins strongly bind F-actin, are localized to actin stress fibers, and synergistically enhance STARS-dependent activation of SRF. Conversely, knockdown of endogenous ABLIM expression utilizing small interfering RNA significantly blunted SRF-dependent transcription in C2C12 skeletal muscle cells. These findings suggest that the members of the novel ABLIM protein family may serve as a scaffold for signaling modules of the actin cytoskeleton and thereby modulate transcription. The actin cytoskeleton controls a wide range of cellular processes, particularly those that require shape changes, such as cell motility, contractility, mitosis and cytokinesis, axon outgrowth, as well as endocytosis and secretion (reviewed in Refs. 1Chen H. Bernstein B.W. Bamburg J.R. Trends Biochem. Sci. 2000; 25: 19-23Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar and 2Carlier M.F. Le Clainche C. Wiesner S. Pantaloni D. Bioessays. 2003; 25: 336-345Crossref PubMed Scopus (124) Google Scholar). Beyond these mechanical functions, actin has also been shown to play a critical role in the regulation of transcription and gene expression, either through its direct association with nuclear chromatin-remodeling proteins (reviewed in Ref. 3Rando O.J. Zhao K. Crabtree G.R. Trends Cell Biol. 2000; 10: 92-97Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar) or indirectly through cytoplasmic changes in cytoskeletal actin dynamics (4Sotiropoulos A. Gineitis D. Copeland J. Treisman R. Cell. 1999; 98: 159-169Abstract Full Text Full Text PDF PubMed Scopus (578) Google Scholar). The latter effects are tightly controlled by the state of actin polymerization, i.e. the equilibrium between incorporation of monomeric actin (G-actin) at the barbed end of a filament and the dissociation of actin from the pointed end of polymerized actin (F-actin). This process, referred to as “actin treadmilling,” is regulated by several signal transduction cascades that converge on actin-binding proteins such as cofilin/actin-depolymerizing factor, profilin, and β-thymosin (reviewed in Ref. 5Wear M.A. Schafer D.A. Cooper J.A. Curr. Biol. 2000; 10: 891-895Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). The Rho family of GTPases, including the best characterized members Rho, Rac, and Cdc42, serve as molecular switches in the regulation of a wide variety of signal transduction pathways (6Sorokina E.M. Chernoff J. J. Cell. Biochem. 2005; 94: 225-231Crossref PubMed Scopus (37) Google Scholar, 7Etienne-Manneville S. Hall A. Nature. 2002; 420: 629-635Crossref PubMed Scopus (3927) Google Scholar), in particular actin polymerization and stress fiber formation (8Ridley A.J. Hall A. Cell. 1992; 70: 389-399Abstract Full Text PDF PubMed Scopus (3892) Google Scholar). Rho GTPases alternate between two conformational states, the active state (bound to GTP) and the inactive state (bound to GDP). This balance is, in turn, carefully regulated by numerous activators (guanine nucleotide exchange factors (GEFs)) 5The abbreviations used are: GEF, guanine nucleotide exchange factor; ABLIM, actin-binding LIM protein; MRTF, myocardin-related transcription factor; SRF, serum-response factor; GST, glutathione S-transferase; siRNA, small interfering RNA; EST, expressed sequence tag; TRITC, tetramethylrhodamine isothiocyanate; PBS, phosphate-buffered saline; BSA, bovine serum albumin. and inactivators (GTPase-activating proteins (GAPs) and guanine nucleotide dissociation inhibitors). Rho effector molecules include the kinase p160 ROCK and the mammalian homologue of diaphanous (mDia), both of which promote stress fiber formation (9Burridge K. Wennerberg K. Cell. 2004; 116: 167-179Abstract Full Text Full Text PDF PubMed Scopus (1539) Google Scholar). Furthermore, RhoA signaling has been shown to stimulate the transcriptional activity of the serum response factor (SRF) via changes in actin dynamics (4Sotiropoulos A. Gineitis D. Copeland J. Treisman R. Cell. 1999; 98: 159-169Abstract Full Text Full Text PDF PubMed Scopus (578) Google Scholar, 10Mack C.P. Somlyo A.V. Hautmann M. Somlyo A.P. Owens G.K. J. Biol. Chem. 2001; 276: 341-347Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar, 11Hill C.S. Wynne J. Treisman R. Cell. 1995; 81: 1159-1170Abstract Full Text PDF PubMed Scopus (1210) Google Scholar). SRF, a MADS (MCM1, Agamous, Deficiens and SRF)-box containing transcription factor, regulates expression of immediate-early genes as well as muscle-specific genes (reviewed in Ref. 12Miano J.M. J. Mol. Cell. Cardiol. 2003; 35: 577-593Abstract Full Text Full Text PDF PubMed Scopus (499) Google Scholar). More recently, the mechanism whereby SRF senses increased levels of polymerized actin has been elucidated (13Miralles F. Posern G. Zaromytidou A.I. Treisman R. Cell. 2003; 113: 329-342Abstract Full Text Full Text PDF PubMed Scopus (1086) Google Scholar, 14Posern G. Miralles F. Guettler S. Treisman R. EMBO J. 2004; 23: 3973-3983Crossref PubMed Scopus (119) Google Scholar). The SRF-co-activator MRTF-A/MAL, a member of the myocardin family of SRF-binding transcription factors (15Wang D.Z. Li S. Hockemeyer D. Sutherland L. Wang Z. Schratt G. Richardson J.A. Nordheim A. Olson E.N. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14855-14860Crossref PubMed Scopus (418) Google Scholar, 16Wang D. Chang P.S. Wang Z. Sutherland L. Richardson J.A. Small E. Krieg P.A. Olson E.N. Cell. 2001; 105: 851-862Abstract Full Text Full Text PDF PubMed Scopus (766) Google Scholar), is sequestered in the cytoplasm of unstimulated cells by association with unpolymerized actin. Upon activation of RhoA, actin becomes polymerized and thus releases MRTF-A/MAL, which translocates to the nucleus to associate with SRF. We previously identified a novel muscle-specific actin-binding protein, STARS (striated muscle activator of Rho signaling), which directly binds actin and co-localizes with actin stress fibers (17Arai A. Spencer J.A. Olson E.N. J. Biol. Chem. 2002; 277: 24453-24459Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). STARS stimulates SRF-dependent transcription through a mechanism that requires actin polymerization and Rho GTPase activation. In muscle cells, STARS regulates the nuclear import of the myocardin-related transcription factors (MRTFs) via depletion of the G-actin pool, thus establishing a mechanism of STARS-dependent SRF activation (18Kuwahara K. Barrientos T. Pipes G.C. Li S. Olson E.N. Mol. Cell. Biol. 2005; 25: 3173-3181Crossref PubMed Scopus (182) Google Scholar). Interestingly, in cardiomyocytes STARS is localized to the sarcomere, thus providing a potential link between contractile function and signaling. In this regard, several recent reports suggest that the sarcomere indeed serves a critical role in sensing biomechanical stress and activation of downstream signaling pathways (19Pyle W.G. Solaro R.J. Circ. Res. 2004; 94: 296-305Crossref PubMed Scopus (208) Google Scholar, 20Frey N. Barrientos T. Shelton J.M. Frank D. Rutten H. Gehring D. Kuhn C. Lutz M. Rothermel B. Bassel-Duby R. Richardson J.A. Katus H.A. Hill J.A. Olson E.N. Nat. Med. 2004; 10: 1336-1343Crossref PubMed Scopus (186) Google Scholar, 21Brancaccio M. Fratta L. Notte A. Hirsch E. Poulet R. Guazzone S. De Acetis M. Vecchione C. Marino G. Altruda F. Silengo L. Tarone G. Lembo G. Nat. Med. 2003; 9: 68-75Crossref PubMed Scopus (251) Google Scholar, 22Knoll R. Hoshijima M. Hoffman H.M. Person V. Lorenzen-Schmidt I. Bang M.L. Hayashi T. Shiga N. Yasukawa H. Schaper W. McKenna W. Yokoyama M. Schork N.J. Omens J.H. McCulloch A.D. Kimura A. Gregorio C.C. Poller W. Schaper J. Schultheiss H.P. Chien K.R. Cell. 2002; 111: 943-955Abstract Full Text Full Text PDF PubMed Scopus (643) Google Scholar). An important function of Rho-dependent signaling in this context (reviewed in Ref. 23Geiger B. Bershadsky A. Cell. 2002; 110: 139-142Abstract Full Text Full Text PDF PubMed Scopus (355) Google Scholar) is further supported by the finding that Rho is not only activated upon pressure overload and biomechanical stress in cardiomyocytes (24Torsoni A.S. Fonseca P.M. Crosara-Alberto D.P. Franchini K.G. Am. J. Physiol. 2003; 284: C1411-C1419Crossref PubMed Scopus (37) Google Scholar) but is also required for the ensuing hypertrophic response (25Hoshijima M. Sah V.P. Wang Y. Chien K.R. Brown J.H. J. Biol. Chem. 1998; 273: 7725-7730Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 26Sah V.P. Hoshijima M. Chien K.R. Brown J.H. J. Biol. Chem. 1996; 271: 31185-31190Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar, 27Hines W.A. Thorburn A. J. Mol. Cell. Cardiol. 1998; 30: 485-494Abstract Full Text PDF PubMed Scopus (55) Google Scholar, 28Pan J. Singh U.S. Takahashi T. Oka Y. Palm-Leis A. Herbelin B.S. Baker K.M. J. Cell. Physiol. 2005; 202: 536-553Crossref PubMed Scopus (89) Google Scholar). Finally, a RhoA-specific GEF, p63 Rho GEF, has been identified that is highly expressed in the heart and also localized to the sarcomere (29Souchet M. Portales-Casamar E. Mazurais D. Schmidt S. Leger I. Javre J.L. Robert P. Berrebi-Bertrand I. Bril A. Gout B. Debant A. Calmels T.P. J. Cell Sci. 2002; 115: 629-640Crossref PubMed Google Scholar), further supporting the hypothesis that sarcomeric Rho signaling plays an important role in striated muscle tissue. In an attempt to further dissect the Rho/STARS/SRF pathway in muscle tissue, we performed a yeast two-hybrid screen of a skeletal muscle cDNA library using STARS as bait. From this screen we identified two novel members of the ABLIM protein family, ABLIM-2 and -3, as STARS-interacting molecules. ABLIM-1 was originally found in human retina as well as in the sarcomeres of murine cardiac tissue and was postulated to regulate actin-dependent signaling (30Roof D.J. Hayes A. Adamian M. Chishti A.H. Li T. J. Cell Biol. 1997; 138: 575-588Crossref PubMed Scopus (110) Google Scholar). Likewise, ABLIM-2 and -3 display distinct tissue-specific expression patterns with the highest expression levels in muscle and neuronal tissue. Moreover, these novel ABLIM proteins are localized to actin stress fibers and show a sarcomeric localization in striated muscle. We show that ABLIM-2 and -3 both strongly bind F-actin and can augment STARS-dependent SRF activation, suggesting that this new protein family serves as a scaffold for signaling modules of the actin cytoskeleton. Yeast Two-hybrid Screen—An amino-terminally truncated mouse STARS cDNA (encoding amino acids 234-375) was fused to the GAL4 DNA binding domain (plasmid Pas1; Clontech) and was used as bait in a yeast two-hybrid screen of ∼1 × 106 clones of a human skeletal muscle cDNA library (Clontech), as described (31Frey N. Olson E.N. J. Biol. Chem. 2002; 277: 13998-14004Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). Briefly, clones displaying differential growth on selective plates, lacking histidine, leucine, and tryptophan, were picked and replated for β-galactosidase assays. Positive clones were grown in selective medium lacking leucine, and plasmid DNA was isolated and subsequently electroporated into Escherichia coli strain DH10B (Invitrogen). The obtained clones were sequenced and retransformed with the original STARS construct to confirm the interaction. Cloning of ABLIM-2 and -3 and Bioinformatics—Partial ABLIM-2 cDNA sequences identified by yeast two-hybrid screens were used to screen the data base (GenBank™) for overlapping expressed sequence tags (EST). After bioinformatic construction of the respective open reading frames, the following primers were used to PCR-clone full-length mouse ABLIM-2 (forward, 5′-ATGAGCGCAGTGTCGCAGCC-3′, and reverse 5′-CTGTCAGAACAGCAAGGCTTTC-3′), human ABLIM-2 (forward, 5′-GACTCCGAGCGGCTGCTGAG-3′, and reverse, 5′-CAGGCTCGCTGGCAGCCGTC-3′), and human ABLIM-3 (forward, 5′-GCAGCCGGGGCCTCCGTATTG-3′, and reverse, 5′-GAGCCTCTGCCTAGAACAGCC-3′) from human and mouse skeletal muscle, cardiac tissue, as well as brain cDNA. In addition, expression constructs were cloned for all three ABLIM proteins, encoding for an amino-terminal fusion with a FLAG tag, as described previously (31Frey N. Olson E.N. J. Biol. Chem. 2002; 277: 13998-14004Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). Human ABLIM-3 was fused with a GST domain (Pgex2T vector) as described (32Frey N. Richardson J.A. Olson E.N. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14632-14637Crossref PubMed Scopus (255) Google Scholar) to allow for GST pulldown experiments. Northern Blot Analysis and Radioactive in Situ Hybridization—Multiple tissue Northern blots (Clontech) containing mouse and human poly(A) RNA were hybridized overnight at 65 °C with [32P]dCTP-labeled (Rediprime II random prime labeling system; Amersham Biosciences) cDNA probes corresponding to the open reading frame of mouse and human ABLIM-2 and -3, respectively. Serial washes were conducted with 2× SSC, 0.1% SDS, and 0.2× SSC, 0.1% SDS at 65 °C. Autoradiography was performed at -80 °C for 24-48 h with an intensifying screen. For radioactive in situ hybridization, RNA probes corresponding to sense and antisense strands of ABLIM-2 and ABLIM-3 cDNAs were prepared, using T7 and T3 RNA polymerase (Roche Applied Science) and 35S-labeled UTP. Sections of mouse embryos at various time points were subjected to in situ hybridization, as described (33Lu J. Richardson J.A. Olson E.N. Mech. Dev. 1998; 73: 23-32Crossref PubMed Scopus (129) Google Scholar). Sense probes were used as negative controls. Generation of an ABLIM-2-specific Antiserum and Western Blot Analysis—A peptide consisting of 15 amino-terminal amino acids (NH2-SQPQAAHAPLEKPAS-OH) of mouse ABLIM-2 was synthesized (Biosynthesis) and used to generate antisera in rabbits. The amino terminus was chosen to generate an isoform-specific antiserum, because this part of the protein does not display significant homology to the other two ABLIM family members (supplemental Fig. 1). Similarly, 14 amino-terminal amino acids of mouse ABLIM-3 (NH2-PYQQSPYSPRGGSN-OH) were utilized to generate an ABLIM-3 antiserum. IgG was purified from rabbit serum using protein A-Sepharose beads (Amersham Biosciences) and subsequently used for Western blotting as well as immunostaining of mouse skeletal muscle cryosections. Immunostaining—The subcellular localization of ABLIM-2 was determined in cryosections of mouse hindlimb skeletal muscle tissue using indirect immunofluorescence. Cryosections were air-dried and fixed in 4% paraformaldehyde for 5 min, followed by three washes with PBS, permeabilization with 0.3% Triton X-100 (Sigma), and blocking in 3% horse serum for 1 h. Primary antibodies were incubated for 1 h at the following dilutions: polyclonal anti-ABLIM-2 1:100, polyclonal anti-STARS 1:50, and monoclonal anti-sarcomeric actinin (Sigma) 1:200. Secondary antibodies conjugated to either fluorescein or TRITC (Vector Laboratories) were also incubated for 1 h at a dilution of 1:250. Transfected C2C12 cells were rinsed with PBS, fixed with 4% paraformaldehyde for 10 min, permeabilized, and blocked with 0.1% Triton X-100 and 2% bovine serum albumin (BSA) in PBS for 30 min at room temperature. Cells were then incubated with primary antibodies (anti-FLAG monoclonal antibody (Sigma), 1:200; anti-ABLIM-2 polyclonal antibody, 1:75) in 2% BSA in PBS for 1 h. Secondary antibodies conjugated to fluorescein or TRITC (Vector Laboratories) were used at 1:200. Vectashield medium with 4′,6′-diamidino-2-phenylindole (Vector Laboratories) was used for mounting. Tissue Culture, Immunoprecipitations, and Reporter Gene Assays—293T cells were maintained in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum, 2 mml-glutamine, and penicillin/streptomycin. 2 × 105 cells were transfected with 1 μg of expression plasmids for full-length ABLIM-1, -2, and -3 as well as a Myc-tagged STARS construct (17Arai A. Spencer J.A. Olson E.N. J. Biol. Chem. 2002; 277: 24453-24459Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar), using FuGENE 6 reagent (Roche Applied Science). Forty eight hours after transfection, cells were harvested in RIPA buffer, containing 10 mm Tris (pH 7.5), 15 mm EDTA, 0.5% sodium deoxycholate, 1% Nonidet P-40, 1 mm dithiothreitol, 0.1% SDS, and a protease inhibitor mixture (Complete; Roche Applied Science). Cells were briefly sonicated, and debris was removed by centrifugation. Tagged proteins were immunoprecipitated for 2-3 h at 4 °C using protein A/G-agarose and 1 μg of the appropriate antibody (monoclonal anti-FLAG (Sigma) and polyclonal anti-Myc (Santa Cruz Biotechnology)). Subsequently, the pellet was washed with ELB buffer and subjected to SDS-PAGE, followed by transfer to polyvinylidene membranes and immunoblotting using anti-FLAG and anti-Myc antibodies, as indicated. COS-7 cells as well as C2C12 cells were transfected with expression plasmids encoding Myc-STARS, FLAG-ABLIM-2 or FLAG-ABLIM-3, and an SM22 promoter-luciferase construct as well as a β-galactosidase construct to normalize for efficiency of transfection, using FuGENE 6 reagent (Roche Applied Science). All experiments were conducted in duplicate and repeated at least three times. Luciferase assays from COS cell lysates were performed using a kit, as suggested by the manufacturer (Promega). RNA Interference and Real Time PCR—C2C12 myoblasts were transfected using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. To verify efficiency of siRNA-mediated knockdown of the ABLIMs 1-3, RNA was prepared using TRIzol reagent (Invitrogen). Reverse transcription of 1 μg of total RNA per sample was carried out using the Superscript III first strand synthesis system for reverse transcription-PCR and random primers (Invitrogen). Real time PCR was performed in an ABI 7700 thermocycler applying the Platinum® SYBR® Green qPCR SuperMix-UDG (Invitrogen). Resulting data were normalized to 18 S rRNA. The siRNAs used were as follows: ABLIM-1, sense 5′-CCAAGCAUUUCCACAUCAA-3′ and antisense 5′-UUGAUGUGGAAAUGCUUGG-3′ (Eurogentec, Belgium). The siRNAs for ABLIM-2 and -3 were obtained from Dharmacon (ONTARGET plus; catalogue numbers 058308 (ABLIM-2) and 041985 (ABLIM-3)). Negative control siRNA duplexes (OR-0030-NEG05) were purchased from Eurogentec. Primers for real time PCR analyses were designed using the primer3 software. All amplicons spanned at least one intron-exon-intron boundary to prevent amplification of contaminating genomic DNA as follows: 18 S rRNA forward 5′-TCAAGAACGAAAGTCGGAGG-3′ and 18 S rRNA reverse 5′-GGACATCTAAGGGCATCAC-3′; ABLIM-1 forward 5′-TGGTTCACCAGGCCATACTA-3′ and ABLIM-1 reverse 5′-CTTCTGCAGATGGAGTTGGA-3′; ABLIM-2 forward 5′-CAGCCAGGACTGAAGACAAA-3′ and ABLIM-2 reverse 5′-AGCAGCCAAGTCCCTGTAGT-3′; and ABLIM-3 forward 5′-TCTGGAGGAGAGGAAGAGGA-3′ and ABLIM-3 reverse 5′-CAGTGAGGCAGATTTGGAGA-3′. GST Pulldown Assays—For GST pulldown experiments, 10 μl of in vitro translated ABLIM-3 was mixed with 500 μl of binding buffer (150 mm NaCl, 20 mm Tris (pH 7.5), 0.5% Nonidet P-40, protease inhibitors (Roche Applied Science)) and added to agarose-GST or agarose-GST-STARS. The mixture was incubated with rotation for 1 h at 4 °C followed by four washes with cold binding buffer. After the washes, 25 μl of protein loading dye was added to the pulldown reaction. The proteins were boiled at 100 °C for 5 min and run on a 10% Tris-acrylamide gel. Actin Co-sedimentation Assay—Full-length cDNA clones of FLAG-ABLIM-2 and -3 were expressed in vitro using the TnT ® T7-coupled reticulocyte lysate system (Promega). Equal expression levels were confirmed by Western blotting. Purified actin, α-actinin, and BSA were purchased from Cytoskeleton Inc. Actin co-sedimentation assays were performed according to the manufacturer’s protocol. Pellets and supernatants were analyzed by SDS-PAGE, Western blotting, and Ponceau S staining. Western blots were performed with the anti-FLAG monoclonal antibody M2 (1 μg/ml; Sigma) and the monoclonal anti-α-actinin antibody (1:1000; Sigma), respectively. Yeast Two-hybrid Screen with STARS and Molecular Cloning of ABLIM-2 and ABLIM-3—To identify novel STARS-interacting proteins in striated muscle tissue, we performed a yeast two-hybrid screen of a human skeletal muscle cDNA library using the evolutionarily conserved carboxyl terminus of STARS (amino acids 234-375) as bait. This region of STARS has been shown to be necessary and sufficient for actin binding and for stimulation of SRF-dependent transcription (17Arai A. Spencer J.A. Olson E.N. J. Biol. Chem. 2002; 277: 24453-24459Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). From this screen, we identified three β-galactosidase-positive clones encoding for skeletal muscle actin (ACTA1), confirming our previous findings that STARS is an actin-binding protein (17Arai A. Spencer J.A. Olson E.N. J. Biol. Chem. 2002; 277: 24453-24459Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). Two additional independent clones encoded a novel open reading frame. Data bank comparisons revealed partial identity of these clones with a previously uncharacterized protein fragment, KIAA1808 (GenBank™ accession number BAB47437). To obtain a full-length clone, we designed primers, using the longest human EST sequences available in the data base, and subsequently cloned the entire open reading frame (supplemental Fig. 1), encoding 649 amino acids (GenBank™ accession number DQ413176). We also identified a shorter splice variant encoding 559 amino acids (Fig. 1C and see supplemental Fig. 1) (GenBank™ accession number DQ413177), as well as the mouse homologue (GenBank™ accession number DQ413175), which displays high homology to the human protein (Fig. 1A). These novel proteins contain four LIM domains as well as a carboxyl-terminal villin domain (Fig. 1A). Further querying of the data base revealed high homology with the actin-binding LIM protein (ABLIM), which had originally been found in human retina (30Roof D.J. Hayes A. Adamian M. Chishti A.H. Li T. J. Cell Biol. 1997; 138: 575-588Crossref PubMed Scopus (110) Google Scholar). Moreover, the data base contained a sequence for another previously uncharacterized putative protein fragment with homology to both ABLIM and KIAA1808 and KIAA0843 (GenBank™ accession number BAA74866). Utilizing the EST data base, we designed primers for the human homologue of this putative novel protein and subcloned the open reading frame from human heart cDNA (Fig. 1A and supplemental Fig. 1) (GenBank™ accession number DQ413174). The identical domain architecture (Fig. 1B), as well as the high homology between these three proteins, suggests that they constitute a novel protein family, and we thus designated the newly identified members ABLIM-2 and ABLIM-3 (Fig. 1). Interestingly, ABLIM proteins are evolutionary conserved with (single) homologues in Caenorhabditis elegans (UNC-115) and Drosophila (D-UNC-115) (Fig. 1, A and B). ABLIM-2 and -3 Are Expressed in a Tissue-specific Pattern—Because STARS is expressed in a highly striated muscle-specific fashion, we examined if ABLIM-2 and -3 might display a similar expression pattern. An adult human multiple tissue Northern blot revealed that ABLIM-2 is highly expressed in skeletal muscle (Fig. 2A, left panel) and at lower levels in brain, spleen, and kidney. In heart and skeletal muscle, at least two distinct bands could be observed, consistent with the existence of splice variants. No significant expression was detected in the heart. Because we identified two possible splice variants of human ABLIM-2 (Fig. 1C), we examined the tissue distribution of the longer splice variant (hABLIMv1), using a probe specific for the 270 nucleotides unique to this isoform. However, we observed the same expression pattern as shown in Fig. 2A (data not shown). In contrast to ABLIM-2, ABLIM-3 is predominantly expressed in human heart and brain, whereas the murine ABLIM-3 homologue displays a somewhat broader tissue distribution that also includes lung and liver. Temporospatial Expression Patterns of ABLIM-2/-3—To analyze the temporospatial expression patterns of ABLIM-2 and ABLIM-3, we performed radioactive in situ hybridization experiments (Fig. 2B). At embryonic day (E) 15.5 of mouse development, ABLIM-2 is predominantly expressed in skeletal muscle tissue, including the diaphragm, and to a lesser extent, in the central nervous system. At the same time point, ABLIM-3 also displays strong expression in skeletal muscle. These findings demonstrate a developmental regulation of ABLIM gene expression, especially of the expression of ABLIM-3, which is highly expressed during embryonic development (Fig. 2B) but down-regulated in adult skeletal muscle (Fig. 2A). ABLIM-2 and-3 Are Expressed in Distinct Regions of the Brain—Given that ABLIM-2 and -3 are both expressed in brain, we sought to further define their expression profiles in central nervous tissue and thus conducted analyses of adult mouse brain sections. Interestingly, these experiments revealed very distinct and nonoverlapping patterns for ABLIM-2 and -3 (Fig. 3, top and bottom). In contrast to ABLIM-3 (panel B2), ABLIM-2 is highly expressed in caudate/putamen (panel A2). Conversely, the olfactory bulb stains strongly for ABLIM-3 (panel B3), whereas ABLIM-2 is only moderately expressed (panel A3). Both genes are expressed in the hippocampus, whereas ABLIM-2 is detected in the CA1, CA2, and CA3 fields of the hippocampus (Fig. 3, panel A4), and ABLIM-3 is found selectively in the CA2 and CA3 fields (panel B4). In cerebellum, Purkinje cells are positive for ABLIM-2 (Fig. 3, panel A5), whereas internal granular cells are selectively positive for ABLIM-3 (panel B5). Protein Expression and Subcellular Localization of ABLIM-2—To analyze the expression pattern of the ABLIM-2 protein, we generated antisera against a synthetic peptide specific for mouse ABLIM-2. Western blots (Fig. 4A) confirmed the expression of ABLIM-2 in skeletal muscle and brain. Interestingly, at least two distinct bands could be detected, suggesting either different splice products of the ablim-2 gene or tissue-specific differential post-translational modifications of the protein. We also generated an antiserum against ABLIM-3 that detects the protein when overexpressed in COS cells (data not shown). However, this antiserum displays a high background when used in tissue preparations and was not useful for further analyses. Staining of mouse skeletal muscle hindlimb cryosections with the anti-ABLIM-2 antibody (Fig. 4B, upper panels) revealed a striated pattern, consistent with a sarcomeric or costameric localization of the protein. Similarly, staining of skeletal muscle cryosections with a polyclonal anti-STARS antibody (17Arai A. Spencer J.A. Olson E.N. J. Biol. Chem. 2002; 277: 24453-24459Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar) revealed the same pattern (Fig. 4B, lower panels), suggesting that ABLIM proteins and STARS co-localize in vivo. ABLIM-2 and -3 Bind F-actin—Both ABLIM-2 and -3 contain a “villin domain” (also called villin headpiece domain) composed of 74 amino acids at their carboxyl termini. This domain is highly conserved among the three different ABLIM isoforms (supplemental Fig. 1), as well as in ABLIM proteins from other species, including C. elegans (UNC-115) and Drosophila (D-UN-115), and has been shown to mediate actin binding (34Vermeulen W. Vanhaesebrouck P. Van Troys M. Verschueren M. Fant F. Goethals M. Ampe C. Martins J.C. Borremans F.A. Protein Sci. 2004; 13: 1276-1287Crossref PubMe

