Dominic P. Del Re

Twelve regulated cell death programs have been described. We review in detail the basic biology of nine including death receptor-mediated apoptosis, death receptor-mediated necrosis (necroptosis), mitochondrial-mediated apoptosis, mitochondrial-mediated necrosis, autophagy-dependent cell death, ferroptosis, pyroptosis, parthanatos, and immunogenic cell death. This is followed by a dissection of the roles of these cell death programs in the major cardiac syndromes: myocardial infarction and heart failure. The most important conclusion relevant to heart disease is that regulated forms of cardiomyocyte death play important roles in both myocardial infarction with reperfusion (ischemia/reperfusion) and heart failure. While a role for apoptosis in ischemia/reperfusion cannot be excluded, regulated forms of necrosis, through both death receptor and mitochondrial pathways, are critical. Ferroptosis and parthanatos are also likely important in ischemia/reperfusion, although it is unclear if these entities are functioning as independent death programs or as amplification mechanisms for necrotic cell death. Pyroptosis may also contribute to ischemia/reperfusion injury, but potentially through effects in non-cardiomyocytes. Cardiomyocyte loss through apoptosis and necrosis is also an important component in the pathogenesis of heart failure and is mediated by both death receptor and mitochondrial signaling. Roles for immunogenic cell death in cardiac disease remain to be defined but merit study in this era of immune checkpoint cancer therapy. Biology-based approaches to inhibit cell death in the various cardiac syndromes are also discussed.

Here we show that Mst1, a proapoptotic kinase, impairs protein quality control mechanisms in the heart through inhibition of autophagy. Stress-induced activation of Mst1 in cardiomyocytes promoted accumulation of p62 and aggresome formation, accompanied by the disappearance of autophagosomes. Mst1 phosphorylated the Thr108 residue in the BH3 domain of Beclin1, which enhanced the interaction between Beclin1 and Bcl-2 and/or Bcl-xL, stabilized the Beclin1 homodimer, inhibited the phosphatidylinositide 3-kinase activity of the Atg14L-Beclin1-Vps34 complex and suppressed autophagy. Furthermore, Mst1-induced sequestration of Bcl-2 and Bcl-xL by Beclin1 allows Bax to become active, thereby stimulating apoptosis. Mst1 promoted cardiac dysfunction in mice subjected to myocardial infarction by inhibiting autophagy, associated with increased levels of Thr108-phosphorylated Beclin1. Moreover, dilated cardiomyopathy in humans was associated with increased levels of Thr108-phosphorylated Beclin1 and signs of autophagic suppression. These results suggest that Mst1 coordinately regulates autophagy and apoptosis by phosphorylating Beclin1 and consequently modulating a three-way interaction among Bcl-2 proteins, Beclin1 and Bax.

Over the last decade, the Rho family GTPases have gained considerable recognition as powerful regulators of actin cytoskeletal organization. As with many high profile signal transducers, these molecules soon attracted the attention of the cardiovascular research community. Shortly thereafter, two prominent members known as RhoA and Rac1 were linked to agonist-induced gene expression and myofilament organization using the isolated cardiomyocyte cell model. Subsequent creation of transgenic mouse lines provided evidence for more complex roles of RhoA and Rac1 signaling. Clues from in vitro and in vivo studies suggest the involvement of numerous downstream targets of RhoA and Rac1 signaling including serum response factor, NF-kappaB, and other transcription factors, myofilament proteins, ion channels, and reactive oxygen species generation. Which of these contribute to the observed phenotypic effects of enhanced RhoA and Rac activation in vivo remain to be determined. Current research efforts with a more translational focus have used statins or Rho kinase blockers to assess RhoA and Rac1 as targets for interventional approaches to blunt hypertrophy or heart failure. Generally, salutary effects on remodeling and ischemic damage are observed, but the broad specificity and multiple cellular targets for these drugs within the myocardium demands caution in interpretation. In this review, we assess the evolution of knowledge related to Rac1 and RhoA in the context of hypertrophy and heart failure and highlight the direction that future exploration will lead.

The Hippo pathway is an evolutionarily conserved regulator of organ size and tumorigenesis that negatively regulates cell growth and survival. Here we report that Yes-associated protein (YAP), the terminal effector of the Hippo pathway, interacts with FoxO1 in the nucleus of cardiomyocytes, thereby promoting survival. YAP and FoxO1 form a functional complex on the promoters of the catalase and manganese superoxide dismutase (MnSOD) antioxidant genes and stimulate their transcription. Inactivation of YAP, induced by Hippo activation, suppresses FoxO1 activity and decreases antioxidant gene expression, suggesting that Hippo signalling modulates the FoxO1-mediated antioxidant response. In the setting of ischaemia/reperfusion (I/R) in the heart, activation of Hippo antagonizes YAP-FoxO1, leading to enhanced oxidative stress-induced cell death through downregulation of catalase and MnSOD. Conversely, restoration of YAP activity protects against I/R injury. These results suggest that YAP is a nuclear co-factor of FoxO1 and that the Hippo pathway negatively affects cardiomyocyte survival by inhibiting the function of YAP-FoxO1. YAP is the terminal effector of the Hippo signalling pathway that regulates cell growth and survival. Shao et al.show that Hippo signalling also stimulates oxidative stress responses in cardiomyocytes, revealing YAP as a regulator of FoxO1-dependent antioxidant gene expression.

Yap1 is an important regulator of cardiomyocyte proliferation and embryonic heart development, yet the function of endogenous Yap1 in the adult heart remains unknown. We studied the role of Yap1 in maintaining basal cardiac function and in modulating injury after chronic myocardial infarction (MI). Cardiomyocyte-specific homozygous inactivation of Yap1 in the postnatal heart (YapF/FCre) elicited increased myocyte apoptosis and fibrosis, dilated cardiomyopathy, and premature death. Heterozygous deletion (Yap+/FCre) did not cause an overt cardiac phenotype compared with YapF/F control mice at base line. In response to stress (MI), nuclear Yap1 was found selectively in the border zone and not in the remote area of the heart. After chronic MI (28 days), Yap+/FCre mice had significantly increased myocyte apoptosis and fibrosis, with attenuated compensatory cardiomyocyte hypertrophy, and further impaired function versus Yap+/F control mice. Studies in isolated cardiomyocytes demonstrated that Yap1 expression is sufficient to promote increased cell size and hypertrophic gene expression and protected cardiomyocytes against H2O2-induced cell death, whereas Yap1 depletion attenuated phenylephrine-induced hypertrophy and augmented apoptosis. Finally, we observed a significant decrease in cardiomyocyte proliferation in Yap+/FCre hearts compared with Yap+/F controls after MI and demonstrated that Yap1 is sufficient to promote cardiomyocyte proliferation in isolated cardiomyocytes. Our findings suggest that Yap1 is critical for basal heart homeostasis and that Yap1 deficiency exacerbates injury in response to chronic MI.Background: Yap1 regulates cardiac development, yet the function of Yap1 in the adult heart remains unknown.Results: Yap1 promotes cardiomyocyte survival, hypertrophy, and proliferation and protects against chronic myocardial infarction (MI).Conclusion: Yap1 is critical for basal heart homeostasis, and Yap1 deficiency exacerbates myocardial injury.Significance: Increasing cardiomyocyte survival and proliferation may afford protection in vivo against MI injury. Yap1 is an important regulator of cardiomyocyte proliferation and embryonic heart development, yet the function of endogenous Yap1 in the adult heart remains unknown. We studied the role of Yap1 in maintaining basal cardiac function and in modulating injury after chronic myocardial infarction (MI). Cardiomyocyte-specific homozygous inactivation of Yap1 in the postnatal heart (YapF/FCre) elicited increased myocyte apoptosis and fibrosis, dilated cardiomyopathy, and premature death. Heterozygous deletion (Yap+/FCre) did not cause an overt cardiac phenotype compared with YapF/F control mice at base line. In response to stress (MI), nuclear Yap1 was found selectively in the border zone and not in the remote area of the heart. After chronic MI (28 days), Yap+/FCre mice had significantly increased myocyte apoptosis and fibrosis, with attenuated compensatory cardiomyocyte hypertrophy, and further impaired function versus Yap+/F control mice. Studies in isolated cardiomyocytes demonstrated that Yap1 expression is sufficient to promote increased cell size and hypertrophic gene expression and protected cardiomyocytes against H2O2-induced cell death, whereas Yap1 depletion attenuated phenylephrine-induced hypertrophy and augmented apoptosis. Finally, we observed a significant decrease in cardiomyocyte proliferation in Yap+/FCre hearts compared with Yap+/F controls after MI and demonstrated that Yap1 is sufficient to promote cardiomyocyte proliferation in isolated cardiomyocytes. Our findings suggest that Yap1 is critical for basal heart homeostasis and that Yap1 deficiency exacerbates injury in response to chronic MI. Background: Yap1 regulates cardiac development, yet the function of Yap1 in the adult heart remains unknown. Results: Yap1 promotes cardiomyocyte survival, hypertrophy, and proliferation and protects against chronic myocardial infarction (MI). Conclusion: Yap1 is critical for basal heart homeostasis, and Yap1 deficiency exacerbates myocardial injury. Significance: Increasing cardiomyocyte survival and proliferation may afford protection in vivo against MI injury.

Trehalose (TRE) is a natural, nonreducing disaccharide synthesized by lower organisms. TRE exhibits an extraordinary ability to protect cells against different kinds of stresses through activation of autophagy. However, the effect of TRE on the heart during stress has never been tested. This study evaluated the effects of TRE administration in a mouse model of chronic ischemic remodeling. Wild-type (WT) or beclin 1+/− mice were subjected to permanent ligation of the left anterior descending artery (LAD) and then treated with either placebo or trehalose (1 mg/g/day intraperitoneally for 48 h, then 2% in the drinking water). After 4 weeks, echocardiographic, hemodynamic, gravimetric, histological, and biochemical analyses were conducted. TRE reduced left ventricular (LV) dilation and increased ventricular function in mice with LAD ligation compared with placebo. Sucrose, another nonreducing disaccharide, did not exert protective effects during post-infarction LV remodeling. Trehalose administration to mice overexpressing GFP-tagged LC3 significantly increased the number of GFP-LC3 dots, both in the presence and absence of chloroquine administration. TRE also increased cardiac LC3-II levels after 4 weeks following myocardial infarction (MI), indicating that it induced autophagy in the heart in vivo. To evaluate whether TRE exerted beneficial effects through activation of autophagy, trehalose was administered to beclin 1+/− mice. The improvement of LV function, lung congestion, cardiac remodeling, apoptosis, and fibrosis following TRE treatment observed in WT mice were all significantly blunted in beclin 1+/− mice. TRE reduced MI-induced cardiac remodeling and dysfunction through activation of autophagy.