Activation of the phosphatase calcineurin and its downstream targets, transcription factors of the NFAT family, results in cardiomyocyte hypertrophy. Recently, it has been shown that the dual specificity tyrosine (Y) phosphorylation-regulated kinase 1A (DYRK1A) is able to antagonize calcineurin signaling by directly phosphorylating NFATs. We thus hypothesized that DYRK1A might modulate the hypertrophic response of cardiomyocytes. In a model of phenylephrine-induced hypertrophy, adenovirus-mediated overexpression of DYKR1A completely abrogated the hypertrophic response and significantly reduced the expression of the natriuretic peptides ANF and BNP. Furthermore, DYRK1A blunted cardiomyocyte hypertrophy induced by overexpression of constitutively active calcineurin and attenuated the induction of the hypertrophic gene program. Conversely, knockdown of DYRK1A, utilizing adenoviruses encoding for a specific synthetic miRNA, resulted in an increase in cell surface area accompanied by up-regulation of ANF- mRNA. Similarly, treatment of cardiomyocytes with harmine, a specific inhibitor of DYRK1A, revealed cardiomyocyte hypertrophy on morphological and molecular level. Moreover, constitutively active calcineurin led to robust induction of an NFAT-dependent luciferase reporter, whereas DYRK1A attenuated calcineurin-induced reporter activation in cardiomyocytes. Conversely, both knockdown and pharmacological inhibition of DYRK1A significantly augmented the effect of calcineurin in this assay. In summary, we identified DYRK1A as a novel negative regulator of cardiomyocyte hypertrophy. Mechanistically, this effect appears to be mediated via inhibition of NFAT transcription factors. Activation of the phosphatase calcineurin and its downstream targets, transcription factors of the NFAT family, results in cardiomyocyte hypertrophy. Recently, it has been shown that the dual specificity tyrosine (Y) phosphorylation-regulated kinase 1A (DYRK1A) is able to antagonize calcineurin signaling by directly phosphorylating NFATs. We thus hypothesized that DYRK1A might modulate the hypertrophic response of cardiomyocytes. In a model of phenylephrine-induced hypertrophy, adenovirus-mediated overexpression of DYKR1A completely abrogated the hypertrophic response and significantly reduced the expression of the natriuretic peptides ANF and BNP. Furthermore, DYRK1A blunted cardiomyocyte hypertrophy induced by overexpression of constitutively active calcineurin and attenuated the induction of the hypertrophic gene program. Conversely, knockdown of DYRK1A, utilizing adenoviruses encoding for a specific synthetic miRNA, resulted in an increase in cell surface area accompanied by up-regulation of ANF- mRNA. Similarly, treatment of cardiomyocytes with harmine, a specific inhibitor of DYRK1A, revealed cardiomyocyte hypertrophy on morphological and molecular level. Moreover, constitutively active calcineurin led to robust induction of an NFAT-dependent luciferase reporter, whereas DYRK1A attenuated calcineurin-induced reporter activation in cardiomyocytes. Conversely, both knockdown and pharmacological inhibition of DYRK1A significantly augmented the effect of calcineurin in this assay. In summary, we identified DYRK1A as a novel negative regulator of cardiomyocyte hypertrophy. Mechanistically, this effect appears to be mediated via inhibition of NFAT transcription factors. Cardiac hypertrophy accompanies a variety of heart diseases and is an independent predictor of cardiovascular morbidity and mortality (1Levy D. Garrison R.J. Savage D.D. Kannel W.B. Castelli W.P. N. Engl. J. Med. 1990; 322: 1561-1566Crossref PubMed Scopus (4935) Google Scholar). Hypertrophy may develop in response to a variety of pathological stimuli, e.g. arterial hypertension or heart valve disease (2Frey N. Olson E.N. Annu. Rev. Physiol. 2003; 65: 45-79Crossref PubMed Scopus (1212) Google Scholar, 3Hill J.A. Olson E.N. N. Engl. J. Med. 2008; 358: 1370-1380Crossref PubMed Scopus (944) Google Scholar, 4Frey N. Katus H.A. Olson E.N. Hill J.A. Circulation. 2004; 109: 1580-1589Crossref PubMed Scopus (702) Google Scholar). While numerous molecular pathways have been implicated in the development of cardiomyocyte hypertrophy, the phosphatase calcineurin and its downstream targets, transcription factors of the nuclear factor of activated T-cells (NFAT) 2The abbreviations used are: NFATnuclear factor of activated T-cellsANFatrial natriuretic factorANPatrial natriuretic peptideBNPB-type natriuretic peptideCnAcalcineurin ADYRK1Adual specificity tyrosine(Y) phosphorylation-regulated kinase 1AET-1endothelin-1moimultiplicity of infectionsNRVMneonatal rat ventricular cardiomyocytesPEphenylephrineRCANregulator of calcineurinDAPI4′,6-diamidino-2-phenylindole. family, appear to play a central role. Activation of calcineurin leads to dephosphorylation of NFATs, subsequent nuclear translocation, and initiation of the hypertrophic gene program as well as marked cardiomyocyte hypertrophy (5Molkentin J.D. Lu J.R. Antos C.L. Markham B. Richardson J. Robbins J. Grant S.R. Olson E.N. Cell. 1998; 93: 215-228Abstract Full Text Full Text PDF PubMed Scopus (2241) Google Scholar). In contrast, genetic ablation of calcineurin Aβ renders the heart unresponsive to hypertrophic stimuli (6Bueno O.F. Wilkins B.J. Tymitz K.M. Glascock B.J. Kimball T.F. Lorenz J.N. Molkentin J.D. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 4586-4591Crossref PubMed Scopus (225) Google Scholar). In addition, inhibitors of calcineurin signaling have been shown to attenuate cardiac hypertrophy (7De Windt L.J. Lim H.W. Bueno O.F. Liang Q. Delling U. Braz J.C. Glascock B.J. Kimball T.F. del Monte F. Hajjar R.J. Molkentin J.D. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3322-3327Crossref PubMed Scopus (181) Google Scholar, 8Frank D. Kuhn C. van Eickels M. Gehring D. Hanselmann C. Lippl S. Will R. Katus H.A. Frey N. Circulation. 2007; 116: 2587-2596Crossref PubMed Scopus (51) Google Scholar, 9Rothermel B.A. McKinsey T.A. Vega R.B. Nicol R.L. Mammen P. Yang J. Antos C.L. Shelton J.M. Bassel-Duby R. Olson E.N. Williams R.S. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3328-3333Crossref PubMed Scopus (269) Google Scholar). nuclear factor of activated T-cells atrial natriuretic factor atrial natriuretic peptide B-type natriuretic peptide calcineurin A dual specificity tyrosine(Y) phosphorylation-regulated kinase 1A endothelin-1 multiplicity of infections neonatal rat ventricular cardiomyocytes phenylephrine regulator of calcineurin 4′,6-diamidino-2-phenylindole. Recently, the dual specificity tyrosine (Y) phosphorylation-regulated kinase 1A (DYRK1A) has been identified as a novel modifier of NFAT transcription factors in Drosophila (10Gwack Y. Sharma S. Nardone J. Tanasa B. Iuga A. Srikanth S. Okamura H. Bolton D. Feske S. Hogan P.G. Rao A. Nature. 2006; 441: 646-650Crossref PubMed Scopus (325) Google Scholar) and vertebrate neurons (11Arron J.R. Winslow M.M. Polleri A. Chang C.P. Wu H. Gao X. Neilson J.R. Chen L. Heit J.J. Kim S.K. Yamasaki N. Miyakawa T. Francke U. Graef I.A. Crabtree G.R. Nature. 2006; 441: 595-600Crossref PubMed Scopus (561) Google Scholar). DYRK1A directly phosphorylates NFATs, thereby promoting their nuclear export followed by a reduction of transcriptional activity. DYRK1A, which belongs to a family of dual specificity kinases, is ubiquitously expressed with high levels in the developing nervous system and the heart (12Okui M. Ide T. Morita K. Funakoshi E. Ito F. Ogita K. Yoneda Y. Kudoh J. Shimizu N. Genomics. 1999; 62: 165-171Crossref PubMed Scopus (72) Google Scholar). All isoforms share the ability to autocatalyze their own phosphorylation at an YXY motif (13Becker W. Weber Y. Wetzel K. Eirmbter K. Tejedor F.J. Joost H.G. J. Biol. Chem. 1998; 273: 25893-25902Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar), while all other substrates are phosphorylated at Ser/Thr residues. DYRK1A, DYRK1B, and the Drosophila homolog, minibrain, are predominantly localized to the nucleus. In addition to NFAT, DYRK1A is capable of phosphorylating other transcription factors, including FKHR/Foxo1 (14Woods Y.L. Rena G. Morrice N. Barthel A. Becker W. Guo S. Unterman T.G. Cohen P. Biochem. J. 2001; 355: 597-607Crossref PubMed Scopus (222) Google Scholar), STAT3 (15Matsuo R. Ochiai W. Nakashima K. Taga T. J. Immunol. Methods. 2001; 247: 141-151Crossref PubMed Scopus (62) Google Scholar), and Gli1 (16Mao J. Maye P. Kogerman P. Tejedor F.J. Toftgard R. Xie W. Wu G. Wu D. J. Biol. Chem. 2002; 277: 35156-35161Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). Furthermore DYRK1A stimulates the phosphorylation of CREB (17Yang E.J. Ahn Y.S. Chung K.C. J. Biol. Chem. 2001; 276: 39819-39824Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). Because the DYRK1A gene is localized to the Down syndrome critical region and DYRK1A is overexpressed in Down syndrome fetal brain, it was proposed to contribute to the phenotype of trisomy 21. Consistent with this notion, transgenic mouse models with overexpression of DYRK1A reveal learning impairment and hyperactivity (18Altafaj X. Dierssen M. Baamonde C. Martí E. Visa J. Guimerà J. Oset M. González J.R. Flórez J. Fillat C. Estivill X. Hum. Mol. Genet. 2001; 10: 1915-1923Crossref PubMed Scopus (332) Google Scholar, 19Branchi I. Bichler Z. Minghetti L. Delabar J.M. Malchiodi-Albedi F. Gonzalez M.C. Chettouh Z. Nicolini A. Chabert C. Smith D.J. Rubin E.M. Migliore-Samour D. Alleva E. J. Neuropathol. Exp. Neurol. 2004; 63: 429-440Crossref PubMed Scopus (83) Google Scholar). In contrast, little is known about the function of DYRK1A in the heart. Because DYRKs were recently recognized as kinases that phosphorylate NFATs, we hypothesized that the cardiac-enriched isoform DYRK1A might modulate cardiac hypertrophy. Here we show for the first time that DYRK1A overexpression potently inhibits cardiomyocyte hypertrophy. Conversely, miRNA-mediated knockdown or pharmacological inhibition of DYRK1A causes a hypertrophic phenotype accompanied by induction of the hypertrophic gene program and increased NFAT activity. Cloning of rat DYRK1A into a pDonRTM201 gateway vector (Invitrogen) was carried out using standard PCR techniques. An adenovirus (AdV) encoding for full-length rat DYRK1A cDNA with a C-terminal V5 tag was generated using the ViraPowerTM Adenoviral Expression System (Invitrogen) according to the manufacturer's protocol. A β-galactosidase V5-encoding adenovirus served as control (Invitrogen). Oligonucleotides encoding for microRNAs specifically targeting rat DYRK1A were designed using the Invitrogen oligo perfect software. Sequences of the inserts, which were subsequently cloned into the pcDNATM6.2-GW/EmGFP-miR vector: miR-544: 5′-TGC TGT TTA ATG GCG ACC CAT TCT TGG TTT TGG CCA CTG ACT GAC CAA GAA TGT CGC CAT TAA A-3′, miR-1331: 5′-TGC TGT CAA GTA GTC AGC TAC AGT GTG TTT TGG CCA CTG ACT GAC ACA CTG TAT GAC TAC TTG A-3′, miR-1732: 5′-TGC TGA ACA GGA TGA GTT TCA ACA GTG TTT TGG CCA CTG ACT GAC ACT GTT GAC TCA TCC TGT T-3′. As negative control, we used the pcDNATM6.2-GW/ EmGFP-miR plasmid, which can form a hairpin structure and is consecutively processed into a mature miRNA, yet is predicted not to target any known mammalian gene (sequence of the insert: 5′-GAA ATG TAC TGC GCG TGG AGA CGT TTT GGC CAC TGA CTG ACG TCT CCA CGC AGT ACA TTT-3′). Adenoviruses encoding for synthetic microRNAs were generated using the ViraPowerTM Adenoviral Expression System (Invitrogen) according to the manufacturer's protocol. Hearts from 1–2-day-old Wistar rats (Charles River) were excised and minced in ADS buffer (120 mmol/liter NaCl, 20 mmol/liter HEPES, 8 mmol/liter NaH2PO4, 6 mmol/liter glucose, 5 mmol/liter KCl, 0.8 mmol/liter MgSO4, pH 7.4). A series of digestion steps was carried out with an enzymatic solution containing collagenase type II (0.5 mg/ml, Worthington) and pancreatin (0.6 mg/ml, Sigma-Aldrich) in sterile ADS buffer. A Percoll (GE Healthcare) gradient centrifugation step was applied to remove contaminating fibroblasts from cardiomyocytes. NRVMs were resuspended and cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum, penicillin/streptomycin, and l-glutamine (PAA) for 24 h. After 24 h, cells were either infected in serum-free medium or serum-starved for 24 h before applying stimulants or inhibitors for the indicated time. Total RNA from NRVMs was isolated using the TRIzol method (Invitrogen) according to the manufacturer's protocol. DNaseI digestion and purification of RNA was carried out on RNeasy columns (Qiagen). DNaseI-digested total RNA of each condition was transcribed into cDNA using the Superscript III first strand kit (Invitrogen). Real-time PCR primer sequences are shown in supplemental Table S1. 18S rRNA served as an internal standard. For quantitative real-time PCR, the Platinum SYBR Green qPCR SuperMix-UDG system (Invitrogen) was used in the ABI Prism 7000 Sequence Detection System (Applied Biosystems). NRVMs were harvested in a lysis buffer containing 20 mmol/liter Tris, pH 7.5, 12.5% (v/v) glycerol, 10 mmol/liter dithiothreitol, 500 mmol/liter NaCl, 1% (v/v) Nonidet P-40 (Sigma-Aldrich), and protease inhibitor mixture tablets (Roche Diagnostics). After up to three brief freeze-and-thaw cycles and a centrifugation step, whole cell lysate was obtained. SDS-PAGE Western blotting was carried out using the WestranTM polyvinylidene difluoride membrane (Schleicher & Schuell). Application of the primary antibody (monoclonal anti-V5 (1:1000, Invitrogen), anti-DYRK1A (1:50, Santa Cruz Biotechnology), polyclonal anti-RCAN1 (1:1000), monoclonal anti-α-tubulin (1:10,000, Sigma-Aldrich)) was followed by incubation with a horseradish peroxidase-coupled secondary antibody (1:10,000, Santa Cruz Biotechnology). Visualization was achieved using a chemiluminescence kit (GE Healthcare). NRVMs were fixed in 4% paraformaldehyde (Sigma-Aldrich) in phosphate-buffered saline (PBS), permeabilized with 0.3% Triton X-100 (Sigma-Aldrich), and blocked for 1 h with 2.5% bovine serum albumin (Sigma-Aldrich) in PBS. The monoclonal antibody against sarcomeric α-actinin (Sigma-Aldrich) was used at a dilution of 1:200. The secondary antibody (Cy3-coupled antimouse antibody, Dianova) was used at 1:200 for 1 h. Nuclei were labeled by DAPI. Cell surface areas of cardiomyocytes were determined applying AxioVision Release 4.4 (Carl Zeiss Vision). 