The small G-protein RhoA regulates the actin cytoskeleton, and its involvement in cell proliferation has also been established. In contrast, little is known about whether RhoA participates in cell survival or apoptosis. In cardiomyocytes in vitro, RhoA induces hypertrophic cell growth and gene expression. In vivo, however, RhoA expression leads to development of heart failure (Sah, V. P., Minamisawa, S., Tam, S. P., Wu, T. H., Dorn, G. W., Ross, J. Jr., Chien, K. R., and Brown, J. H. (1999) J. Clin. Investig. 103, 1627–1634), a condition widely associated with cardiomyocyte apoptosis. We demonstrate here that adenoviral overexpression of activated RhoA in cardiomyocytes induces hypertrophy, which transitions over time to apoptosis, as evidenced by caspase activation and nucleosomal DNA fragmentation. The Rho kinase inhibitors Y-27632 and HA-1077 and expression of a dominant negative Rho kinase block these responses. Caspase-9, but not caspase-8, is activated, and its inhibition prevents DNA fragmentation, consistent with involvement of a mitochondrial death pathway. Interestingly, RhoA expression induces a 3–4-fold up-regulation of the proapoptotic Bcl-2 family protein Bax. RhoA also increases levels of activated Bax and the amount of Bax protein localized at mitochondria. Bax mRNA is increased by RhoA, indicating transcriptional regulation, and the ability of a dominant negative p53 mutant to block Bax up-regulation implicates p53 in this response. The involvement of Bax in RhoA-induced apoptosis was examined by treatment with a Bax-inhibitory peptide, which was found to significantly attenuate DNA fragmentation and caspase-9 and -3 activation. The dominant negative p53 also prevents RhoA-induced apoptosis. We conclude that RhoA/Rho kinase activation up-regulates Bax through p53 to induce a mitochondrial death pathway and cardiomyocyte apoptosis.

Rationale: In Drosophila , the Hippo signaling pathway negatively regulates organ size by suppressing cell proliferation and survival through the inhibition of Yorkie, a transcriptional cofactor. Yes-associated protein (YAP), the mammalian homolog of Yorkie, promotes cardiomyocyte growth and survival in postnatal hearts. However, the underlying mechanism responsible for the beneficial effect of YAP in cardiomyocytes remains unclear. Objectives: We investigated whether miR-206, a microRNA known to promote hypertrophy in skeletal muscle, mediates the effect of YAP on promotion of survival and hypertrophy in cardiomyocytes. Methods and Results: Microarray analysis indicated that YAP increased miR-206 expression in cardiomyocytes. Increased miR-206 expression induced cardiac hypertrophy and inhibited cell death in cultured cardiomyocytes, similar to that of YAP. Downregulation of endogenous miR-206 in cardiomyocytes attenuated YAP-induced cardiac hypertrophy and survival, suggesting that miR-206 plays a critical role in mediating YAP function. Cardiac-specific overexpression of miR-206 in mice induced hypertrophy and protected the heart from ischemia/reperfusion injury, whereas suppression of miR-206 exacerbated ischemia/reperfusion injury and prevented pressure overload-induced cardiac hypertrophy. miR-206 negatively regulates Forkhead box protein P1 expression in cardiomyocytes and overexpression of Forkhead box protein P1 attenuated miR-206–induced cardiac hypertrophy and survival, suggesting that Forkhead box protein P1 is a functional target of miR-206. Conclusions: YAP increases the abundance of miR-206, which in turn plays an essential role in mediating hypertrophy and survival by silencing Forkhead box protein P1 in cardiomyocytes.

Mammalian sterile 20-like kinase 1 (Mst1) is a mammalian homolog of Drosophila Hippo, the master regulator of cell death, proliferation, and organ size in flies. It is the chief component of the mammalian Hippo pathway and promotes apoptosis and inhibits compensatory cardiac hypertrophy, playing a critical role in mediating heart failure. How Mst1 is regulated, however, remains unclear. Using genetically altered mice in which expression of the tumor suppressor Ras-association domain family 1 isoform A (Rassf1A) was modulated in a cell type–specific manner, we demonstrate here that Rassf1A is an endogenous activator of Mst1 in the heart. Although the Rassf1A/Mst1 pathway promoted apoptosis in cardiomyocytes, thereby playing a detrimental role, the same pathway surprisingly inhibited fibroblast proliferation and cardiac hypertrophy through both cell-autonomous and autocrine/paracrine mechanisms, playing a protective role during pressure overload. In cardiac fibroblasts, the Rassf1A/Mst1 pathway negatively regulated TNF-α, a key mediator of hypertrophy, fibrosis, and resulting cardiac dysfunction. These results suggest that the functional consequence of activating the proapoptotic Rassf1A/Mst1 pathway during pressure overload is cell type dependent in the heart and that suppressing this mechanism in cardiac fibroblasts could be detrimental.

The mTOR and Hippo pathways have recently emerged as the major signaling transduction cascades regulating organ size and cellular homeostasis. However, direct crosstalk between two pathways is yet to be determined. Here, we demonstrate that mTORC2 is a direct negative regulator of the MST1 kinase, a key component of the Hippo pathway. mTORC2 phosphorylates MST1 at serine 438 in the SARAH domain, thereby reducing its homodimerization and activity. We found that Rictor/mTORC2 preserves cardiac structure and function by restraining the activity of MST1 kinase. Cardiac-specific mTORC2 disruption through Rictor deletion leads to a marked activation of MST1 that, in turn, promotes cardiac dysfunction and dilation, impairing cardiac growth and adaptation in response to pressure overload. In conclusion, our study demonstrates the existence of a direct crosstalk between mTORC2 and MST1 that is critical for cardiac cell survival and growth.

The Hippo pathway, evolutionarily conserved from flies to mammals, promotes cell death and inhibits cell proliferation to regulate organ size. The core component of this cascade, Mst1 in mammalian cells, is sufficient to promote apoptosis. However, the mechanisms underlying both its activation and its ability to elicit cell death remain largely undefined. We here identify a signaling cassette in cardiac myocytes consisting of K-Ras, the scaffold RASSF1A, and Mst1 that is localized to mitochondria and promotes Mst1 activation in response to oxidative stress. Activated Mst1 phosphorylates Bcl-xL at Ser14, which resides in the BH4 domain, thereby antagonizing Bcl-xL-Bax binding. This, in turn, causes activation of Bax and subsequent mitochondria-mediated apoptotic death. Our findings demonstrate mitochondrial localization of Hippo signaling and identify Bcl-xL as a target that is directly modified to promote apoptosis.

Rationale: NF2 (neurofibromin 2) is an established tumor suppressor that promotes apoptosis and inhibits growth in a variety of cell types, yet its function in cardiomyocytes remains largely unknown. Objective: We sought to determine the role of NF2 in cardiomyocyte apoptosis and ischemia/reperfusion (I/R) injury in the heart. Methods and Results: We investigated the function of NF2 in isolated cardiomyocytes and mouse myocardium at baseline and in response to oxidative stress. NF2 was activated in cardiomyocytes subjected to H 2 O 2 and in murine hearts subjected to I/R. Increased NF2 expression promoted the activation of Mst1 (mammalian sterile 20–like kinase 1) and the inhibition of Yap (Yes-associated protein), whereas knockdown of NF2 attenuated these responses after oxidative stress. NF2 increased the apoptosis of cardiomyocytes that appeared dependent on Mst1 activity. Mice deficient for NF2 in cardiomyocytes, NF2 cardiomyocyte-specific knockout (CKO), were protected against global I/R ex vivo and showed improved cardiac functional recovery. Moreover, NF2 cardiomyocyte-specific knockout mice were protected against I/R injury in vivo and showed the upregulation of Yap target gene expression. Mechanistically, we observed nuclear association between NF2 and its activator MYPT-1 (myosin phosphatase target subunit 1) in cardiomyocytes, and a subpopulation of stress-induced nuclear Mst1 was diminished in NF2 CKO hearts. Finally, mice deficient for both NF2 and Yap failed to show protection against I/R indicating that Yap is an important target of NF2 in the adult heart. Conclusions: NF2 is activated by oxidative stress in cardiomyocytes and mouse myocardium and facilitates apoptosis. NF2 promotes I/R injury through the activation of Mst1 and inhibition of Yap, thereby regulating Hippo signaling in the adult heart.

Rationale: The Hippo pathway plays an important role in determining organ size through regulation of cell proliferation and apoptosis. Hippo inactivation and consequent activation of YAP (Yes-associated protein), a transcription cofactor, have been proposed as a strategy to promote myocardial regeneration after myocardial infarction. However, the long-term effects of Hippo deficiency on cardiac function under stress remain unknown. Objective: We investigated the long-term effect of Hippo deficiency on cardiac function in the presence of pressure overload (PO). Methods and Results: We used mice with cardiac-specific homozygous knockout of WW45 (WW45cKO), in which activation of Mst1 (Mammalian sterile 20-like 1) and Lats2 (large tumor suppressor kinase 2), the upstream kinases of the Hippo pathway, is effectively suppressed because of the absence of the scaffolding protein. We used male mice at 3 to 4 month of age in all animal experiments. We subjected WW45cKO mice to transverse aortic constriction for up to 12 weeks. WW45cKO mice exhibited higher levels of nuclear YAP in cardiomyocytes during PO. Unexpectedly, the progression of cardiac dysfunction induced by PO was exacerbated in WW45cKO mice, despite decreased apoptosis and activated cardiomyocyte cell cycle reentry. WW45cKO mice exhibited cardiomyocyte sarcomere disarray and upregulation of TEAD1 (transcriptional enhancer factor) target genes involved in cardiomyocyte dedifferentiation during PO. Genetic and pharmacological inactivation of the YAP-TEAD1 pathway reduced the PO-induced cardiac dysfunction in WW45cKO mice and attenuated cardiomyocyte dedifferentiation. Furthermore, the YAP-TEAD1 pathway upregulated OSM (oncostatin M) and OSM receptors, which played an essential role in mediating cardiomyocyte dedifferentiation. OSM also upregulated YAP and TEAD1 and promoted cardiomyocyte dedifferentiation, indicating the existence of a positive feedback mechanism consisting of YAP, TEAD1, and OSM. Conclusions: Although activation of YAP promotes cardiomyocyte regeneration after cardiac injury, it induces cardiomyocyte dedifferentiation and heart failure in the long-term in the presence of PO through activation of the YAP-TEAD1-OSM positive feedback mechanism.

Fibrotic remodeling of the heart in response to injury contributes to heart failure, yet therapies to treat fibrosis remain elusive. Yes-associated protein (YAP) is activated in cardiac fibroblasts by myocardial infarction, and genetic inhibition of fibroblast YAP attenuates myocardial infarction–induced cardiac dysfunction and fibrosis. YAP promotes myofibroblast differentiation and associated extracellular matrix gene expression through engagement of TEA domain transcription factor 1 and subsequent de novo expression of myocardin-related transcription factor A. Thus, fibroblast YAP is a promising therapeutic target to prevent fibrotic remodeling and heart failure.