24 h after plating, neonatal cardiomyocytes were infected by an adenovirus (AdNFAT-luc, 20 moi) carrying a firefly luciferase construct under the transcriptional control of three consensus NFAT binding sites (Biomyx) and serum-starved for another 24 h. Simultaneous to the infection with the reporter virus, cells were infected by adenoviruses as indicated. To apply an identical virus load per condition, we used an adenovirus encoding for luciferase without NFAT binding sites (Adluc), which had been tested to not activate the reporter. Luciferase assays were performed according to the manufacturer's instructions (Promega). A β-galactosidase assay was performed in a 0.1 mol/liter sodium phosphate buffer (pH 7.3) with 1 mmol/liter MgCl2 as an internal control of infection. 2-Nitrophenyl-β-d-galactopyranoside (30 μg/ml) served as substrate. After incubation for 30 min, a photometric analysis was performed. Inhibitor assays were carried out using the DYRK1A inhibitor harmine (Fluka) dissolved in ethanol. Control cells received the vehicle only. All results are shown as the means ± standard error of the mean (S.E.) unless stated otherwise. Real-time PCR data analyses were carried out using the ΔΔct method. Statistical analyses of the data were carried out using one-way analysis of variance (ANOVA) followed by Student-Newman-Keuls post-hoc tests. If appropriate, the Student's t test (two sided) was employed. p values <0.05 were considered statistically significant. Recently it has been shown that DYRK1A inhibits the activation of NFAT transcription factors by phosphorylation (10Gwack Y. Sharma S. Nardone J. Tanasa B. Iuga A. Srikanth S. Okamura H. Bolton D. Feske S. Hogan P.G. Rao A. Nature. 2006; 441: 646-650Crossref PubMed Scopus (325) Google Scholar, 11Arron J.R. Winslow M.M. Polleri A. Chang C.P. Wu H. Gao X. Neilson J.R. Chen L. Heit J.J. Kim S.K. Yamasaki N. Miyakawa T. Francke U. Graef I.A. Crabtree G.R. Nature. 2006; 441: 595-600Crossref PubMed Scopus (561) Google Scholar). We thus hypothesized that DYRK1A might have an influence on cardiomyocyte hypertrophy. First, we asked whether the expression level of DYRK1A is modulated in established models of cardiomyocyte hypertrophy. Neonatal rat ventricular cardiomyocytes (NRVM) were stimulated with ET-1 (100 nmol/liter) or phenylephrine (PE, 50 μmol/liter) for 24 h, and DYRK1A protein expression was found to be significantly induced by either hypertrophic agent (PE: 1.6 ± 0.05-fold, ET-1: 1.4 ± 0.03-fold, p < 0.001, n = 6, Fig. 1, A and B). To further elucidate the role of DYRK1A in cardiomyocyte hypertrophy, we utilized adenoviral gene transfer for overexpression of DYRK1A (AdDYRK1A) in NRVMs treated with prohypertrophic agents. Infection of NRVM led to a dose-dependent overexpression of DYRK1A on mRNA and protein level (data not shown). NRVMs were infected by AdDYRK1A or by a control virus overexpressing β-galactosidase (AdβGal) and treated with the hypertrophic agonist phenylephrine. While stimulation with PE (100 μmol/liter, 24 h) led to a robust 1.5 ± 0.04-fold increase in cardiomyocyte surface area in control cells (p < 0.001, 50 cells per condition, n = 3), AdDYRK1A treatment completely abrogated cardiomyocyte hypertrophy induced by PE (p < 0.001, Fig. 2, A and B). Because cellular hypertrophy is accompanied by activation of specific genes (e.g. Nppa/ANF/ANP, Nppb/BNP, β-MyHC), we searched for expression changes of these members of the hypertrophic gene program by DYRK1A. DYRK1A blunted PE-mediated up-regulation of the mRNAs encoding for the natriuretic peptides ANF by 67.8% (p < 0.01, n = 6, Fig. 2C) and BNP by 75.8% (p < 0.001, n = 6, Fig. 2D). Interestingly, a catalytically inactive mutant (20Kentrup H. Becker W. Heukelbach J. Wilmes A. Schürmann A. Huppertz C. Kainulainen H. Joost H.G. J. Biol. Chem. 1996; 271: 3488-3495Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar) of DYRK1A (AdK188R) failed to inhibit PE-induced hypertrophy (50 cells per condition, n = 3, Fig. 2E). The cell surface area of PE-stimulated cardiomyocytes overexpressing DYRK1A-K188R did not differ from PE-treated cells overexpressing the control virus, suggesting that DYRK1A′s kinase activity is required for its antihypertrophic effect. Moreover, the overexpression of DYRK1A-K188R led to significant cellular hypertrophy (1.3 ± 0.04-fold, p < 0.001). To dissect the underlying pathway we utilized a constitutively active mutant of calcineurin A (CnA), a well known inducer of cardiomyocyte hypertrophy (21De Windt L.J. Lim H.W. Taigen T. Wencker D. Condorelli G. Dorn 2nd, G.W. Kitsis R.N. Molkentin J.D. Circ. Res. 2000; 86: 255-263Crossref PubMed Scopus (200) Google Scholar). Whereas overexpression of CnA led to a significant increase in cardiomyocyte surface area (1.6 ± 0.04-fold, p < 0.001, 100 cells per condition, n = 3, Fig. 3, A and B), DYRK1A inhibited the CnA-induced increase by 78% (p < 0.001). This difference in cell size was accompanied by an attenuated induction of the natriuretic peptides ANF and BNP. Both members of the hypertrophic gene program have been shown to be responsive to the calcineurin/NFAT pathway (5Molkentin J.D. Lu J.R. Antos C.L. Markham B. Richardson J. Robbins J. Grant S.R. Olson E.N. Cell. 1998; 93: 215-228Abstract Full Text Full Text PDF PubMed Scopus (2241) Google Scholar, 21De Windt L.J. Lim H.W. Taigen T. Wencker D. Condorelli G. Dorn 2nd, G.W. Kitsis R.N. Molkentin J.D. Circ. Res. 2000; 86: 255-263Crossref PubMed Scopus (200) Google Scholar, 22Yang J. Rothermel B. Vega R.B. Frey N. McKinsey T.A. Olson E.N. Bassel-Duby R. Williams R.S. Circ. Res. 2000; 87: E61-E68Crossref PubMed Google Scholar). DYRK1A caused a reduction of CnA-induced ANF-mRNA by −60% (p < 0.05, n = 3, Fig. 3C) and BNP-mRNA by −56% (p < 0.05, n = 3, Fig. 3D). Taken together, these data suggest that DYRK1A is sufficient to inhibit cardiomyocyte hypertrophy as well as the hypertrophic gene program. Next, we evaluated whether down-regulation of DYRK1A might in turn facilitate the development of cardiomyocyte hypertrophy. Thus, adenoviruses encoding for three different synthetic miRNAs targeting DYRK1A (AdmiR-544, -1331, -1732) were generated, and the most potent one was chosen for further analyses (AdmiR-1732/AdmiR-DYRK1A: −70.1% DYRK1A protein, Fig. 4, A and B). Treatment with 10 moi AdmiR-DYRK1A led to a reduction of −76.9% DYRK1A mRNA (p < 0.01, Fig. 4C). Knockdown of DYRK1A resulted in a significant increase of cardiomyocyte surface area (1.8 ± 0.05-fold, p < 0.001, 75 cells per condition, n = 3, Fig. 4, D and E), accompanied by a significant up-regulation of ANF expression (9.8 ± 2.8-fold, p < 0.05, n = 8, Fig. 4F), suggesting that DYRK1A is not only sufficient but also required for the inhibition of cardiomyocyte hypertrophy. To further confirm the antihypertrophic properties of DYRK1A, we utilized harmine, a specific inhibitor of DYRK1A (23Bain J. Plater L. Elliott M. Shpiro N. Hastie C.J. McLauchlan H. Klevernic I. Arthur J.S. Alessi D.R. Cohen P. Biochem. J. 2007; 408: 297-315Crossref PubMed Scopus (2141) Google Scholar, 24Sitz J.H. Baumgärtel K. Hämmerle B. Papadopoulos C. Hekerman P. Tejedor F.J. Becker W. Lutz B. Neuroscience. 2008; 157: 596-605Crossref PubMed Scopus (61) Google Scholar, 25Seifert A. Allan L.A. Clarke P.R. Febs J. 2008; 275: 6268-6280Crossref PubMed Scopus (107) Google Scholar). Consistent with the knockdown data, treatment of NRVMs with 1 μmol/liter harmine for 24 h resulted in a significant increase in cell size (1.6 ± 0.04-fold, p < 0.001, 100 cells per condition, n = 3, Fig. 5, A and B) compared with cardiomyocytes treated with vehicle only. Furthermore, harmine caused an induction of ANF expression up to 2.2 ± 0.2-fold (p < 0.05, Fig. 5C). To answer the question whether DYRK1A modulates the activity of NFAT transcription factors in NRVMs we performed luciferase reporter assays employing a reporter under transcriptional control of three NFAT binding sites. Infection of NRVMs by a constitutively active variant of calcineurin A (AdCnA; 30 moi) led to marked induction of the reporter (3.6 ± 0.1-fold, p < 0.001), whereas DYRK1A significantly and dose-dependently (AdDYRK1A; 30, 60, 90 moi) attenuated AdCnA-induced reporter activity by up to 50.1% (n = 3, Fig. 6A), indicating a negative regulation of NFAT activity by DYRK1A. Conversely, miRNA-mediated knockdown of DYRK1A (5, 10, 25 moi) resulted in an up to 4.3 ± 0.5-fold increase of luciferase activity (p < 0.001, Fig. 6B). Interestingly, DYRK1A knockdown further augmented NFAT induction by calcineurin (30 moi) up to 2.0-fold (p < 0.001, n = 12–18, Fig. 6B). Likewise, the inhibition of DYRK1A by 0.1 μmol/liter harmine markedly exacerbated the effect of calcineurin (+53%, p < 0.01, n = 9, Fig. 6C). Finally, we examined the regulation of the RCAN isoform 1–4 which is expressed from the RCAN1 locus by an alternative promoter (22Yang J. Rothermel B. Vega R.B. Frey N. McKinsey T.A. Olson E.N. Bassel-Duby R. Williams R.S. Circ. Res. 2000; 87: E61-E68Crossref PubMed Google Scholar, 26Davies K.J. Ermak G. Rothermel B.A. Pritchard M. Heitman J. Ahnn J. Henrique-Silva F. Crawford D. Canaider S. Strippoli P. Carinci P. Min K.T. Fox D.S. Cunningham K.W. Bassel-Duby R. Olson E.N. Zhang Z. Williams R.S. Gerber H.P. Pérez-Riba M. Seo H. Cao X. Klee C.B. Redondo J.M. Maltais L.J. Bruford E.A. Povey S. Molkentin J.D. McKeon F.D. Duh E.J. Crabtree G.R. Cyert M.S. de la Luna S. Estivill X. Faseb J. 2007; 21: 3023-3028Crossref PubMed Scopus (147) Google Scholar). This promoter is activated via binding of transcription factors of the NFAT family, whereas the promoter of RCAN1–1 is not sensitive to calcineurin/NFAT signaling. RCAN1–4 was significantly up-regulated in harmine-treated cardiomyocytes (Fig. 6D), again indicating that inhibition of DYRK1A leads to increased calcineurin/NFAT activity. Taken together, these data suggest that the antihypertrophic effect of DYRK1A is at least in part mediated via inhibition of NFAT activity. Here we show for the first time that DYRK1A plays a significant role in the regulation of cardiomyocyte hypertrophy. Our results suggest that DYRK1A is sufficient to completely inhibit agonist- as well as calcineurin-induced hypertrophy. Conversely, both down-regulation and pharmacological inhibition of DYRK1A leads to a hypertrophic phenotype in cardiomyocytes. It is well established that agonists of Gq/11-protein-coupled receptors, such as PE, mediate their effect through activation of the calcineurin/NFAT cascade (27Taigen T. De Windt L.J. Lim H.W. Molkentin J.D. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1196-1201Crossref PubMed Scopus (263) Google Scholar). Activated calcineurin dephosphorylates NFATs in the cytoplasm which results in their nuclear translocation and subsequent activation of prohypertrophic target genes. In contrast, DYRK1A is capable of phosphorylating transcription factors of the NFAT family leading to their nuclear export (10Gwack Y. Sharma S. Nardone J. Tanasa B. Iuga A. Srikanth S. Okamura H. Bolton D. Feske S. Hogan P.G. Rao A. Nature. 2006; 441: 646-650Crossref PubMed Scopus (325) Google Scholar, 11Arron J.R. Winslow M.M. Polleri A. Chang C.P. Wu H. Gao X. Neilson J.R. Chen L. Heit J.J. Kim S.K. Yamasaki N. Miyakawa T. Francke U. Graef I.A. Crabtree G.R. Nature. 2006; 441: 595-600Crossref PubMed Scopus (561) Google Scholar). Thus, the underlying mechanism of the antihypertrophic properties of DYRK1A is likely to be a reduction of NFAT activity (Fig. 7). Consistent with this notion DYRK1A directly reduces calcineurin-induced NFAT reporter activity in cardiomyocytes, while its baseline activity is markedly augmented by knockdown or inhibition of DYRK1A. NFAT transcription factors serve as integrators of multiple signaling pathways. Several kinases have been identified that can antagonize calcineurin-signaling by phosphorylating NFATs, including glycogen synthase kinase 3 (GSK3), c-Jun N-terminal kinase (JNK), and p38 MAP kinases (28Wilkins B.J. Molkentin J.D. Biochem. Biophys. Res. Commun. 2004; 322: 1178-1191Crossref PubMed Scopus (371) Google Scholar). Interestingly, GSK3 phosphorylates Ser/Thr residues only after a priming phosphorylation reaction (29Sugden P.H. Fuller S.J. Weiss S.C. Clerk A. Br. J. Pharmacol. 2008; 153: S137-S153Crossref PubMed Scopus (168) Google Scholar). Because DYRK1A targets a serine in NFATc4 in close proximity to the GSK3 phosphorylation site and DYRK1A synergizes with GSK3 to inhibit NFAT activity (11Arron J.R. Winslow M.M. Polleri A. Chang C.P. Wu H. Gao X. Neilson J.R. Chen L. Heit J.J. Kim S.K. Yamasaki N. Miyakawa T. Francke U. Graef I.A. Crabtree G.R. Nature. 2006; 441: 595-600Crossref PubMed Scopus (561) Google Scholar), DYRK1A represents a good candidate for a physiological priming kinase. Of note, adenovirally overexpressed GSK3β blocks the hypertrophic response to pharmacological inducers of hypertrophy such as PE or endothelin-1 (30Haq S. Choukroun G. Kang Z.B. Ranu H. Matsui T. Rosenzweig A. Molkentin J.D. Alessandrini A. Woodgett J. Hajjar R. Michael A. Force T. J. Cell Biol. 2000; 151: 117-130Crossref PubMed Scopus (341) Google Scholar). Likewise, cardiac hypertrophy induced by adrenergic stimulation or pressure overload is diminished by overexpression of constitutively active GSK3β (31Antos C.L. McKinsey T.A. Frey N. Kutschke W. McAnally J. Shelton J.M. Richardson J.A. Hill J.A. Olson E.N. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 907-912Crossref PubMed Scopus (418) Google Scholar). It will thus be interesting to see whether GSK3β is also required for the antihypertrophic properties of DYKR1A. Finally, we cannot exclude that phosphorylation of other targets than NFATs by DYRK1A may contribute to the antihypertrophic effects of DYRK1A. In summary, we identified DYRK1A as a novel regulator of cardiomyocyte hypertrophy. Whereas DYRK1A inhibited pharmacological agonist- and calcineurin-induced hypertrophy, the absence or inhibition of DYRK1A caused a hypertrophic phenotype. Mechanistically, the antihypertrophic effect of DYRK1A is likely mediated via increased phosphorylation of NFAT transcription factors. We thank Walter Becker for DYRK1A expression constructs and Ulrike Oehl and Jutta Krebs for excellent technical assistance. Download .pdf (.06 MB) Help with pdf files