Cardiovascular disease (CVD) remains the leading cause of death globally, and heart failure is a major component of CVD-related morbidity and mortality. The development of cardiac hypertrophy in response to hemodynamic overload is initially considered to be beneficial; however, this adaptive response is limited and, in the presence of prolonged stress, will transition to heart failure. Yes-associated protein (YAP), the central downstream effector of the Hippo signaling pathway, regulates proliferation and survival in mammalian cells. Our previous work demonstrated that cardiac-specific loss of YAP leads to increased cardiomyocyte (CM) apoptosis and impaired CM hypertrophy during chronic myocardial infarction (MI) in the mouse heart. Because of its documented cardioprotective effects, we sought to determine the importance of YAP in response to acute pressure overload (PO). Our results indicate that endogenous YAP is activated in the heart during acute PO. YAP activation that depended upon RhoA was also observed in CMs subjected to cyclic stretch. To examine the function of endogenous YAP during acute PO, <i>Yap</i>^+/<i>^flox;Cre</i>^α-MHC (YAP-CHKO) and <i>Yap</i>^+/<i>^flox</i> mice were subjected to transverse aortic constriction (TAC). We found that YAP-CHKO mice had attenuated cardiac hypertrophy and significant increases in CM apoptosis and fibrosis that correlated with worsened cardiac function after 1 week of TAC. Loss of CM YAP also impaired activation of the cardioprotective kinase Akt, which may underlie the YAP-CHKO phenotype. Together, these data indicate a prohypertrophic, prosurvival function of endogenous YAP and suggest a critical role for CM YAP in the adaptive response to acute PO.

Despite significant improvements in reperfusion strategies, acute coronary syndromes all too often culminate in a myocardial infarction (MI). The consequent MI can, in turn, lead to remodeling of the left ventricle (LV), the development of LV dysfunction, and ultimately progression to heart failure (HF). Accordingly, an improved understanding of the underlying mechanisms of MI remodeling and progression to HF is necessary. One common approach to examine MI pathology is with murine models that recapitulate components of the clinical context of acute coronary syndrome and subsequent MI. We evaluated the different approaches used to produce MI in mouse models and identified opportunities to consolidate methods, recognizing that reperfused and nonreperfused MI yield different responses. The overall goal in compiling this consensus statement is to unify best practices regarding mouse MI models to improve interpretation and allow comparative examination across studies and laboratories. These guidelines will help to establish rigor and reproducibility and provide increased potential for clinical translation. Listen to another corresponding podcast at https://ajpheart.podbean.com/e/guidelines-for-in-vivo-mouse-models-of-myocardial-infarction/ .

RhoA a small G-protein that has an established role in cell growth and in regulation of the actin cytoskeleton. Far less is known about whether RhoA can modulate cell fate. We previously reported that sustained RhoA activation induces cardiomyocyte apoptosis (Del Re, D. P., Miyamoto, S., and Brown, J. H. (2007) <i>J. Biol. Chem.</i> 282, 8069–8078). Here we demonstrate that less chronic RhoA activation affords a survival advantage, protecting cardiomyocytes from apoptotic insult induced by either hydrogen peroxide treatment or glucose deprivation. Under conditions where RhoA is protective, we observe Rho kinase-dependent cytoskeletal rearrangement and activation of focal adhesion kinase (FAK). Activation of endogenous cardiomyocyte FAK leads to its increased association with the p85 regulatory subunit of phosphatidylinositol-3-kinase (PI3K) and to concomitant activation of Akt. Treatment of isolated perfused hearts with sphingosine 1-phosphate recapitulates this response. The pathway by which RhoA mediates cardiomyocyte Akt activation is demonstrated to require Rho kinase, FAK and PI3K, but not Src, based on studies with pharmacological inhibitors (Y-27632, LY294002, PF271 and PP2) and inhibitory protein expression (FAK-related nonkinase). Inhibition of RhoA-mediated Akt activation at any of these steps, including inhibition of FAK, prevents RhoA from protecting cardiomyocytes against apoptotic insult. We further demonstrate that stretch of cardiomyocytes, which activates endogenous RhoA, induces the aforementioned signaling pathway, providing a physiologic context in which RhoA-mediated FAK phosphorylation can activate PI3K and Akt. We suggest that RhoA-mediated effects on the cardiomyocyte cytoskeleton provide a novel mechanism for protection from apoptosis.

The small GTPase RhoA serves as a nodal point for signaling through hormones and mechanical stretch. However, the role of RhoA signaling in cardiac pathophysiology is poorly understood. To address this issue, we generated mice with cardiomyocyte-specific conditional expression of low levels of activated RhoA (CA-RhoA mice) and demonstrated that they exhibited no overt cardiomyopathy. When challenged by in vivo or ex vivo ischemia/reperfusion (I/R), however, the CA-RhoA mice exhibited strikingly increased tolerance to injury, which was manifest as reduced myocardial lactate dehydrogenase (LDH) release and infarct size and improved contractile function. PKD was robustly activated in CA-RhoA hearts. The cardioprotection afforded by RhoA was reversed by PKD inhibition. The hypothesis that activated RhoA and PKD serve protective physiological functions during I/R was supported by several lines of evidence. In WT mice, both RhoA and PKD were rapidly activated during I/R, and blocking PKD augmented I/R injury. In addition, cardiac-specific RhoA-knockout mice showed reduced PKD activation after I/R and strikingly decreased tolerance to I/R injury, as shown by increased infarct size and LDH release. Collectively, our findings provide strong support for the concept that RhoA signaling in adult cardiomyocytes promotes survival. They also reveal unexpected roles for PKD as a downstream mediator of RhoA and in cardioprotection against I/R.

The discovery of the Hippo pathway can be traced back to two areas of research. Genetic screens in fruit flies led to the identification of the Hippo pathway kinases and scaffolding proteins that function together to suppress cell proliferation and tumor growth. Independent research, often in the context of muscle biology, described Tead (TEA domain) transcription factors, which bind CATTCC DNA motifs to regulate gene expression. These two research areas were joined by the finding that the Hippo pathway regulates the activity of Tead transcription factors mainly through phosphorylation of the transcriptional coactivators Yap and Taz, which bind to and activate Teads. Additionally, many other signal transduction proteins crosstalk to members of the Hippo pathway forming a Hippo signal transduction network. We discuss evidence that the Hippo signal transduction network plays important roles in myogenesis, regeneration, muscular dystrophy, and rhabdomyosarcoma in skeletal muscle, as well as in myogenesis, organ size control, and regeneration of the heart. Understanding the role of Hippo kinases in skeletal and heart muscle physiology could have important implications for translational research.

Abstract Fibrosis is a hallmark of heart disease independent of etiology and is thought to contribute to impaired cardiac dysfunction and development of heart failure. However, the underlying mechanisms that regulate the differentiation of fibroblasts to myofibroblasts and fibrotic responses remain incompletely defined. As a result, effective treatments to mitigate excessive fibrosis are lacking. We recently demonstrated that the Hippo pathway effector Yes-associated protein (YAP) is an important mediator of myofibroblast differentiation and fibrosis in the infarcted heart. Yet, whether YAP activation in cardiac fibroblasts is sufficient to drive fibrosis, and how fibroblast YAP affects myocardial inflammation, a significant component of adverse cardiac remodeling, are largely unknown. In this study, we leveraged adeno-associated virus (AAV) to target cardiac fibroblasts and demonstrate that chronic YAP expression upregulated indices of fibrosis and inflammation in the absence of additional stress. YAP occupied the Ccl2 gene and promoted Ccl2 expression, which was associated with increased macrophage infiltration, pro-inflammatory cytokine expression, collagen deposition, and cardiac dysfunction in mice with cardiac fibroblast-targeted YAP overexpression. These results are consistent with other recent reports and extend our understanding of YAP function in modulating fibrotic and inflammatory responses in the heart.

Acute myocardial infarction (MI) occurs when blood flow to the myocardium is restricted, leading to cardiac damage and massive loss of viable cardiomyocytes. Timely restoration of coronary flow is considered the gold standard treatment for MI patients and limits infarct size; however, this intervention, known as reperfusion, initiates a complex pathological process that somewhat paradoxically also contributes to cardiac injury. Despite being a sterile environment, ischemia/reperfusion (I/R) injury triggers inflammation, which contributes to infarct expansion and subsequent cardiac remodeling and wound healing. The immune response is comprised of subsets of both myeloid and lymphoid-derived cells that act in concert to modulate the pathogenesis and resolution of I/R injury. Multiple mechanisms, including altered metabolic status, regulate immune cell activation and function in the setting of acute MI, yet our understanding remains incomplete. While numerous studies demonstrated cardiac benefit following strategies that target inflammation in preclinical models, therapeutic attempts to mitigate I/R injury in patients were less successful. Therefore, further investigation leveraging emerging technologies is needed to better characterize this intricate inflammatory response and elucidate its influence on cardiac injury and the progression to heart failure.

Mst1 is a central Ser-Thr kinase in the Hippo pathway, which promotes apoptosis and inhibits cell proliferation. We have shown previously that, in cardiomyocytes, oxidative stress activates Mst1 at mitochondria, where Mst1 phosphorylates Bcl-xL at Ser14, inducing dissociation of Bcl-xL from Bax and thereby promoting apoptosis. However, the functional significance of Ser14 phosphorylation of endogenous Bcl-xL in vivo remains elusive. We generated knockin (KI) mice in which Ser14 of Bcl-xL is replaced with Ala. KI mice were born at the expected Mendelian ratio, and adult KI mice exhibited normal cardiac morphology and function at baseline. However, KI mice were protected from myocardial ischemia/reperfusion (I/R) injury and exhibited reduced cardiomyocyte apoptosis. Although suppression of endogenous Mst1 also reduced I/R injury, there was no additive protective effect when Mst1 was inhibited in KI mice. The development of dilated cardiomyopathy induced by cardiac-specific overexpression of Mst1 was also ameliorated in KI mice. Lats2 and YAP, two other key components of the Hippo pathway, were not affected in KI mice. These results suggest that Ser14 phosphorylation of Bcl-xL plays an essential role in mediating both cardiomyocyte apoptosis and myocardial injury by acting as a key downstream mediator of Mst1 independently of the canonical Hippo pathway.

Inflammation is an integral component of cardiovascular disease and is thought to contribute to cardiac dysfunction and heart failure. While ischemia-induced inflammation has been extensively studied in the heart, relatively less is known regarding cardiac inflammation during non-ischemic stress. Recent work has implicated a role for Yes-associated protein (YAP) in modulating inflammation in response to ischemic injury; however, whether YAP influences inflammation in the heart during non-ischemic stress is not described. We hypothesized that YAP mediates a pro-inflammatory response during pressure overload (PO)-induced non-ischemic injury, and that targeted YAP inhibition in the myeloid compartment is cardioprotective. In mice, PO elicited myeloid YAP activation, and myeloid-specific YAP knockout mice (YAPF/F;LysMCre) subjected to PO stress had better systolic function, and attenuated pathological remodeling compared to control mice. Inflammatory indicators were also significantly attenuated, while pro-resolving genes including Vegfa were enhanced, in the myocardium, and in isolated macrophages, of myeloid YAP KO mice after PO. Experiments using bone marrow-derived macrophages (BMDMs) from YAP KO and control mice demonstrated that YAP suppression shifted polarization toward a resolving phenotype. We also observed attenuated NLRP3 inflammasome priming and function in YAP deficient BMDMs, as well as in myeloid YAP KO hearts following PO, indicating disruption of inflammasome induction. Finally, we leveraged nanoparticle-mediated delivery of the YAP inhibitor verteporfin and observed attenuated PO-induced pathological remodeling compared to DMSO nanoparticle control treatment. These data implicate myeloid YAP as an important molecular nodal point that facilitates cardiac inflammation and fibrosis during PO stress and suggest that selective inhibition of YAP may prove a novel therapeutic target in non-ischemic heart disease.

The neonatal rat ventricular myocyte model of hypertrophy has provided tremendous insight with regard to signaling pathways regulating cardiac growth and gene expression. Many mediators thus discovered have been successfully extrapolated to the in vivo setting, as assessed using genetically engineered mice and physiological interventions. Studies in neonatal rat ventricular myocytes demonstrated a role for the small G-protein RhoA and its downstream effector kinase, Rho-associated coiled-coil containing protein kinase (ROCK), in agonist-mediated hypertrophy. Transgenic expression of RhoA in the heart does not phenocopy this response, however, nor does genetic deletion of ROCK prevent hypertrophy. Pharmacologic inhibition of ROCK has effects most consistent with roles for RhoA signaling in the development of heart failure or responses to ischemic damage. Whether signals elicited downstream of RhoA promote cell death or survival and are deleterious or salutary is, however, context and cell-type dependent. The concepts discussed above are reviewed, and the hypothesis that RhoA might protect cardiomyocytes from ischemia and other insults is presented. Novel RhoA targets including phospholipid regulated and regulating enzymes (Akt, PI kinases, phospholipase C, protein kinases C and D) and serum response element-mediated transcriptional responses are considered as possible pathways through which RhoA could affect cardiomyocyte survival.

Patients with diabetes are more prone to developing heart failure in the presence of high blood pressure than those without diabetes. Yes-associated protein (YAP), a key effector of the Hippo signaling pathway, is persistently activated in diabetic hearts, and YAP plays an essential role in mediating the exacerbation of heart failure in response to pressure overload in the hearts of mice fed a high-fat diet. YAP induced dedifferentiation of cardiomyocytes through activation of transcriptional enhancer factor 1 (TEAD1), a transcription factor. Thus, YAP and TEAD1 are promising therapeutic targets for diabetic patients with high blood pressure to prevent the development of heart failure.

Heart disease is a major cause of clinical morbidity and mortality, and a significant health and economic burden worldwide. The loss of functional cardiomyocytes, often a result of myocardial infarction, leads to impaired cardiac output and ultimately heart failure. Therefore, efforts to improve cardiomyocyte viability and stimulate cardiomyocyte proliferation remain attractive therapeutic goals. Originally identified in Drosophila, the Hippo signaling pathway is highly conserved from flies to humans and regulates organ size through modulation of both cell survival and proliferation. This is particularly relevant to the heart, an organ with limited regenerative ability. Recent work has demonstrated a critical role for this signaling cascade in determining heart development, homeostasis, injury and the potential for regeneration. Here we review the function of canonical and non-canonical Hippo signaling in cardiomyocytes, with a particular focus on proliferation and survival, and how this impacts the stressed adult heart.

Following myocardial infarction (MI), myocardial inflammation plays a crucial role in the pathogenesis of MI injury and macrophages are among the key cells activated during the initial phases of the host response regulating the healing process. While macrophages have emerged as attractive effectors in tissue injury and repair, the contribution of macrophages on cardiac cell function and survival is not fully understood due to complexity of the in vivo inflammatory microenvironment. Understanding the key cells involved and how they communicate with one another is of paramount importance for the development of effective clinical treatments. Here, novel in vitro myocardial inflammation models were developed to examine how both direct and indirect interactions with polarized macrophage subsets present in the post-MI microenvironment affect cardiomyocyte function. The indirect model using conditioned medium from polarized macrophage subsets allowed examination of the effects of macrophage-derived factors on stem cell-derived cardiomyocyte function for up to 3 days. The results from the indirect model demonstrated that pro-inflammatory macrophage-derived factors led to a significant downregulation of cardiac troponin T (cTnT) and sarcoplasmic/endoplasmic reticulum calcium ATPase (Serca2) gene expression. It also demonstrated that inhibition of macrophage-secreted matricellular protein, osteopontin (OPN), led to a significant decrease in cardiomyocyte store-operated calcium entry (SOCE). In the direct model, stem cell-derived cardiomyocytes were co-cultured with polarized macrophage subsets for up to 3 days. It was demonstrated that anti-inflammatory macrophages significantly increased cardiomyocyte Ca2+ fractional release while macrophages independent of their subtypes led to significant downregulation of SOCE response in cardiomyocytes. This study describes simplified and controlled in vitro myocardial inflammation models, which allow examination of potential beneficial and deleterious effects of macrophages on cardiomyocytes and vise versa. This can lead to our improved understanding of the inflammatory microenvironment post-MI, otherwise difficult to directly investigate in vivo or by using currently available in vitro models.

The Hippo pathway plays a wide variety of roles in response to stress in the heart. Lats2, a component of the Hippo pathway, is phosphorylated by Mst1/2 and, in turn, phosphorylates YAP, causing inactivation of YAP. Lats2 stimulates apoptosis and negatively affects hypertrophy in cardiomyocytes. However, the role of Lats2 during cardiac stress is poorly understood in vivo. Lats2 is activated in the mouse heart in response to transverse aortic constriction (TAC). We used systemic Lats2 +/- mice to elucidate the role of endogenous Lats2. Cardiac hypertrophy and dysfunction induced by 4 weeks of TAC were attenuated in Lats2 +/- mice, and interstitial fibrosis and apoptosis were suppressed. Although TAC upregulated the Bcl-2 family proapoptotic (Bax and Bak) and anti-apoptotic (Bcl-2 and Bcl-xL) molecules in non-transgenic mice, TAC-induced upregulation of Bax and Bak was alleviated and that of Bcl-2 was enhanced in Lats2 +/- mice. TAC upregulated p53, but this upregulation was abolished in Lats2 +/- mice. Lats2-induced increases in apoptosis and decreases in survival in cardiomyocytes were inhibited by Pifithrin-α, a p53 inhibitor, suggesting that Lats2 stimulates apoptosis via a p53-dependent mechanism. In summary, Lats2 is activated by pressure overload, thereby promoting heart failure by stimulating p53-dependent mechanisms of cell death.

Myocardial infarction (MI) is a major cause of morbidity and mortality worldwide, especially in aging and metabolically unhealthy populations. A major target of regenerative tissue engineering is the restoration of viable cardiomyocytes to preserve cardiac function and circumvent the progression to heart failure post-MI. Amelioration of ischemia is a crucial component of such restorative strategies. Angiogenic β-sheet peptides can self-assemble into thixotropic nanofibrous hydrogels. These syringe aspiratable cytocompatible gels were loaded with stem cells and showed excellent cytocompatibility and minimal impact on the storage and loss moduli of hydrogels. Gels with and without cells were delivered into the myocardium of a mouse MI model (LAD ligation). Cardiac function and tissue remodeling were evaluated up to 4 weeks in vivo. Injectable peptide hydrogels synergized with loaded murine embryonic stem cells to demonstrate enhanced survival after intracardiac delivery during the acute phase post-MI, especially at 7 days. This approach shows promise for post-MI treatment and potentially functional cardiac tissue regeneration and warrants large-scale animal testing prior to clinical translation.

The evolutionarily conserved Hippo pathway plays a pivotal role in governing a variety of biological processes. Heart failure (HF) is a major global health problem with a significant risk of mortality. This review provides a contemporary understanding of the Hippo pathway in regulating different cell types during HF. Through a systematic analysis of each component's regulatory mechanisms within the Hippo pathway, we elucidate their specific effects on cardiomyocytes, fibroblasts, endothelial cells, and macrophages in response to various cardiac injuries. Insights gleaned from both in vitro and in vivo studies highlight the therapeutic promise of targeting the Hippo pathway to address cardiovascular diseases, particularly HF.

Cardiac injury initiates a tissue remodeling process in which aberrant fibrosis plays a significant part, contributing to impaired contractility of the myocardium and the progression to heart failure. Fibrotic remodeling is characterized by the activation, proliferation, and differentiation of quiescent fibroblasts to myofibroblasts, and the resulting effects on the extracellular matrix and inflammatory milieu. Molecular mechanisms underlying fibroblast fate decisions and subsequent cardiac fibrosis are complex and remain incompletely understood. Emerging evidence has implicated the Hippo-Yap signaling pathway, originally discovered as a fundamental regulator of organ size, as an important mechanism that modulates fibroblast activity and adverse remodeling in the heart, while also exerting distinct cell type-specific functions that dictate opposing outcomes on heart failure. This brief review will focus on Hippo-Yap signaling in cardiomyocytes, cardiac fibroblasts, and other non-myocytes, and present mechanisms by which it may influence the course of cardiac fibrosis and dysfunction.

Initially identified inDrosophila melanogaster, the Hippo signaling pathway regulates organ size through modulation of cell proliferation, survival and differentiation. This pathway is evolutionarily conserved and canonical signaling involves a kinase cascade that phosphorylates and inhibits the downstream effector Yes-associated protein (YAP). Recent research has demonstrated a fundamental role of Hippo signaling in cardiac development, homeostasis, injury and regeneration, and remains the subject of intense investigation. However, 2 prominent members of this pathway, RASSF1A and Mst1, have been shown to influence heart function and stress responses through YAP-independent mechanisms. This review summarizes non-canonical targets of RASSF1A and Mst1 and discusses their role in the context of cardiac hypertrophy, autophagy, apoptosis and function. (Circ J 2016; 80: 1504–1510)