Left ventricular non-compaction (LVNC) is caused by mutations in multiple genes. It is still unclear whether LVNC is the primary determinant of cardiomyopathy or rather a secondary phenomenon with intrinsic cardiomyocyte dysfunction being the actual cause of the disease. Here, we describe a family with LVNC due to a novel missense mutation, pE96K, in the cardiac troponin T gene (TNNT2).The novel mutation was identified in the index patient and all affected relatives, but not in 430 healthy control individuals. Mutations in known LVNC-associated genes were excluded. To investigate the pathophysiological implications of the mutation, we generated transgenic mice expressing human wild-type cTNT (hcTNT) or a human troponin T harbouring the pE96K mutation (mut cTNT). Animals were characterized by echocardiography, histology, and gene expression analysis. Mut cTNT mice displayed an impaired left ventricular function and induction of marker genes of heart failure. Remarkably, left ventricular non-compaction was not observed.Familial co-segregation and the cardiomyopathy phenotype of mut cTNT mice strongly support a causal relationship of the pE96K mutation and disease in our index patient. In addition, our data suggest that a non-compaction phenotype is not required for the development of cardiomyopathy in this specific TNNT2 mutation leading to LVNC.

Aims Tumour necrosis factor (TNF)‐like weak inducer of apoptosis (sTWEAK) is a multifunctional cytokine that has recently been implicated in cardiovascular disease. The aim of this study was to define the plasma levels of sTWEAK in patients with stable chronic heart failure and evaluate the possibility of a prognostic impact of sTWEAK. Methods and results sTWEAK levels in plasma samples from 364 patients with systolic heart failure were compared with 36 control patients. The median levels of sTWEAK in heart failure patients were significantly lower than those of the control group (217 pg/mL, interquartile range 136–311 vs. 325 pg/mL, interquartile range 250–394 pg/mL). Moreover, sTWEAK levels were lower in patients with ischaemic cardiomyopathy vs. dilated cardiomyopathy and correlated significantly with functional NYHA class. Patients with plasma levels below a ROC‐derived cut‐off value of 227 pg/mL had a significantly higher mortality rate after 4 years. Upon univariate and multivariate analyses, sTWEAK levels below 227 pg/mL emerged as an independent predictor of subsequent death. Conclusion In contrast to other cytokines shown to be increased in heart failure patients, plasma levels of sTWEAK are significantly reduced in chronic stable heart failure. In addition, lower plasma levels of sTWEAK predict an adverse prognosis independent of established risk markers such as NT‐proBNP.