HomeCirculation ResearchVol. 123, No. 1Beyond the Cardiomyocyte Free AccessArticle CommentaryPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessArticle CommentaryPDF/EPUBBeyond the CardiomyocyteConsideration of HIPPO Pathway Cell-Type Specificity Dominic P. Del Re Dominic P. Del ReDominic P. Del Re From the Department of Cell Biology and Molecular Medicine, Cardiovascular Research Institute, Rutgers New Jersey Medical School, Newark. Search for more papers by this author Originally published22 Jun 2018https://doi.org/10.1161/CIRCRESAHA.118.313383Circulation Research. 2018;123:30–32Increasing interest in the HIPPO signaling pathway has stemmed largely from its ability to modulate cardioprotection and heart regeneration after injury, responses originating from manipulation within the cardiomyocyte. This viewpoint discusses the need for definitive understanding of HIPPO pathway function in cardiac nonmyocytes and highlights the necessity of considering cell-type specificity for effective therapeutic targeting of the HIPPO pathway in cardiovascular disease.The HIPPO pathway is a highly conserved kinase cascade that determines organ size by regulating cell survival, proliferation, and differentiation. On activation, the core components of the pathway, MST (mammalian sterile 20-like kinase) 1/2, SAV1 (salvador 1), LATS (large tumor suppressor kinase) 1/2, and MOB1 (Mps one binder kinase activator-like 1), serve to phosphorylate and repress activity of the transcriptional cofactors YAP (Yes-associated protein) and TAZ (transcriptional coactivator with a PDZ-binding domain). When HIPPO is inhibited, YAP/TAZ cytosolic retention is relinquished, allowing for nuclear localization and association with multiple transcription factors, the most established being the TEAD (TEA domain) family (Figure 1). Investigation of the HIPPO pathway in the heart has garnered increased attention during the past decade and justifiably so. HIPPO is fundamental in maintaining adult heart homeostasis and modulating responses to injury. To date, the majority of studies investigating HIPPO signaling in the heart have focused on the cardiomyocyte. In response to myocardial infarction or pressure overload stress, the core HIPPO kinase MST1 is activated in cardiomyocytes. MST1 signals through canonical (to restrain YAP) and noncanonical (to antagonize BCL-XL [B-cell lymphoma-extra large] and Beclin1) mechanisms resulting in the inhibition of cardiomyocyte proliferation and autophagy and the enhancement of cell death.1–3 Conversely, HIPPO pathway inhibition limits injury and promotes tissue regeneration through YAP-dependent increases in cardiomyocyte proliferation and survival.4 As a result, interventions that activate cardiomyocyte YAP have become attractive and shown therapeutic potential.5Download figureDownload PowerPointFigure 1. A simplified representation of the HIPPO pathway. On pathway activation, the core kinase MST (mammalian sterile 20-like kinase) 1/2 facilitates canonical signaling through phosphorylation and activation of LATS (large tumor suppressor kinase) 1/2 and the subsequent phosphorylation and inhibition of the transcriptional cofactors, YAP and TAZ. MST1 can also directly phosphorylate BCL-XL, leading to apoptosis, and Beclin1, which attenuates autophagy in cardiomyocytes. MOB1 indicates Mps one binder kinase activator-like 1; NF2, neurofibromin 2; and SAV1, salvador 1.In contrast to the cardiomyocyte, HIPPO-YAP function in other cell types known to impact heart injury remains much less understood. It is clear that nonmyocytes outnumber cardiomyocytes in the heart and play essential roles in cardiac growth, homeostasis, and disease. Therefore, a more complete understanding of HIPPO-YAP function in nonmyocytes is imperative to furthering our comprehension, and improving the treatment, of cardiovascular disease.HIPPO Pathway Function in Immune CellsAfter stress, such as myocardial infarction, a sterile inflammatory response is rapidly activated in the myocardium. This is necessary to clear debris and promote wound healing after injury. However, excessive inflammation can augment matrix degradation, cause greater cardiomyocyte loss, increase fibrosis, and worsen heart function. Therefore, a balanced response is critical to provide maximum cardioprotection and optimal wound healing.The HIPPO pathway can modulate both adaptive and innate immune responses. MST1 influences T cell survival, adhesion, chemotaxis, and proliferation—all important determinants of the immune response.6 Recent work demonstrated that TAZ regulates T cell differentiation. TAZ promoted the development of TH17 cells—a proinflammatory subtype that contributes to autoimmunity, while attenuating Treg cell production.7 TAZ directly bound to RORγt (retinoic acid-related orphan receptor gamma t) to promote the TH17 subset and potentiated autoimmune disease, implicating HIPPO pathway as a negative regulator of adaptive immune responses. In contrast, data from 2 recent studies suggest that activation of HIPPO signaling, and inhibition of YAP, is necessary for proper innate antiviral immunity. In macrophages, YAP directly interacted with IRF3 (interferon regulatory factor 3) to prevent IRF3 dimerization and nuclear translocation, thereby attenuating antiviral defense.8 Additionally, YAP bound to TBK1 (TANK-binding kinase 1), preventing its association with signaling adapters and impeding IRF3 activation.9 Taken together, these findings suggest that HIPPO-YAP/TAZ signaling may have opposing effects depending on immune cell subtype. Importantly, because HIPPO signaling impacts adaptive and innate immune cell function, it is likely to modulate both pathogen-triggered responses, for example, myocarditis, as well as sterile inflammation resulting from injury, for example, myocardial infarction. To what extent HIPPO function in immune cells impacts heart injury and healing remains unknown.HIPPO Pathway in Vascular CellsEndothelial and smooth muscle cells are critical components of the vasculature in the heart, and a functional role for HIPPO-YAP/TAZ has been demonstrated in both. Endothelial cell behavior is fundamentally influenced by blood flow-induced sheer stress. Laminar flow prevents leukocyte adhesion, inflammation, and atherosclerotic plaque formation, whereas sites of disturbed flow promote inflammation and are atheroprone. YAP/TAZ is activated in endothelial cells by disturbed flow and stimulates proliferation, inflammation, and promotes lesion formation.10 Inhibition of YAP/TAZ in endothelial cells was shown to protect against the development of atherosclerosis and suggests that activation of HIPPO may be advantageous. However, endothelial YAP/TAZ has also been shown to promote vascular barrier formation and MYC-dependent proliferation to mediate angiogenesis in the developing nervous system.11 Whether YAP/TAZ also promotes angiogenic responses after injury to the adult heart remains unknown. If so, there may be therapeutic potential in targeting YAP/TAZ activation (or HIPPO inhibition) to stimulate vessel formation during ischemia to enhance cardiomyocyte survival. Importantly though, any predicted benefit must be weighed against the potential for atherosclerotic progression. It has also been shown that thromboxane A2—a mediator of platelet aggregation—causes the repression of HIPPO, and the subsequent activation of YAP/TAZ, in vascular smooth muscle cells.12 Inhibition of YAP/TAZ prevented proliferation and migration. Therefore, enhancing YAP/TAZ activation in this context could lead to unintended outcomes, for example, promoting restenosis, further increasing the complexity of HIPPO-YAP/TAZ signaling in the vasculature.HIPPO Signaling in FibroblastsRecent work beautifully demonstrated that deficiency in HIPPO signaling (LATS1/2 kinase inhibition) causes the arrest of cardiac fibroblast differentiation during heart development—a response that is mediated by YAP/TAZ activation.13 These findings indicate that, at least in the embryonic heart, aberrant YAP/TAZ function is sufficient to restrain cardiac fibroblast maturation from epicardial precursors, thereby disrupting proper extracellular matrix composition and subsequent coronary vasculature formation. In the setting of idiopathic pulmonary fibrosis, where tissue stiffness is augmented, YAP/TAZ was activated in lung fibroblasts and drove proliferation, profibrotic gene expression, and pathological matrix remodeling.14 Importantly, little is known about the function of cardiac fibroblast HIPPO-YAP/TAZ in the adult heart. Although the latter study is not cardiac, it is possible that YAP/TAZ promotes a similar feed-forward fibrotic cycle to remodel the myocardium in response to injury.Intercellular communication integrates signaling to modulate heart responses through autocrine and paracrine mechanisms. We previously examined the function of RASSF1A (Ras association domain family 1A)—a HIPPO pathway activator—in cardiomyocytes and cardiac fibroblasts in the context of pressure overload-induced hypertrophy and heart failure.15 Interestingly, RASSF1A promoted HIPPO signaling in both cell types, but pathway engagement showed opposing outcomes for heart pathology and function. Systemic, but not cardiomyocyte-restricted, deletion of RASSF1A resulted in augmented cardiac fibrosis, and inhibition of RASSF1A-MST1 in cardiac fibroblasts increased proliferation, NF-κB (nuclear factor kappa B) activation, and TNFα (tumor necrosis factor alpha) secretion. Neutralization of TNFα in RASSF1A knockout mice was sufficient to prevent the hypertrophic and fibrotic phenotype, thereby demonstrating an important paracrine function downstream of HIPPO signaling that originates in the cardiac fibroblast. Although these studies offer a glimpse into HIPPO-YAP fibroblast function in the adult heart, additional studies that leverage fibroblast-selective genetic manipulation in vivo are necessary to expand our understanding of cardiac fibrosis.Concluding RemarksThe heart is a sophisticated organ consisting of multiple cell types that work in concert to move blood against resistance. In looking forward, it will be imperative to remain cognizant that these cell types fundamentally influence one another to maintain heart homeostasis and respond to challenges. Although much has been learned about HIPPO-YAP signaling in the cardiomyocyte, and efforts to manipulate YAP activation have shown promise, optimal interventions will likely require cell-type specificity because YAP activation in nonmyocytes may lead to adverse outcomes and negate cardiomyocyte benefit (Figure 2). Therefore, the ability to selectively modulate HIPPO-YAP activity by cell type may offer unique opportunities for future therapeutic development.Download figureDownload PowerPointFigure 2. An abbreviated summary of phenotypes resulting from HIPPO inactivation or YAP/TAZ activation in cell types relevant to the adult heart. Generally, YAP/TAZ is restrained by basal HIPPO activity under physiological conditions. Further HIPPO activation can elicit opposite phenotypes of those noted, although this has not been validated in all cell types. To date, the majority of studies that focused on nonmyocyte HIPPO-YAP signaling were performed in noncardiac systems.Sources of FundingD.P. Del Re is supported by grants from the National Institutes of Health (R01 HL127339 and R15 HL135726).DisclosuresNone.FootnotesThe opinions expressed in this article are not necessarily those of the editors or of the American Heart Association.Correspondence to Dominic P. Del Re, PhD, Department of Cell Biology and Molecular Medicine, Cardiovascular Research Institute, Rutgers New Jersey Medical School, 185 S Orange Ave, MSB G-609, Newark, NJ 07103-2714. E-mail [email protected]References1. Maejima Y, Kyoi S, Zhai P, Liu T, Li H, Ivessa A, Sciarretta S, Del Re DP, Zablocki DK, Hsu CP, Lim DS, Isobe M, Sadoshima J. Mst1 inhibits autophagy by promoting the interaction between Beclin1 and Bcl-2.Nat Med. 2013; 19:1478–1488. doi: 10.1038/nm.3322.CrossrefMedlineGoogle Scholar2. Del Re DP, Matsuda T, Zhai P, Maejima Y, Jain MR, Liu T, Li H, Hsu CP, Sadoshima J. Mst1 promotes cardiac myocyte apoptosis through phosphorylation and inhibition of Bcl-xL.Mol Cell. 2014; 54:639–650. doi: 10.1016/j.molcel.2014.04.007.CrossrefMedlineGoogle Scholar3. Matsuda T, Zhai P, Sciarretta S, Zhang Y, Jeong JI, Ikeda S, Park J, Hsu CP, Tian B, Pan D, Sadoshima J, Del Re DP. NF2 activates hippo signaling and promotes ischemia/reperfusion injury in the heart.Circ Res. 2016; 119:596–606. doi: 10.1161/CIRCRESAHA.116.308586.LinkGoogle Scholar4. Leach JP, Heallen T, Zhang M, Rahmani M, Morikawa Y, Hill MC, Segura A, Willerson JT, Martin JF. Hippo pathway deficiency reverses systolic heart failure after infarction.Nature. 2017; 550:260–264. doi: 10.1038/nature24045.CrossrefMedlineGoogle Scholar5. Lin Z, von Gise A, Zhou P, Gu F, Ma Q, Jiang J, Yau AL, Buck JN, Gouin KA, van Gorp PR, Zhou B, Chen J, Seidman JG, Wang DZ, Pu WT. Cardiac-specific YAP activation improves cardiac function and survival in an experimental murine MI model.Circ Res. 2014; 115:354–363. doi: 10.1161/CIRCRESAHA.115.303632.LinkGoogle Scholar6. Zhou D, Medoff BD, Chen L, Li L, Zhang XF, Praskova M, Liu M, Landry A, Blumberg RS, Boussiotis VA, Xavier R, Avruch J. The Nore1B/Mst1 complex restrains antigen receptor-induced proliferation of naïve T cells.Proc Natl Acad Sci USA. 2008; 105:20321–20326. doi: 10.1073/pnas.0810773105.CrossrefMedlineGoogle Scholar7. Geng J, Yu S, Zhao H, et al. The transcriptional coactivator TAZ regulates reciprocal differentiation of TH17 cells and treg cells.Nat Immunol. 2017; 18:800–812. doi: 10.1038/ni.3748.CrossrefMedlineGoogle Scholar8. Wang S, Xie F, Chu F, Zhang Z, Yang B, Dai T, Gao L, Wang L, Ling L, Jia J, van Dam H, Jin J, Zhang L, Zhou F. YAP antagonizes innate antiviral immunity and is targeted for lysosomal degradation through IKKε-mediated phosphorylation.Nat Immunol. 2017; 18:733–743. doi: 10.1038/ni.3744.CrossrefMedlineGoogle Scholar9. Zhang Q, Meng F, Chen S, et al. Hippo signalling governs cytosolic nucleic acid sensing through YAP/TAZ-mediated TBK1 blockade.Nat Cell Biol. 2017; 19:362–374. doi: 10.1038/ncb3496.CrossrefMedlineGoogle Scholar10. Wang L, Luo JY, Li B, et al. Integrin-yap/taz-jnk cascade mediates atheroprotective effect of unidirectional shear flow.Nature. 2016; 540:579–582. doi: 10.1038/nature20602.CrossrefMedlineGoogle Scholar11. Kim J, Kim YH, Kim J, Park DY, Bae H, Lee DH, Kim KH, Hong SP, Jang SP, Kubota Y, Kwon YG, Lim DS, Koh GY. YAP/TAZ regulates sprouting angiogenesis and vascular barrier maturation.J Clin Invest. 2017; 127:3441–3461. doi: 10.1172/JCI93825.CrossrefMedlineGoogle Scholar12. Feng X, Liu P, Zhou X, Li MT, Li FL, Wang Z, Meng Z, Sun YP, Yu Y, Xiong Y, Yuan HX, Guan KL. Thromboxane A2 activates YAP/TAZ protein to induce vascular smooth muscle cell proliferation and migration.J Biol Chem. 2016; 291:18947–18958. doi: 10.1074/jbc.M116.739722.CrossrefMedlineGoogle Scholar13. Xiao Y, Hill MC, Zhang M, Martin TJ, Morikawa Y, Wang S, Moise AR, Wythe JD, Martin JF. Hippo signaling plays an essential role in cell state transitions during cardiac fibroblast development.Dev Cell. 2018; 45:153.e6–169.e6. doi: 10.1016/j.devcel.2018.03.019.CrossrefGoogle Scholar14. Liu F, Lagares D, Choi KM, et al. Mechanosignaling through YAP and TAZ drives fibroblast activation and fibrosis.Am J Physiol Lung Cell Mol Physiol. 2015; 308:L344–L357. doi: 10.1152/ajplung.00300.2014.CrossrefMedlineGoogle Scholar15. Del Re DP, Matsuda T, Zhai P, Gao S, Clark GJ, Van Der Weyden L, Sadoshima J. Proapoptotic Rassf1A/Mst1 signaling in cardiac fibroblasts is protective against pressure overload in mice.J Clin Invest. 2010; 120:3555–3567. doi: 10.1172/JCI43569.CrossrefMedlineGoogle Scholar Previous Back to top Next FiguresReferencesRelatedDetailsCited By Zhang Q, Wang L, Wang S, Cheng H, Xu L, Pei G, Wang Y, Fu C, Jiang Y, He C and Wei Q (2022) Signaling pathways and targeted therapy for myocardial infarction, Signal Transduction and Targeted Therapy, 10.1038/s41392-022-00925-z, 7:1, Online publication date: 1-Dec-2022. Patel N, Nassal D, Gratz D and Hund T (2020) Emerging therapeutic targets for cardiac arrhythmias: role of STAT3 in regulating cardiac fibroblast function, Expert Opinion on Therapeutic Targets, 10.1080/14728222.2021.1849145, 25:1, (63-73), Online publication date: 2-Jan-2021. Manno G, Filorizzo C, Fanale D, Brando C, Di Lisi D, Lunetta M, Bazan V, Russo A and Novo G (2021) Role of the HIPPO pathway as potential key player in the cross talk between oncology and cardiology, Critical Reviews in Oncology/Hematology, 10.1016/j.critrevonc.2021.103246, 159, (103246), Online publication date: 1-Mar-2021. Sweaad W, Stefanizzi F, Chamorro-Jorganes A, Devaux Y and Emanueli C (2021) Relevance of N6-methyladenosine regulators for transcriptome: Implications for development and the cardiovascular system, Journal of Molecular and Cellular Cardiology, 10.1016/j.yjmcc.2021.05.006, 160, (56-70), Online publication date: 1-Nov-2021. Zhang C, Wang F, Gao Z, Zhang P, Gao J and Wu X (2020) Regulation of Hippo Signaling by Mechanical Signals and the Cytoskeleton, DNA and Cell Biology, 10.1089/dna.2019.5087, 39:2, (159-166), Online publication date: 1-Feb-2020. Gong L, Wang S, Shen L, Liu C, Shenouda M, Li B, Liu X, Shaw J, Wineman A, Yang Y, Xiong D, Eichmann A, Evans S, Weiss S and Si M (2020) SLIT3 deficiency attenuates pressure overload–induced cardiac fibrosis and remodeling, JCI Insight, 10.1172/jci.insight.136852, 5:12, Online publication date: 18-Jun-2020. Cho H, Kim J, Ahn J, Hong Y, Mäkinen T, Lim D and Koh G (2018) YAP and TAZ Negatively Regulate Prox1 During Developmental and Pathologic Lymphangiogenesis, Circulation Research, 124:2, (225-242), Online publication date: 18-Jan-2019. Meng F, Xie B and Martin J (2021) Targeting the Hippo pathway in heart repair, Cardiovascular Research, 10.1093/cvr/cvab291 June 22, 2018Vol 123, Issue 1 Advertisement Article InformationMetrics © 2018 American Heart Association, Inc.https://doi.org/10.1161/CIRCRESAHA.118.313383PMID: 29929975 Originally publishedJune 22, 2018 Keywordscardiovascular diseasesfibroblastsmyocardial infarctioninflammationregenerationPDF download Advertisement SubjectsCell Signaling/Signal TransductionHeart FailureMyocardial Infarction

Inflammation is a central feature of cardiovascular disease, including myocardial infarction and heart failure. Reperfusion of the ischemic myocardium triggers a complex inflammatory response that can exacerbate injury and worsen heart function, as well as prevent myocardial rupture and mediate wound healing. Therefore, a more complete understanding of this process could contribute to interventions that properly balance inflammatory responses for improved outcomes. In this study, we leveraged several approaches, including global and regional ischemia/reperfusion (I/R), genetically modified mice, and primary cell culture, to investigate the cell type–specific function of the tumor suppressor Ras association domain family member 1 isoform A (RASSF1A) in cardiac inflammation. Our results revealed that genetic inhibition of RASSF1A in cardiomyocytes affords cardioprotection, whereas myeloid-specific deletion of RASSF1A exacerbates inflammation and injury caused by I/R in mice. Cell-based studies revealed that RASSF1A negatively regulates NF-κB and thereby attenuates inflammatory cytokine expression. These findings indicate that myeloid RASSF1A antagonizes I/R-induced myocardial inflammation and suggest that RASSF1A may be a promising target in immunomodulatory therapy for the management of acute heart injury. Inflammation is a central feature of cardiovascular disease, including myocardial infarction and heart failure. Reperfusion of the ischemic myocardium triggers a complex inflammatory response that can exacerbate injury and worsen heart function, as well as prevent myocardial rupture and mediate wound healing. Therefore, a more complete understanding of this process could contribute to interventions that properly balance inflammatory responses for improved outcomes. In this study, we leveraged several approaches, including global and regional ischemia/reperfusion (I/R), genetically modified mice, and primary cell culture, to investigate the cell type–specific function of the tumor suppressor Ras association domain family member 1 isoform A (RASSF1A) in cardiac inflammation. Our results revealed that genetic inhibition of RASSF1A in cardiomyocytes affords cardioprotection, whereas myeloid-specific deletion of RASSF1A exacerbates inflammation and injury caused by I/R in mice. Cell-based studies revealed that RASSF1A negatively regulates NF-κB and thereby attenuates inflammatory cytokine expression. These findings indicate that myeloid RASSF1A antagonizes I/R-induced myocardial inflammation and suggest that RASSF1A may be a promising target in immunomodulatory therapy for the management of acute heart injury.

T here is no cure for ischemic heart disease; therefore, limiting damage and salvaging viable myocardium are the current gold standard for patient care.In recent years, much interest has surrounded the idea of cell-based myocardial regeneration as a therapeutic alternative to conventional treatment regimens.However, success in human trials using a variety of stem cells or progenitor-like cells has been limited. 1 This has left the field to ponder how this approach can be improved.It seems as though the ideal candidate cell type should effectively maintain the stem cell/progenitor population and efficiently differentiate into functional cardiomyocytes.Admittedly, this is no easy task.The reprogramming of somatic cells to induced pluripotent stem cells, 2 or even directly to cardiomyocyte-like cells, 3 is remarkable and is expected to have lasting effects on stem cell biology and medicine in general.Yet this reprogramming approach to organ regeneration remains limited in its current state at least partly because of the 1:1 ratio between starting material and finished product.

Abstract Control of cell number and organ size is critical for appropriate development and tissue homeostasis. Studies in both Drosophila and mammals have established the Hippo signaling pathway as an important modulator of organ size and tumorigenesis. Upon activation, this kinase cascade modulates gene expression through the phosphorylation and inhibition of transcription co‐activators that are involved in cell proliferation, differentiation, growth and apoptosis. Hippo signaling serves to limit organ size and suppress malignancies, and has been implicated in tissue regeneration following injury. These outcomes highlight the important role that Hippo signaling plays in regulating both physiologic and pathologic processes. In this review, an overview of the signaling pathway will be discussed as well as recent work that has investigated its role in cardiac development, regeneration and disease.

In general, Ras proteins are thought to promote cardiac hypertrophy, an important risk factor for cardiovascular disease and heart failure. However, the contribution of different Ras isoforms has not been investigated. The objective of this study was to define the role of H- and K-Ras in modulating stress-induced myocardial hypertrophy and failure.We used H- and K-Ras gene knockout mice and subjected them to pressure overload to induce cardiac hypertrophy and dysfunction. We observed a worsened cardiac phenotype in Hras-/- mice, while outcomes were improved in Kras+/- mice. We also used a neonatal rat cardiomyocyte culture system to elucidate the mechanisms underlying these observations. Our findings demonstrate that H-Ras, but not K-Ras, promotes cardiomyocyte hypertrophy both in vivo and in vitro. This response was mediated in part through the phosphoinositide 3-kinase-AKT signaling pathway. Adeno-associated virus-mediated increase in AKT activation improved the cardiac function in pressure overloaded Hras null hearts in vivo. These findings further support engagement of the phosphoinositide 3-kinase-AKT signaling axis by H-Ras.Taken together, these findings indicate that H- and K-Ras have divergent effects on cardiac hypertrophy and heart failure in response to pressure overload stress.