Excessive stress, e.g. due to biomechanical overload or ischemia/reperfusion is a potent inductor of cardiomyocyte apoptosis, which contributes to maladaptive remodeling. Despite substantial progress in the understanding of the molecular pathophysiology, many components of the signaling pathways underlying remodeling in general and apoptosis in particular still remain unknown. Recent evidence suggests that microRNAs (miRs) play an important role in the heart's response to increased cardiac stress. To identify novel modulators of stress-dependent remodeling, we conducted a genome-wide miR-screen of mechanically stretched neonatal rat cardiomyocytes (NRCM). Out of 351 miRs, eight were significantly regulated by biomechanical stress, including microRNA-20a, which is part of the miR17–92 cluster. Interestingly, further expression analyses also revealed upregulation of microRNA-20a in an in vitro hypoxia/“reperfusion” model. Given the potential apoptosis-modulating properties of the miR17–92 cluster, we subjected NRCM to hypoxia and subsequent reoxygenation. AdmiR-20a significantly inhibited hypoxia-mediated apoptosis in a dose-dependent fashion, while targeted knockdown of miR-20a in NRCM induced cardiomyocyte apoptosis. Mechanistically, the antiapoptotic effect of miR-20a appears to be mediated through direct targeting and subsequent downregulation of the proapoptotic factor Egln3. Thus, miR-20a is upregulated in acute biomechanical stress as well as hypoxia and inhibits apoptosis in cardiomyocytes. These properties reveal miR-20a as a cardioprotective micro-RNA and a potential target for novel therapeutic strategies to prevent cardiac remodeling.

Chronic heart failure is a complex clinical syndrome with different underlying molecular mechanisms. Nitric oxide (NO) is an important modulator of cardiac function and dysregulation of the NO pathway has been shown to be involved in the pathogenesis of chronic heart failure [ 1 Massion P.B. Feron O. Dessy C. Balligand J.-L. Nitric oxide and cardiac function: ten years after, and continuing. Circ Res. 2003; 93: 388-398 Crossref PubMed Scopus (503) Google Scholar , 2 Paulus W.J. Bronzwaer J.G.F. Nitric oxide's role in the heart: control of beating or breathing?. Am J Physiol Heart Circ Physiol. 2004; 287: H8-H13 Crossref PubMed Scopus (65) Google Scholar , 3 Seddon M. Shah A.M. Casadei B. Cardiomyocytes as effectors of nitric oxide signalling. Cardiovasc Res. 2007; 75: 315-326 Crossref PubMed Scopus (177) Google Scholar ]. Due to its structural similarity to the NO synthase (NOS) substrate, l-arginine, the amino acid homoarginine has been proposed to act as an alternative substrate for the formation of NO [ [4] Moali C. Boucher J.L. Sari M.A. Stuehr D.J. Mansuy D. Substrate specificity of NO synthases: detailed comparison of l-arginine, homo-l-arginine, their N omega-hydroxy derivatives, and N omega-hydroxynor-l-arginine. Biochemistry. 1998; 37: 10453-10460 Crossref PubMed Scopus (180) Google Scholar ]. Clinical data support an association of homoarginine to cerebro- and cardiovascular diseases. In patients subjected to coronary angiography, low homoarginine plasma concentrations were found to be associated with cardiovascular (CV) and all-cause mortality [ [5] März W. Meinitzer A. Drechsler C. et al. Homoarginine, cardiovascular risk, and mortality. Circulation. 2010; 122: 967-975 Crossref PubMed Scopus (170) Google Scholar ]. The aim of the present study was to analyze plasma homoarginine in patients with chronic heart failure either due to dilated (DCM) or ischemic (ICM) cardiomyopathy in order to assess its prognostic value.

The emerging role of the ubiquitin-proteasome system in cardiomyocyte function and homeostasis implies the necessity of tight regulation of protein degradation. However, little is known about cardiac components of this machinery.We sought to determine whether molecules exist that control turnover of cardiac-specific proteins.Using a bioinformatic approach to identify novel cardiac-enriched sarcomere proteins, we identified F-box and leucine-rich repeat protein 22 (Fbxl22). Tissue-specific expression was confirmed by multiple tissue Northern and Western Blot analyses as well as quantitative reverse-transcriptase polymerase chain reaction on a human cDNA library. Immunocolocalization experiments in neonatal and adult rat ventricular cardiomyocytes as well as murine heart tissue located Fbxl22 to the sarcomeric z-disc. To detect cardiac protein interaction partners, we performed a yeast 2-hybrid screen using Fbxl22 as bait. Coimmunoprecipitation confirmed the identified interactions of Fbxl22 with S-phase kinase-associated protein 1 and Cullin1, 2 critical components of SCF (Skp1/Cul1/F-box) E3- ligases. Moreover, we identified several potential substrates, including the z-disc proteins α-actinin and filamin C. Consistently, in vitro overexpression of Fbxl22-mediated degradation of both substrates in a dose-dependent fashion, whereas proteasome inhibition with MG-132 markedly attenuated degradation of both α-actinin and filamin C. Finally, targeted knockdown of Fbxl22 in rat cardiomyocytes as well as zebrafish embryos results in the accumulation of α-actinin associated with severely impaired contractile function and cardiomyopathy in vivo.These findings reveal the previously uncharacterized cardiac-specific F-box protein Fbxl22 as a component of a novel cardiac E3 ligase. Fbxl22 promotes the proteasome-dependent degradation of key sarcomeric proteins, such as α-actinin and filamin C, and is essential for maintenance of normal contractile function in vivo.

Bioresorbable vascular scaffolds (BVS) have been available on the European market since November 2011. The ASSURE registry aims to investigate the safety and efficacy of the Absorb everolimus-eluting bioresorbable vascular scaffold in a real-world setting.Patients with de novo coronary artery disease were consecutively enrolled at six German centres in this prospective registry. Outcomes were procedural success, cardiovascular death, myocardial infarction, and ischaemia-driven target lesion revascularisation (TLR). Angiographic parameters were assessed quantitatively and visual estimates of lesion dimensions were studied. One hundred and eighty-three patients were treated. In 128 (64.7%) lesions a complex ACC/AHA morphology was present. Procedural success was achieved in all patients. Acute gain was 1.54±0.51 mm, resulting in a final minimal lumen diameter (MLD), which met the baseline reference vessel diameter (RVD), although visual estimates overrated the RVD by 0.5±0.5 mm. Up to 12 months, one patient (0.5%) had died from gastrointestinal bleeding, three (1.7%) non-target vessel myocardial infarctions occurred, and five (2.8%) TLR had become necessary because of restenosis.One-year results suggest that bioresorbable vascular scaffolds for de novo coronary artery disease are associated with favourable clinical and functional outcomes in routine clinical practice despite a visually overestimated RVD.

Emergency departments (EDs) routinely struggle with gaps in information when providing patient care. A point to point health information exchange (HIE) model has the potential to effectively fill those gaps.To examine the utility, perceived and actual, of a point-to-point HIE tool called Care Everywhere (CE) and its impact on patient care in the ED.This mixed methods study was performed at four large hospital EDs between January 2012 and November 2012. Retrospective data was extracted from the electronic health record (EHR) to evaluate CE utilization since implementation. ED notes data were extracted from ED visits occurring between January 2012 and June 2012 and were reviewed to evaluate the impact of exchanged information on patient care.Per focus group discussions, physicians thought the information received via CE was of value to patient care, particularly laboratory results, imaging, medication lists, discharge summaries and ECG interpretations. They feel the greatest impact of HIE is the avoidance of duplicative diagnostic testing and the identification of drug-seeking behavior. Nursing and ancillary staff expressed somewhat less enthusiasm but still felt HIE positively impacted patient care. Over a period of six months, CE was used in approximately 1.46% of ED encounters. A review of ED provider notes over that time period revealed CE use resulted in 560 duplicate diagnostic procedures being avoided and 28 cases of drug seeking behavior identified.Our study provides insight into the perceived value of HIE from the point of view of our ED physicians and staff. It also demonstrates that a point-to-point HIE tool such as Epic System's Care Everywhere has the potential to generate greater efficiencies within the ED and impact to patient care through elimination of duplicative diagnostic imaging or testing and resource utilization associated with those procedures.

Aims To evaluate the influence of endomyocardial biopsy (EMB)‐proven intramyocardial inflammation on mortality in patients with cardiac transthyretin amyloid (ATTR) or amyloid light‐chain (AL) amyloidosis. Methods and results We included 54 consecutive patients (mean age 68.83 ± 9.59 years; 45 men) with EMB‐proven cardiac amyloidosis. We followed up patients from first diagnostic biopsy to as long as 36 months (mean 11.5 ± 12 months) and compared their outcome with information on all‐cause mortality with or without proof of inflammation on EMB. Intramyocardial inflammation was assessed by quantitative immunohistology. Patients suffering from amyloidosis revealed a significant poor prognosis with proof of intramyocardial inflammation in contrast to those without inflammation (log‐rank P = 0.019). Re‐grouping of patients indicated AL amyloidosis to have a significant impact on all‐cause mortality (log‐rank P = 0.012). The detailed subgroup analysis showed that patients suffering from AL amyloidosis with intramyocardial inflammation have a significantly worse prognosis compared with AL amyloidosis without inflammation and ATTR with or without inflammation, respectively (log‐rank P = 0.014, contingency Fisher's exact test, P = 0.008). Conclusion Our study reports for the first time a high incidence (48.1%) of intramyocardial inflammation in a series of patients with EMB‐proven cardiac amyloidosis and could show that in patients with AL amyloidosis, intramyocardial inflammation correlated significantly with increased mortality. Our data have a direct clinical impact because one can hypothesize that additional immunomodulating/anti‐inflammatory treatment regimens in patients with biopsy‐proven inflammation of heart muscle tissue could be beneficial for patients suffering from cardiac AL amyloidosis.

AimsDown syndrome-associated dual-specificity tyrosine-(Y)-phosphorylation-regulated kinase 1A (DYRK1A) is a ubiquitously expressed protein kinase. Up to date a variety of targets have been identified, establishing a key role for Dyrk1a in selected signalling pathways. In cardiomyocytes, Dyrk1a acts as a negative regulator of hypertrophy by phosphorylating transcription factors of the NFAT family, but its mechanistic function in the heart remains poorly understood. This study was designed to investigate a potential protective role of Dyrk1a in cardiac hypertrophy in vivo.

Inflammatory, autoimmune and metabolic disorders have been associated with alterations in osteopontin (OPN) serum levels. Furthermore, elevated serum levels of OPN were reported from a small cohort of patients with sepsis. We therefore analyzed OPN serum concentrations in a large cohort of critically ill medical patients.A total of 159 patients (114 with sepsis, 45 without sepsis) were studied prospectively upon admission to the medical intensive care unit (ICU) as well as after 3 days of ICU treatment and compared to 50 healthy controls. Clinical data, various laboratory parameters as well as investigational inflammatory cytokine profiles were assessed. Patients were followed for approximately 1 year.We found significantly elevated serum levels of OPN at admission to the ICU and after 3 days of treatment in critically ill patients compared to healthy controls. OPN concentrations were related to disease severity and significantly correlated with established prognosis scores and classical as well as experimental markers of inflammation and multi-organ failure. In the total cohort, OPN levels decreased from admission to day 3 of ICU treatment. However, persistently elevated OPN levels at day 3 of ICU treatment were a strong independent predictor for an unfavorable prognosis, with similar or better diagnostic accuracy than routinely used markers of organ failure or prognostic scoring systems such as SAPS2 or APACHE II score.Persistently elevated OPN serum concentrations are associated with an unfavourable outcome in patients with critical illness, independent of the presence of sepsis. Besides a possible pathogenic role of OPN in critical illness, our study indicates a potential value for OPN as a prognostic biomarker in critically ill patients during the early course of ICU treatment.

The purpose of this study was to evaluate the outcomes of the novel Fantom coronary bioresorbable scaffold at 6 months. The Fantom sirolimus-eluting bioresorbable scaffold incorporates a unique proprietary iodinated, polycarbonate copolymer of tyrosine analogs that is radiopaque, with thin struts (125 μm) that facilitate device delivery and precise target lesion treatment. The 6-month outcomes and performance of the Fantom scaffold were evaluated in 117 patients with single de novo native coronary artery lesions of length ≤20 mm and reference vessel diameter 2.5 to 3.5 mm. The primary angiographic endpoint was mean late lumen loss at 6 months measured by quantitative coronary angiography. Procedural outcomes were categorized as short-term technical success, short-term procedural success, and clinical procedural success. The primary clinical endpoint was major adverse cardiac events at 6 months, the composite of cardiac death, myocardial infarction (MI), or clinically driven target lesion revascularization (TLR). Short-term technical success, short-term procedural success, and clinical procedural success were achieved in 96.6%, 99.1%, and 99.1% of patients, respectively. Mean 6-month in-stent late lumen loss was 0.25 ± 0.40 mm (n = 100). Binary restenosis was present in 2 patients (2.0%). Major adverse cardiac events within 6 months occurred in 3 patients (2.6%), including no deaths, 2 MIs, and 2 TLRs (1 patient had both an MI and TLR). Scaffold thrombosis occurred in 1 patient (0.9%). The clinical results from 117 patients enrolled in cohort A of the multicenter FANTOM II (Safety & Performance Study of the FANTOM Sirolimus-Eluting Bioresorbable Coronary Scaffold) study demonstrate favorable 6-month outcomes of this novel device in the treatment of noncomplex coronary artery disease.

In heart failure (HF), enhanced persistent Na+ current (INaL) exerts detrimental effects on cellular electrophysiology and can induce arrhythmias. However, the underlying regulatory mechanisms remain unclear. Our aim was to potentially investigate the regulation and electrophysiological contribution of neuronal sodium channel NaV1.8 in failing human heart and eventually to reveal a novel anti-arrhythmic therapy.By western blot, we found that NaV1.8 protein expression is significantly up-regulated, while of the predominant cardiac isoform NaV1.5 is inversely reduced in human HF. Furthermore, to investigate the relation of NaV1.8 regulation with the cellular proarrhythmic events, we performed comprehensive electrophysiology recordings and explore the effect of NaV1.8 on INaL, action potential duration (APD), Ca2+ spark frequency, and arrhythmia induction in human failing cardiomyocytes. NaV1.8 inhibition with the specific blockers A-803467 and PF-01247324 decreased INaL, abbreviated APD and reduced cellular-spontaneous Ca2+-release and proarrhythmic events in human failing cardiomyocytes. Consistently, in mouse cardiomyocytes stressed with isoproterenol, pharmacologic inhibition and genetically knockout of NaV1.8 (SCN10A-/-), were associated with reduced INaL and abbreviated APD.We provide first evidence of differential regulation of NaV1.8 and NaV1.5 in the failing human myocardium and their contribution to arrhythmogenesis due to generation of INaL. We propose inhibition of NaV1.8 thus constitutes a promising novel approach for selective anti-arrhythmic therapy in HF.

Abstract Background Receptor selectivity of sodium-glucose cotransporter-2 inhibitors (SGLT2i) varies greatly between agents. The overall improvement of cardiovascular (CV) outcomes in heart failure (HF) patients varies between trials. We, therefore, evaluated the comparative efficacy of individual SGLT2i and the influence of their respective receptor selectivity thereon. Methods We identified randomized controlled trials investigating the use of SGLT2i in patients with HF—either as the target cohort or as a subgroup of it. Comparators included placebo or any other active treatment. The primary endpoint was the composite of hospitalization for HF or CV death. Secondary outcomes included all-cause mortality, CV mortality, hospitalization for HF, worsening renal function (RF), and the composite of worsening RF or CV death. Evidence was synthesized using network meta-analysis. In addition, the impact of receptor selectivity on outcomes was analysed using meta-regression. Results We identified 18,265 patients included in 22 trials. Compared to placebo, selective and non-selective SGLT2i improved fatal and non-fatal HF events. Head-to-head comparisons suggest superior efficacy with sotagliflozin as compared to dapagliflozin, empagliflozin or ertugliflozin. No significant difference was found between canagliflozin and sotagliflozin. Meta-regression analyses show a decreasing benefit on HF events with increasing receptor selectivity of SGLT2i. In contrast, receptor selectivity did not affect mortality and renal endpoints and no significant difference between individual SGLT2i was noted. Conclusion Our data point towards a class-effect of SGLT2i on mortality and renal outcomes. However, non-selective SGLT2i such as sotagliflozin may be superior to highly selective SGLT2i in terms of HF outcomes.

An update of the first description of quality indicators and structural requirements for Cardiac Arrest Centers from 2017 based on first experiences and certifications is presented. Criteria were adjusted, substantiated and in some parts redefined for feasibility in everyday clinical use.

With more than 25 million people affected, heart failure (HF) is a global threat. As energy production pathways are known to play a pivotal role in HF, we sought here to identify key metabolic changes in ischemic- and non-ischemic HF by using a multi-OMICS approach. Serum metabolites and mRNAseq and epigenetic DNA methylation profiles were analyzed from blood and left ventricular heart biopsy specimens of the same individuals. In total we collected serum from n = 82 patients with Dilated Cardiomyopathy (DCM) and n = 51 controls in the screening stage. We identified several metabolites involved in glycolysis and citric acid cycle to be elevated up to 5.7-fold in DCM (p = 1.7 × 10−6). Interestingly, cardiac mRNA and epigenetic changes of genes encoding rate-limiting enzymes of these pathways could also be found and validated in our second stage of metabolite assessment in n = 52 DCM, n = 39 ischemic HF and n = 57 controls. In conclusion, we identified a new set of metabolomic biomarkers for HF. We were able to identify underlying biological cascades that potentially represent suitable intervention targets.