The anti-apoptotic function of Bcl-xL in the heart during ischemia/reperfusion is diminished by K-Ras-Mst1-mediated phosphorylation of Ser14, which allows dissociation of Bcl-xL from Bax and promotes cardiomyocyte death. Here we show that Ser14 phosphorylation of Bcl-xL is also promoted by hemodynamic stress in the heart, through the H-Ras-ERK pathway. Bcl-xL Ser14 phosphorylation-resistant knock-in male mice develop less cardiac hypertrophy and exhibit contractile dysfunction and increased mortality during acute pressure overload. Bcl-xL Ser14 phosphorylation enhances the Ca2+ transient by blocking the inhibitory interaction between Bcl-xL and IP3Rs, thereby promoting Ca2+ release and activation of the calcineurin-NFAT pathway, a Ca2+-dependent mechanism that promotes cardiac hypertrophy. These results suggest that phosphorylation of Bcl-xL at Ser14 in response to acute pressure overload plays an essential role in mediating compensatory hypertrophy by inducing the release of Bcl-xL from IP3Rs, alleviating the negative constraint of Bcl-xL upon the IP3R-NFAT pathway.

HomeCirculationVol. 120, No. 10Optimizing Cell-Based Therapy for Cardiac Regeneration Free AccessEditorialPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessEditorialPDF/EPUBOptimizing Cell-Based Therapy for Cardiac Regeneration Dominic P. Del Re and Junichi Sadoshima Dominic P. Del ReDominic P. Del Re From the Cardiovascular Research Institute, Department of Cell Biology and Molecular Medicine, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark. Search for more papers by this author and Junichi SadoshimaJunichi Sadoshima From the Cardiovascular Research Institute, Department of Cell Biology and Molecular Medicine, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark. Search for more papers by this author Originally published24 Aug 2009https://doi.org/10.1161/CIRCULATIONAHA.109.887836Circulation. 2009;120:831–834Other version(s) of this articleYou are viewing the most recent version of this article. Previous versions: August 24, 2009: Previous Version 1 Long thought to be a terminally differentiated organ, recent findings suggest that the adult mammalian heart is a slowly regenerating organ1 and home to a resident population of cardiac progenitor cells (CPCs) that renew cardiomyocytes and have the potential to differentiate into multiple cell types within the myocardium.2 In spite of this, the regenerative capacity of the mammalian heart is inadequate compared with the resulting damage caused by ischemic episodes. In light of the challenges faced by resident progenitor cells, many studies have focused on delivery of exogenously prepared stem or progenitor cell types to the damaged heart. CPCs are an ideal candidate for cardiac cell-based therapy because they are programmed to differentiate into cardiomyocytes, endothelial cells, and smooth muscle cells, thus providing not only contractile benefit but also increased vascularization. However, the ischemic myocardium is a hostile microenvironment, and multiple factors contribute to prevent cardiac regeneration, including ischemia, inflammation, and fibrosis. Therefore, it is critical to provide a complementary approach that promotes CPC survival, proliferation, and differentiation.Article see p 876In the study by Padin-Iruegas et al3 in this issue of Circulation, insulin-like growth factor 1 (IGF-1), an endogenous peptide that is activated in response to cardiac injury,4 is delivered to the heart using nanofiber tethering (NF-IGF-1) both alone and in combination with CPCs. In vivo administration of CPCs plus NF-IGF-1 led to significantly more CPC-derived cardiomyocytes that were larger, more differentiated, and incorporated electrically and mechanically into the host myocardium. This combination treatment also increased endogenous CPC proliferation and differentiation to cardiomyocytes, endothelial cells, and smooth muscle cells. Combination therapy led to significant improvement in both ventricular performance and morphological parameters compared with CPC or NF-IGF-1 treatment alone, emphasizing the therapeutic benefit of prolonged exposure of CPCs to IGF-1.One of the biggest hurdles facing cell-based therapies in the ischemic heart is maintaining the survival of newly implanted cells. Reports have demonstrated cell survival rates broadly ranging from 1% to 32% 1 week after injection into injured hearts.5 Current thinking supposes 3 main causes of cell death: loss of matrix support, ischemia, and inflammation. Cell contacts with the extracellular environment and subsequent receptor engagement initiate protective signaling pathways that are important for survival. Preparing progenitor cells for therapeutic intervention inevitably leads to decreased adhesion-related survival signals and programmed cell death.6 Recent evidence suggests that inhibition of Rho-associated kinase can increase survival of human embryonic stem cells after dissociation,7 possibly by preventing anoikis, providing an opportunity to further improve cell viability through pharmacological means. More importantly, however, death also results from the ischemic milieu, characterized by increased reactive oxygen species production and mitochondrial dysfunction. This harsh environment is also highly inflamed after an ischemic episode. IGF-1 stimulates cell-protective mechanisms against oxidative stress3,8 and inflammation,9 thereby protecting CPCs from death. IGF-1 also facilitates survival of CPCs, possibly through local upregulation of vascular endothelial growth factor and consequent stimulation of angiogenesis.10 Because both CPCs8 and endogenous cardiomyocytes11 possess IGF-1 receptor, the expression of which is upregulated during myocardial repair after myocardial injury,11 locally applied IGF-1 should act on both CPCs and cardiomyocytes.Another obstacle hampering cell-based cardiac repair is the relatively low number of endogenous stem/progenitor cells available in the heart. Previous work has demonstrated a plateau in the effectiveness of increasing the number of stem cells to be injected.5 Therefore, increasing in situ proliferation of CPCs should lead to better outcomes. In this work, Padin-Iruegas et al have shown that IGF-1 has cell-autonomous proliferative effects on CPCs.3 IGF-1 is an effective promoter of cell proliferation in pluripotent stem cells, including human embryonic stem cells.12 IGF-1 also promotes proliferation in human stem cell–derived cardiomyocytes13 and adult cardiomyocytes.14 The proliferative effect of IGF-1 in the cardiomyocyte lineage is unique in that progenitor cells may differentiate without surrendering their ability to proliferate. Work by Engert et al15 using the locally acting isoform mIGF-1 has demonstrated that IGF-1 stimulates the proliferation of myoblasts initially and promotes differentiation later in skeletal muscle. This property of IGF-1 appears to be in contrast to the effect of Wnt/β-catenin signaling on ISL1+ cells, another type of CPC, in that Wnt/β-catenin stimulates proliferation but inhibits differentiation of ISL1+ cells.16 In this sense, IGF-1 is an ideal growth factor for cell therapy in the heart.The signaling mechanism competent for both proliferation and differentiation of CPCs is of great interest. In human embryonic stem cell–derived cardiomyocytes, IGF-1–induced proliferation is PI-3 kinase/Akt dependent but ERK independent.13 Activation of Akt in the nucleus causes proliferation of CPCs.17 Because IGF-1 enhances nuclear phospho-Akt in cardiomyocytes8 and because increased nuclear phospho-Akt is observed in the surviving myocardium in the study by Padin-Iruegas et al, if the same effect is seen in CPCs, it may mediate IGF-1–induced proliferation of CPCs. An important point, however, is that the NF-IGF-1–treated CPCs were larger than the untreated CPCs. Because nuclear Akt is thought to antagonize hypertrophy,18 additional signaling mechanisms should be activated in NF-IGF-1–treated CPCs, and further study is warranted to elucidate their role in mediating CPC proliferation.Ensuring that progenitor cells differentiate into the desired cell type(s) is an equally important challenge. Treatment of CPCs with IGF-1 alone or in combination with hepatocyte growth factor gives rise to endothelial cells, smooth muscle cells, and cardiomyocytes.3,19 It would be interesting to test whether IGF-1 is sufficient to stimulate differentiation of CPC clones in a cell-autonomous fashion in vitro. Whether IGF-1 stimulates differentiation rather than proliferation depends on the signaling mechanism mediated through the C-terminal structure of the IGF-1 receptor, as well as the lack of insulin receptor substrate-1 phosphorylation in hematopoietic cells.20 Because nuclear activation of Akt increases the number of cardiomyocyte-committed CPCs,17 testing whether IGF-1 activates nuclear Akt is of interest. In skeletal muscle, Akt is involved in differentiation of satellite cells through p300 phosphorylation and subsequent chromatin remodeling.21 Recent work by Zhu et al22 showed, however, that insulin-like growth factor binding protein-4 (IGFBP-4) plays an important role in mediating cardiomyocyte differentiation during development, when the cardiogenic effect of IGFBP-4 is inhibited by IGF-1 through IGFBP-4 sequestration. Thus, it is possible that IGF-1 may not stimulate all aspects of cardiomyocyte differentiation if CPCs use similar mechanisms to differentiate. Elucidating the downstream signaling mechanisms by which IGF-1 stimulates differentiation of CPCs may allow us to identify a better therapeutic intervention to facilitate CPC differentiation into cardiomyocytes. Padin-Iruegas et al showed that many myocytes in the area of regeneration are BrdU positive. Thus, although they are functionally competent, they are not identical to existing cardiomyocytes. Clinically, it is important to track these new myocytes and to determine to what extent they continue to proliferate and to what extent IGF-1 treatment induces further differentiation of CPC-derived myocytes.There are additional important effects of IGF-1 on endogenous progenitor cells that may translate to CPCs. Recent work has shown the necessity of IGF-1 for stem cell self-renewal.23 Furthermore, IGF-1 promotes the migration of mesenchymal stem cells from bone marrow and improves their localization to the site of infarct.24 The SDF-1:CXCR4 signaling axis is critical to paracrine signaling and homing of resident stem cells for cardiac repair and can be upregulated by IGF-110 (see the Figure). Download figureDownload PowerPointFigure. The diverse effects of IGF-1 on CPCs. IGF-1 has the potential to elicit various cellular effects in pluripotent cells, ranging from differentiation to proliferation and self-renewal. IGF-1 can also promote cell survival by inhibiting apoptosis and acting as a potent antiinflammatory intermediate.The true novelty of the work by Padin-Iruegas et al is the finding that IGF-1, when applied in the right location in the heart for a sufficient period of time, enhances cardiac regeneration by CPCs. Although injection of ex vivo–modified mesenchymal stem cells overexpressing IGF-110 may achieve the same goal, it requires ex vivo manipulation of the stem cell genome before injection, giving rise to the fundamental concerns of gene therapy, which may preclude the use of ex vivo manipulation for immediate clinical application. Nanofibers consisting of short peptides can be injected into the myocardium, where they reassemble to create a stable microenvironment25 and serve as a source of humoral factors. Prior work has shown that whereas IGF-1 tethered to nanofibers remained present 84 days later, untethered IGF-1 was no longer detectable 7 days after injection into the heart.25 This technique not only enhances survival and function of exogenous CPCs but also stimulates recruitment of endogenous CPCs and promotes angiogenesis to improve ischemia. Synergistic enhancement of cardiac regeneration by CPCs and NF-IGF-1 indicates that by optimizing the combination of the right cell population and the right humoral factor, one may further improve the efficiency of cell-based therapies.This technology can easily be applied to additional growth factors/cytokines and even small molecules. An excellent system to discover novel and potentially important endogenous candidates that promote cardiac regeneration is the zebrafish. Zebrafish hearts have the remarkable capacity to regenerate when up to 20% of the ventricle is removed.26 A recent genetic screen for endogenous growth factors revealed 662 genes, including vegfc, pdgf-a, igf2, and thymocin β4, that are differentially expressed during zebrafish heart regeneration.27 These targets may represent powerful endogenous factors that should be explored further if they indeed do translate to the mammalian heart. Using a small-molecule library screen for activators of nkx2.5, Sadek et al28 recently discovered a family of small molecules that can trigger cardiac gene expression in a variety of stem/progenitor cells, underscoring the potential for small molecules to promote differentiation of stem cells to cardiomyocytes for therapeutic use.Recent progress shows that cardiomyocytes can be generated successfully from human induced pluripotent stem cells,29 indicating the possibility that we can make our own cardiomyocytes from fibroblasts. However, the fact that CPCs are genetically programmed to generate the exact constituents of the heart makes them more attractive as a source of cell therapy for the heart. With the recent establishment of a genetic method to purify another type of CPCs,30 we have an enhanced choice of CPCs for cell-based therapies. We expect that combinatorial efforts of molecular biology and bioengineering should further enhance the efficiency of heart regeneration and improve LV function in patients after myocardial infarction.The opinions expressed in this article are not necessarily those of the editors or of the American Heart Association.We thank Daniela Zablocki for critical reading of the manuscript.Sources of FundingThis work was in part supported by US Public Health Service Grants HL059139, HL067724, HL069020, AG023039, AG027211, and HL91469.DisclosuresNone.FootnotesCorrespondence to Junichi Sadoshima, Cardiovascular Research Institute, New Jersey Medical School, Medical Science Bldg G-609, University of Medicine and Dentistry of New Jersey, 185 S Orange Ave, Newark, NJ 07103. E-mail [email protected] References 1 Bergmann O, Bhardwaj RD, Bernard S, Zdunek S, Barnabe-Heider F, Walsh S, Zupicich J, Alkass K, Buchholz BA, Druid H, Jovinge S, Frisen J. Evidence for cardiomyocyte renewal in humans. 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Gao L, Zhang N, Ding Q, Chen H, Hu X, Jiang S, Li T, Chen Y, Wang Z, Ye Y and Zhu Z (2013) Common Expression of Stemness Molecular Markers and Early Cardiac Transcription Factors in Human Wharton's Jelly-Derived Mesenchymal Stem Cells and Embryonic Stem Cells, Cell Transplantation, 10.3727/096368912X662444, 22:10, (1883-1900), Online publication date: 1-Oct-2013. Lee H and Bae H (2013) Forming vascular networks within functional cardiac tissue constructs, Biomedical Engineering Letters, 10.1007/s13534-013-0106-y, 3:3, (138-143), Online publication date: 1-Sep-2013. Liu J, van Mil A, Vrijsen K, Zhao J, Gao L, Metz C, Goumans M, Doevendans P and Sluijter J (2010) MicroRNA-155 prevents necrotic cell death in human cardiomyocyte progenitor cells via targeting RIP1, Journal of Cellular and Molecular Medicine, 10.1111/j.1582-4934.2010.01104.x, 15:7, (1474-1482), Online publication date: 1-Jul-2011. Mishra A, Velotta J, Brinton T, Wang X, Chang S, Palmer O, Sheikh A, Chung J, Yang P, Robbins R and Fischbein M (2011) RevaTen platelet-rich plasma improves cardiac function after myocardial injury, Cardiovascular Revascularization Medicine, 10.1016/j.carrev.2010.08.005, 12:3, (158-163), Online publication date: 1-May-2011. Kellar R, Lancaster J, Goldman S, McAllister T and L'Heureux N (2011) Cardiovascular Tissue Engineering Comprehensive Biomaterials, 10.1016/B978-0-08-055294-1.00177-X, (361-376), . Parodi G and Antoniucci D (2010) Left ventricular remodeling after primary percutaneous coronary intervention, American Heart Journal, 10.1016/j.ahj.2010.10.010, 160:6, (S11-S15), Online publication date: 1-Dec-2010. September 8, 2009Vol 120, Issue 10 Advertisement Article InformationMetrics https://doi.org/10.1161/CIRCULATIONAHA.109.887836PMID: 19704092 Originally publishedAugust 24, 2009 Keywordssignal transductionmyocardial infarctionEditorialsPDF download Advertisement SubjectsCell Signaling/Signal TransductionGrowth Factors/CytokinesIschemia