Background Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia. However, underlying molecular mechanisms are insufficiently understood. Previous studies suggested that microRNA (miRNA) dependent gene regulation plays an important role in the initiation and maintenance of AF. The 2-pore-domain potassium channel TASK-1 (tandem of P domains in a weak inward rectifying K+ channel-related acid sensitive K+ channel 1) is an atrial-specific ion channel that is upregulated in AF. Inhibition of TASK-1 current prolongs the atrial action potential duration to similar levels as in patients with sinus rhythm. Here, we hypothesize that miRNAs might be responsible for the regulation of KCNK3 that encodes for TASK-1. Methods and Results We selected miRNAs potentially regulating KCNK3 and studied their expression in atrial tissue samples obtained from patients with sinus rhythm, paroxysmal AF, or permanent/chronic AF. MiRNAs differentially expressed in AF were further investigated for their ability to regulate KCNK3 mRNA and TASK-1 protein expression in human induced pluripotent stem cells, transfected with miRNA mimics or inhibitors. Thereby, we observed that miR-34a increases TASK-1 expression and current and further decreases the resting membrane potential of Xenopus laevis oocytes, heterologously expressing hTASK-1. Finally, we investigated associations between miRNA expression in atrial tissues and clinical parameters of our patient cohort. A cluster containing AF stage, left ventricular end-diastolic diameter, left ventricular end-systolic diameter, left atrial diameter, atrial COL1A2 (collagen alpha-2(I) chain), and TASK-1 protein level was associated with increased expression of miR-25, miR-21, miR-34a, miR-23a, miR-124, miR-1, and miR-29b as well as decreased expression of miR-9 and miR-485. Conclusions These results suggest an important pathophysiological involvement of miRNAs in the regulation of atrial expression of the TASK-1 potassium channel in patients with atrial cardiomyopathy.

Endoplasmic Reticulum (ER) stress and oxidative stress have been highly implicated in the pathogenesis of cardiac hypertrophy and heart failure (HF). However, the mechanisms involved in the interplay between these processes in the heart are not fully understood. The present study sought to determine a causative link between Pak2-dependent UPR activation and oxidative stress via Nrf2 regulation under pathological ER stress. We report that sustained ER stress and Pak2 deletion in cardiomyocytes enhance Nrf2 expression. Conversely, AAV9 mediated Pak2 delivery in the heart leads to a significant decrease in Nrf2 levels. Pak2 overexpression enhances the XBP1-Hrd1 UPR axis and ameliorates tunicamycin induced cardiac apoptosis and dysfunction in mice. We found that Pak2 deletion and altered proteostasis render Nrf2 detrimental by switching from its antioxidant role to renin-angiotensin aldosterone system (RAAS) gene regulator. Mechanistically, Pak2 mediated Hrd1 expression targets Nrf2 for ubiquitination and degradation thus preventing its aberrant activation. Moreover, we find a significant increase in Nrf2 with a decrease in Pak2 in human myocardium of dilated heart disease. Using human-induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), we find that Pak2 is able to ameliorate Nrf2 induced RAAS activation under ER stress. These findings demonstrate that Pak2 is a novel Nrf2 regulator in the stressed heart. Activation of XBP1-Hrd1 is attributed to prevent ER stress-induced Nrf2 RAAS component upregulation. This mechanism explains the functional dichotomy of Nrf2 in the stressed heart. Thus, Pak2 regulation of Nrf2 homeostasis may present as a potential therapeutic route to alleviate detrimental ER stress and heart failure.

Growing evidence suggests an altered gut microbiome in patients with heart failure (HF). However, the exact interrelationship between microbiota, HF, and its consequences on the metabolome are still unknown. We thus aimed here to decipher the association between the severity and progression of HF and the gut microbiome composition and circulating metabolites. Using a mouse model of transverse aortic constriction (TAC), gut bacterial diversity was found to be significantly lower in mice as early as day 7 post-TAC compared to Sham controls (p = 0.03), with a gradual progressive decrease in alpha-diversity on days 7, 14, and 42 (p = 0.014, p = 0.0016, p = 0.0021) compared to day 0, which coincided with compensated hypertrophy, maladaptive hypertrophy, and overtly failing hearts, respectively. Strikingly, segregated analysis based on the severity of the cardiac dysfunction (EF < 40% vs. EF 40−55%) manifested marked differences in the abundance and the grouping of several taxa. Multivariate analysis of plasma metabolites and bacterial diversity produced a strong correlation of metabolic alterations, such as reduced short-chain fatty acids and an increase in primary bile acids, with a differential abundance of distinct bacteria in HF. In conclusion, we showed that HF begets HF, likely via a vicious cycle of an altered microbiome and metabolic products.

Background: The human heart has limited capacity to generate new cardiomyocytes and this capacity declines with age. Because loss of cardiomyocytes may contribute to heart failure, it is crucial to explore stimuli of endogenous cardiac regeneration to favorably shift the balance between loss of cardiomyocytes and the birth of new cardiomyocytes in the aged heart. We have previously shown that cardiomyogenesis can be activated by exercise in the young adult mouse heart. Whether exercise also induces cardiomyogenesis in aged hearts, however, is still unknown. Here, we aim to investigate the effect of exercise on the generation of new cardiomyocytes in the aged heart. Methods: Aged (20-month-old) mice were subjected to an 8-week voluntary running protocol, and age-matched sedentary animals served as controls. Cardiomyogenesis in aged hearts was assessed on the basis of 15 N-thymidine incorporation and multi-isotope imaging mass spectrometry. We analyzed 1793 cardiomyocytes from 5 aged sedentary mice and compared these with 2002 cardiomyocytes from 5 aged exercised mice, followed by advanced histology and imaging to account for ploidy and nucleation status of the cell. RNA sequencing and subsequent bioinformatic analyses were performed to investigate transcriptional changes induced by exercise specifically in aged hearts in comparison with young hearts. Results: Cardiomyogenesis was observed at a significantly higher frequency in exercised compared with sedentary aged hearts on the basis of the detection of mononucleated/diploid 15 N-thymidine–labeled cardiomyocytes. No mononucleated/diploid 15 N-thymidine–labeled cardiomyocyte was detected in sedentary aged mice. The annual rate of mononucleated/diploid 15 N-thymidine–labeled cardiomyocytes in aged exercised mice was 2.3% per year. This compares with our previously reported annual rate of 7.5% in young exercised mice and 1.63% in young sedentary mice. Transcriptional profiling of young and aged exercised murine hearts and their sedentary controls revealed that exercise induces pathways related to circadian rhythm, irrespective of age. One known oscillating transcript, however, that was exclusively upregulated in aged exercised hearts, was isoform 1.4 of regulator of calcineurin, whose regulation and functional role were explored further. Conclusions: Our data demonstrate that voluntary running in part restores cardiomyogenesis in aged mice and suggest that pathways associated with circadian rhythm may play a role in physiologically stimulated cardiomyogenesis.

Abstract RNA–protein interactions are central to cardiac function, but how activity of individual RNA-binding protein is regulated through signaling cascades in cardiomyocytes during heart failure development is largely unknown. The mechanistic target of rapamycin kinase is a central signaling hub that controls mRNA translation in cardiomyocytes; however, a direct link between mTOR signaling and RNA-binding proteins in the heart has not been established. Integrative transcriptome and translatome analysis revealed mTOR dependent translational upregulation of the RNA binding protein Ybx1 during early pathological remodeling independent of mRNA levels. Ybx1 is necessary for pathological cardiomyocyte growth by regulating protein synthesis. To identify the molecular mechanisms how Ybx1 regulates cellular growth and protein synthesis, we identified mRNAs bound to Ybx1. We discovered that eucaryotic elongation factor 2 (Eef2) mRNA is bound to Ybx1, and its translation is upregulated during cardiac hypertrophy dependent on Ybx1 expression. Eef2 itself is sufficient to drive pathological growth by increasing global protein translation. Finally, Ybx1 depletion in vivo preserved heart function during pathological cardiac hypertrophy. Thus, activation of mTORC1 links pathological signaling cascades to altered gene expression regulation by activation of Ybx1 which in turn promotes translation through increased expression of Eef2.

Soziale Medien in der Kardiologie bieten sowohl Chancen als auch Gefahren. Sie ermöglichen einerseits eine schnelle Verbreitung von wissenschaftlichen Erkenntnissen, die Vernetzung von Fachleuten und bieten eine Plattform für den interaktiven Austausch. Andererseits fehlen bislang klare Qualitätskriterien für Inhalte. Ebenso gestaltet sich die Überprüfung von Autoren und deren Informationen schwierig. Für einen rechtskonformen Gebrauch sozialer Medien in der Kardiologie sollten vor einer Nutzung die damit einhergehenden juristischen Fallstricke bekannt sein. Auch wäre wünschenswert, dass sich die medizinischen Fachgesellschaften in Zukunft auf ihren Kongressveranstaltungen referenten- wie auch zuhörerseitig für die Schaffung eines rechtssicheren Rahmens einsetzen. Insgesamt erfordert ein professioneller Umgang mit Social Media fortwährend eine inhaltlich kritische Bewertung geteilter Beiträge, die Beachtung von Verhaltensregeln sowie eine bewusste Themenauswahl.

Abstract Background High‐altitude pulmonary hypertension (HAPH) has a prevalence of approximately 10%. Changes in cardiac morphology and function at high altitude, compared to a population that does not develop HAPH are scarce. Methods Four hundred twenty‐one subjects were screened in a hypoxic chamber inspiring a FiO 2 = 12% for 2 h. In 33 subjects an exaggerated increase in systolic pulmonary artery pressure (sPAP) could be confirmed in two independent measurements. Twenty nine of these, and further 24 matched subjects without sPAP increase were examined at 4559 m by Doppler echocardiography including global longitudinal strain (GLS). Results SPAP increase was higher in HAPH subjects (∆ = 10.2 vs. ∆ = 32.0 mm Hg, p &lt; .001). LV eccentricity index (∆ = .15 vs. ∆ = .31, p = .009) increased more in HAPH. D‐shaped LV (0 [0%] vs. 30 [93.8%], p = .00001) could be observed only in the HAPH group, and only in those with a sPAP ≥50 mm Hg. LV‐EF (∆ = 4.5 vs. ∆ = 6.7%, p = .24) increased in both groups. LV‐GLS (∆ = 1.2 vs. ∆ = 1.1 –%, p = .60) increased slightly. RV end‐diastolic (∆ = 2.20 vs. ∆ = 2.7 cm 2 , p = .36) and end‐systolic area (∆ = 2.1 vs. ∆ = 2.7 cm 2 , p = .39), as well as RA end‐systolic area index (∆ = −.9 vs. ∆ = .3 cm 2 /m 2 , p = .01) increased, RV‐FAC (∆ = −2.9 vs. ∆ = −4.7%, p = .43) decreased, this was more pronounced in HAPH, RV‐GLS (∆ = 1.6 vs. ∆ = −.7 –%, p = .17) showed marginal changes. Conclusions LV and LA dimensions decrease and left ventricular function increases at high‐altitude in subjects with and without HAPH. RV and RA dimensions increase, and RV longitudinal strain increases or remains unchanged in subjects with HAPH. Changes are negligible in those without HAPH.

Assessment of cardiovascular risk is critical for patients with cancer. Previous retrospective studies suggest potential cardiotoxicity of CAR T cell therapies. We aimed to prospectively assess cardiotoxicity and the predictive value of cardiac biomarkers and classical risk factors (age, cardiac function, diabetes, arterial hypertension, smoking) for cardiac events and all-cause mortality (ACM).In this prospective cohort study, all patients treated with CAR T cell constructs (axi-cel, tisa-cel, brexu-cel, ide-cel, or the 3rd generation CAR HD-CAR-1) from Oct 1, 2018, to Sept 30, 2022 at the University Hospital Heidelberg were included. Surveillance included cardiac assessment with biomarkers (high-sensitive Troponin T (hs-cTnT), N-terminal brain natriuretic peptide (NT-proBNP)), 12-lead-ECG, and 2D echocardiography. ACM was defined as the primary study endpoint, while cardiotoxicity, defined by clinical syndromes of heart failure or decline in ejection fraction, served as a secondary endpoint.Overall, 137 patients (median age 60, range 20-83, IQR 16), were included in the study. 46 patients died during the follow up period (median 0.75 years, range 0.02-4.33, IQR 0.89) 57 month, with a median survival of 0.57 years (range 0.03-2.38 years, IQR 0.79). A septal wall thickness above 11 mm (HR 2.48, 95%-CI = 1.10-5.67, p = 0.029) was associated with an increased risk of ACM, with a trend seen for reduced left ventricular ejection fraction prior to therapy (LVEF <40%; HR 9.17, 95%-CI = 1.30-183.11, p = 0.051). Secondary endpoint was reached by 93 patients while no baseline parameter was able to predict an elevated risk. However, hs-cTnT change from baseline of 50% or more during the first 14 days after CAR infusion predicted ACM (HR 3.81, 95%-CI = 1.58-9.45; p = 0.003). None of the baseline characteristics was able to predict the incidence of cardiac events.Reduced pre-lymphodepletion ejection fraction and early post-infusion biomarker kinetics may be associated with increased ACM and cardiotoxicity events. These findings may help to identify patients who could benefit from intensified cardio-oncological surveillance.The German Center for Cardiovascular Research, German Research Foundation, and the Federal Ministry of Education and Research.

The identification of genetic mutations underlying familial structural heart disease has provided exciting new insights into how alterations in structural components of the cardiomyocyte lead to different forms of cardiomyopathy. Specifically, mutations in components of the sarcomere are frequently associated with hypertrophic cardiomyopathy, whereas mutations in cytoskeletal proteins lead to dilated cardiomyopathy. In addition, extrinsic stresses such as hypertension and valvular disease can produce myocardial remodeling that is very similar to that observed in genetic cardiomyopathy. For myocardial remodeling to occur, changes in gene expression must occur; therefore, changes in contractile function or wall stress must be communicated to the nucleus via signal transduction pathways. The identity of these signaling pathways has become a key question in molecular biology. Numerous signaling molecules have been implicated in the development of hypertrophy and failure, including the beta-adrenergic receptor, G alpha(q) and downstream effectors, mitogen-activated protein kinase pathways, and the Ca(2+)-regulated phosphatase, calcineurin. In the past it has been difficult to discern which signaling molecules actually contributed to disease progression in vivo; however, the development of numerous transgenic and knockout mouse models of cardiomyopathy is now allowing the direct testing of stimulatory and inhibitory molecules in the mouse heart. From this work it has been possible to identify signaling molecules and pathways that are required for different aspects of disease progression in vivo. In particular, a number of signaling pathways have now been identified that may be key regulators of changes in myocardial structure and function in response to mutations in structural components of the cardiomyocyte. Myocardial structure and signal transduction are now merging into a common field of research that will lead to a more complete understanding of the molecular mechanisms that underly heart disease.

Heterotrimeric G proteins are pivotal regulators of myocardial contractility. In addition to the receptor-induced GDP/GTP exchange, G protein alpha subunits can be activated by a phosphate transfer via a plasma membrane-associated complex of nucleoside diphosphate kinase B (NDPK B) and G protein betagamma-dimers (Gbetagamma). To investigate the physiological role of this phosphate transfer in cardiomyocytes, we generated a Gbeta1gamma2-dimer carrying a single amino acid exchange at the intermediately phosphorylated His-266 in the beta1 subunit (Gbeta1H266Lgamma2). Recombinantly expressed Gbeta1H266Lgamma2 were integrated into heterotrimeric G proteins in rat cardiomyocytes but were deficient in intermediate Gbeta phosphorylation. Compared with wild-type Gbeta1gamma2 (Gbeta1WTgamma2), overexpression of Gbeta1H266Lgamma2 suppressed basal cAMP formation up to 55%. A similar decrease in basal cAMP production occurred when the formation of NDPK B/Gbetagamma complexes was attenuated by siRNA-mediated NDPK B knockdown. In adult rat cardiomyocytes expressing Gbeta1H266Lgamma2, the basal contractility was suppressed by approximately 50% which correlated to similarly reduced basal cAMP levels and reduced Ser16-phosphorylation of phospholamban. In the presence of the beta-adrenoceptor agonist isoproterenol, the total cAMP formation and contractility were significantly lower in Gbeta1H266Lgamma2 than in Gbeta1WTgamma2 expressing cardiomyocytes. However, the relative isoproterenol-induced increased was not affected by Gbeta1H266Lgamma2. We conclude that the receptor-independent activation of G proteins via NDPK B/Gbetagamma complexes requires the intermediate phosphorylation of G protein beta subunits at His-266. Our results highlight the histidine kinase activity of NDPK B for Gbeta and demonstrate its contribution to the receptor-independent regulation of cAMP synthesis and contractility in intact cardiomyocytes.

Rationale : The intercalated disc (ID) is a highly specialized cell-cell contact structure that ensures mechanical and electric coupling of contracting cardiomyocytes. Recently, the ID has been recognized to be a hot spot of cardiac disease, in particular inherited cardiomyopathy. Objective : Given its complex structure and function we hypothesized that important molecular constituents of the ID still remain unknown. Methods and Results : Using a bioinformatics screen, we discovered and cloned a previously uncharacterized 54 kDa cardiac protein which we termed Myozap (Myocardium-enriched zonula occludens-1–associated protein). Myozap is strongly expressed in the heart and lung. In cardiac tissue it localized to the ID and directly binds to desmoplakin and zonula occludens-1. In a yeast 2-hybrid screen for additional binding partners of Myozap we identified myosin phosphatase–RhoA interacting protein (MRIP), a negative regulator of Rho activity. Myozap, in turn, strongly activates SRF-dependent transcription through its ERM (Ezrin/radixin/moesin)-like domain in a Rho-dependent fashion. Finally, in vivo knockdown of the Myozap ortholog in zebrafish led to severe contractile dysfunction and cardiomyopathy. Conclusions : Taken together, these findings reveal Myozap as a previously unrecognized component of a Rho-dependent signaling pathway that links the intercalated disc to cardiac gene regulation. Moreover, its subcellular localization and the observation of a severe cardiac phenotype in zebrafish, implicate Myozap in the pathogenesis of cardiomyopathy.

Soluble TWEAK has recently been introduced as a potential mediator of cardiovascular disease. In this retrospective pilot study we thus sought to evaluate serum levels of soluble TWEAK (sTWEAK) patients with acute ST-elevation myocardial infarction (STEMI).Blood samples of 173 patients admitted to our hospital with acute STEMI (<24 h after symptom onset) were evaluated for their sTWEAK serum levels immediately at the time of admission and compared to those of patients with stable coronary artery disease (CAD) and healthy controls. Moreover, patients with STEMI were analyzed for their 30-day short-term outcome after acute STEMI. Adverse events were defined as the combined endpoint of cardiovascular death, resuscitation>24 h after reperfusion, cardiogenic shock or need for vasopressor therapy, repeated target vessel revascularization/myocardial infarction and stroke/TIA.Patients with STEMI showed significantly higher levels of sTWEAK on admission compared to control patients or patients with chronic stable coronary artery disease (p<0.0001). Moreover, sTWEAK levels were higher in female patients. Additionally, sTWEAK levels were related to C-reactive protein levels and inversely correlated with the time between symptom onset and admission. Soluble TWEAK levels above the ROC-defined cutoff (>1286 pg/ml) significantly predicted an adverse short-term outcome in patients with STEMI after 30 days (p=0.0032). In this pilot study there was no significant relation between serum levels of sTWEAK and common risk factors like diabetes, hypertension, active smoking and age, white blood count or indices of myocardial function and damage like ejection fraction and infarct size in STEMI patients. Moreover, no significant relation was found between peak troponin T levels and sTWEAK on admission.Our retrospective pilot study shows for the first time that sTWEAK is significantly elevated in patients with acute myocardial infarction compared to healthy controls and patients with stable coronary artery disease. Moreover, in our study sTWEAK levels on admission were associated with an adverse short-term outcome in STEMI patients. Further work is needed to precisely define the potential role of sTWEAK as a prognostic marker in myocardial infarction.

The M-band represents a transverse structure in the center of the sarcomeric A-band and provides an anchor for the myosin-containing thick filaments. In contrast to other sarcomeric structures, eg, the Z-disc, only few M-band-specific proteins have been identified to date, and its exact molecular composition remains unclear.Using a bioinformatic approach to identify novel heart- and muscle-specific genes, we found a leucine rich protein, myomasp (Myosin-interacting, M-band-associated stress-responsive protein)/LRRC39. RT-PCR and Northern and Western blot analyses confirmed a cardiac-enriched expression pattern, and immunolocalization of myomasp revealed a strong and specific signal at the sarcomeric M-band. Yeast 2-hybrid screens, as well as coimmunoprecipitation experiments, identified the C terminus of myosin heavy chain (MYH)7 as an interaction partner for myomasp. Knockdown of myomasp in neonatal rat ventricular myocytes (NRVCMs) led to a significant upregulation of the stretch-sensitive genes GDF-15 and BNP. Conversely, the expression of MYH7 and the M-band proteins myomesin-1 and -2 was found to be markedly reduced. Mechanistically, knockdown of myomasp in NRVCM led to a dose-dependent suppression of serum response factor-dependent gene expression, consistent with earlier observations linking the M-band to serum response factor-mediated signaling. Finally, downregulation of myomasp/LRRC39 in spontaneously beating engineered heart tissue constructs resulted in significantly lower force generation and reduced fractional shortening. Likewise, knockdown of the myomasp/LRRC39 ortholog in zebrafish resulted in severely impaired heart function and cardiomyopathy in vivo.These findings reveal myomasp as a previously unrecognized component of an M-band-associated signaling pathway that regulates cardiomyocyte gene expression in response to biomechanical stress.

Asymmetric dimethylarginine (ADMA) is associated with increased mortality in patients with chronic heart failure but it remains unclear if the etiology of heart failure influences the prognostic value of dimethylarginines.L-Arginine, ADMA, and symmetric dimethylarginine (SDMA) were measured by liquid chromatography-tandem mass spectrometry in 341 patients with chronic heart failure due to dilated cardiomyopathy (DCM; n = 226) or ischemic cardiomyopathy (ICM; n = 115). Median (interquartile range [IQR]) ADMA and SDMA plasma levels were higher, L-arginine and the L-arginine-ADMA ratio were lower in patients with severe forms of heart failure (New York Heart Association (NYHA) functional class III or IV) compared with milder forms (NYHA functional class I or II) (ADMA 0.57 (0.14) μmol/L vs 0.54 (0.12) μmol/L [P < .001]; SDMA 0.47 (0.27) μmol/L vs 0.37 (0.13) μmol/L [P < .001]; L-arginine 81.8 (39.1) μmol/L vs 92.6 (39.3) μmol/L [P < .01]), but no significant differences were observed between the different etiologies. The L-arginine-ADMA ratio was associated with outcome only in patients with DCM. In multivariate analysis, the mortality risk of DCM patients was significantly lower for those in the highest quartile compared with the lowest quartile during a median observation time of 3.3 years (hazard ratio 0.31, 95% CI 0.11-0.88; P = .028, adjusted for other risk factors).DCM patients with unfavourable L-arginine-ADMA ratio are at increased risk for death.

This prospective, observational all-comers registry assessed the safety and efficacy of a Drug Coated Balloon-only strategy (DCB-only) in patients with coronary lesions.Data regarding the performance of a DCB-only approach, especially in patients with previously untreated de-novo coronary artery disease (CAD), are still limited.This study was conducted as an international, multicenter registry primarily enrolling patients with de-novo CAD. However, it was also possible to include patients with in-stent restenosis (ISR). The primary endpoint was the rate of clinically driven target lesion revascularization (TLR) after 9 months.A total of 1,025 patients with a mean age of 64.0 ± 11.2 years were enrolled. The majority of treated lesions were de-novo (66.9%), followed by drug-eluting-stent ISR (DES-ISR; 22.6%) and bare-metal-stent ISR (BMS-ISR; 10.5%). The TLR rate was lower in the de-novo group (2.3%) when compared to BMS- (2.9%) and DES-ISR (5.8%) (P = 0.049). Regarding MACE, there was a trend toward fewer events in the de-novo group (5.6%) than in the BMS- (7.8%) and DES-ISR cohort (9.6%) (P = 0.131). Subgroup analyses revealed that lesion type (95% CI 1.127-6.587); P = 0.026) and additional stent implantation (95% CI 0.054-0.464; P = 0.001) were associated with higher TLR rates.Our results show that DCB-only angioplasty of de-novo coronary lesions is associated with low MACE and TLR rates. Thus, DCBs appear to be an attractive alternative for the interventional, stentless treatment of suitable de-novo coronary lesions.

Contemporary data on patients with previously undiagnosed severe aortic stenosis (AS) are scarce. We aimed to address this gap by gathering data from consecutive patients diagnosed with severe AS on echocardiography.This was a prospective, multicentre, multinational, registry in 23 tertiary care hospitals across 9 European countries. Patients with a diagnosis of severe AS were included using echocardiography (aortic valve area (AVA) <1 cm2, indexed AVA <0.6 cm2/m2, maximum jet-velocity (Vmax) >4 m/s and/or mean transvalvular gradient >40 mm Hg).The 2171 participants had a mean age of 77.9 years and 48.0% were female. The mean AVA was 0.73 cm2, Vmax4.3 m/s and mean gradient 47.1 mm Hg; 62.1% had left ventricular hypertrophy and 27.3% an ejection fraction (EF) <50%. 1743 patients (80.3%) were symptomatic (shortness-of-breath 91.0%; dizziness 30.2%, chest pain 28.9%). Patients had a EuroSCORE II of 4.0; 25.3% had a creatinine clearance <50 mL/min, and 3.2% had an EF <30%. Symptomatic patients were older and had more comorbidities than asymptomatic patients. Despite European Society of Cardiology 2017 valvular heart disease guideline class I recommendation, in only 76.2% a decision was made for an intervention (transcatheter 50.4%, surgical aortic valve replacement 25.8%). In asymptomatic patients, 57.7% with a class I/IIa indication were scheduled for a procedure, while 36.3% patients without an indication had their valve replaced.The majority of patients with severe AS presented at an advanced disease stage. Management of severe AS remained suboptimal in a significant proportion of contemporary patients with severe AS.NCT02241447;Results.

Haemorrhoidal disease (HEM) affects a large and silently suffering fraction of the population but its aetiology, including suspected genetic predisposition, is poorly understood. We report the first genome-wide association study (GWAS) meta-analysis to identify genetic risk factors for HEM to date.We conducted a GWAS meta-analysis of 218 920 patients with HEM and 725 213 controls of European ancestry. Using GWAS summary statistics, we performed multiple genetic correlation analyses between HEM and other traits as well as calculated HEM polygenic risk scores (PRS) and evaluated their translational potential in independent datasets. Using functional annotation of GWAS results, we identified HEM candidate genes, which differential expression and coexpression in HEM tissues were evaluated employing RNA-seq analyses. The localisation of expressed proteins at selected loci was investigated by immunohistochemistry.We demonstrate modest heritability and genetic correlation of HEM with several other diseases from the GI, neuroaffective and cardiovascular domains. HEM PRS validated in 180 435 individuals from independent datasets allowed the identification of those at risk and correlated with younger age of onset and recurrent surgery. We identified 102 independent HEM risk loci harbouring genes whose expression is enriched in blood vessels and GI tissues, and in pathways associated with smooth muscles, epithelial and endothelial development and morphogenesis. Network transcriptomic analyses highlighted HEM gene coexpression modules that are relevant to the development and integrity of the musculoskeletal and epidermal systems, and the organisation of the extracellular matrix.HEM has a genetic component that predisposes to smooth muscle, epithelial and connective tissue dysfunction.