The RASSF proteins are a family of polypeptides, each containing a conserved Ras association domain, suggesting that these scaffold proteins may be effectors of activated Ras or Ras-related small GTPases. RASSF proteins are characterized by their ability to inhibit cell growth and proliferation while promoting cell death. RASSF1 isoform A is an established tumor suppressor and is frequently silenced in a variety of tumors and human cancer cell lines. However, our understanding of its function in terminally differentiated cell types, such as cardiac myocytes, is relatively nascent. Herein, we review the role of RASSF1A in cardiac physiology and disease and highlight signaling pathways that mediate its function.

Ischemic heart disease is a leading cause of morbidity and mortality worldwide [...].

Embryonic stem cells have the capacity to differentiate into a wide range of cell types. We previously described that blastocyst injection of wild type (WT) embryonic stem cells (ESCs) into various knockout (KO) mouse models of human disease prevents disease from occurring. In this study we ask if the blastocyst approach can also correct defects in a mouse model of transgenic (Tg) overexpression of a pro-apoptotic factor. We injected ROSA26 (LacZ-marked) WT ESCs into human mammalian sterile 20 like-kinase 1 (Mst1) Tg blastocysts. Mst1 Tg mice overexpress Mst1, a pro-apoptotic factor, in a cardiac-specific manner. As a result, Mst1 Tg mice develop adult dilated cardiomyopathy driven by apoptosis, reduction in cell density and no hypertrophic compensation. Incorporation of WT ESCs generated WT/Mst1 chimeric mice with normal hearts at histological and functional levels. Accordingly, apoptosis and cell density parameters were normalized. The experiments suggest that an adult-onset cardiac myopathy induced by overexpression of the pro-apoptotic Mst1 can be reversed by developmental incorporation of WT ESCs. The findings also suggest that since forced expression of the Mst1 transgene is not abolished in the rescued chimeras, the WT ES-derived cells normalize pathways that lie downstream of Mst1. The results expand the therapeutic capability of the ESCs to mouse models that overproduce detrimental proteins.

OAE Publishing Inc. is an international scholarly publisher specializing in peer-reviewed academic journals. To promote academic exchange and knowledge sharing, OAE provides an outstanding academic platform for biomedical experts and scholars all over the world.

Abstract Inhibitor of DNA binding (Id) proteins play important roles in regulating cardiac development via paracrine signaling. Id1/Id3 knockout mice die at mid-gestation with multiple cardiac defects. Single Id knockout studies have not reported cardiomyopathies. To bypass embryonic lethality we used Tie2CRE-mediated recombination to conditionally delete Id1 against global Id3 ablation (Id cDKOs), which develops adult-onset dilated cardiomyopathy. We confirm upregulation of thrombospondin-1 (TSP1) in Id cDKO hearts. Colocalization studies reveal increased TSP1 expression in the vicinity of endothelial cells and near regions of endocardial fibrosis/disruption. Downstream fibrotic molecules were upregulated. Endocardial capillary density was reduced with evidence of vascular distention. Treatment of Id cDKO cardiac explants with LSKL, a peptide antagonist of TSP1 activation of TGFβ, reversed the increased expression of fibrotic molecules. We conducted bone marrow transplant experiments in which we transferred bone marrow cells from Id cDKO mice into lethally irradiated WT mice. The majority of WT recipients of Id cDKO bone marrow cells phenocopied Id cDKO cardiac fibrosis 4 months post-transplantation. Injection of LSKL into adult Id cDKO mice led to downregulation of fibrotic molecules. The results prompt caution when bone marrow transfers from individuals potentially carrying mutations in the Id axis are applied in clinical settings.

Inflammation is an integral component of cardiovascular disease and is thought to contribute to cardiac dysfunction and heart failure. While ischemia-induced inflammation has been extensively studied in the heart, relatively less is known regarding cardiac inflammation during non-ischemic stress. Recent work has implicated a role for Yes-associated protein (YAP) in modulating inflammation in response to ischemic injury; however, whether YAP influences inflammation in the heart during non-ischemic stress is not described. We hypothesized that YAP mediates a pro-inflammatory response during pressure overload (PO)-induced non-ischemic injury, and that targeted YAP inhibition in the myeloid compartment is cardioprotective. In mice, PO elicited myeloid YAP activation, and myeloid-specific YAP knockout mice (YAP F/F ;LysM Cre ) subjected to PO stress had preserved systolic function, and attenuated pathological remodeling compared to control mice. Inflammatory indicators were also significantly attenuated, while pro-resolving genes including Vegfa were enhanced, in the myocardium of myeloid YAP KO mice after PO. Experiments using bone marrow-derived macrophages (BMDMs) from YAP KO and control mice demonstrated that YAP suppression shifted polarization toward a resolving phenotype. We also observed attenuated NLRP3 inflammasome priming and function in YAP deficient BMDMs, as well as in myeloid YAP KO hearts following PO, indicating disruption of inflammasome induction. Studies in RAW264.7 cells demonstrated the effectiveness of pharmacological YAP inhibition using verteporfin to attenuate pro-inflammatory gene expression, and augment VEGFA levels. Finally, we leveraged nanoparticle-mediated delivery of verteporfin and observed attenuated PO-induced pathological remodeling and inflammation compared to DMSO nanoparticle control treatment. These data implicate myeloid YAP as an important molecular nodal point that facilitates cardiac inflammation and fibrosis during PO stress and suggest that selective inhibition of YAP may prove a novel therapeutic target in non-ischemic heart disease.

Department of Cell Biology and Molecular Medicine, New Jersey Medical School, Rutgers University, Newark, NJ 07103.Neurofibromin 2 (NF2) is a tumor suppressor that can engage the Hippo signaling pathway and modulate cell proliferation and survival. We previously demonstrated that NF2 mediates cardiomyocyte apoptosis and injury caused by acute myocardial infarction. However, the function of NF2 in the heart remains largely uncharacterized. Our current study sought to determine whether NF2 modulates heart failure due to chronic stress. We used a transverse aortic constriction (TAC) model in WT C57BL/6J mice to generate chronic pressure overload (PO) stress, which elicits cardiac remodeling and failure. We found that NF2 is transiently upregulated in mouse myocardium in response to early phase of PO, and is downregulated during late phase PO. We generated cardiomyocyte-specific NF2 knockout (cKO) mice, which had normal cardiac morphology and function at baseline. Following TAC, NF2 cKO hearts unexpectedly showed worsened cardiac function, assessed by echocardiography and hemodynamic analysis, compared to controls. RNAseq analysis indicated downregulation of several metabolic pathways including oxidative phosphorylation and fatty acid oxidation in NF2 cKO hearts at baseline. Additionally, we observed reduced ATP content in baseline NF2 cKO hearts compared to controls. Fractionation experiments indicated that nuclear NF2 is enriched following PO stress. RNAseq revealed the downregulation of ERRβ and ERRγ in NF2 cKO hearts, which was confirmed by qPCR. Experiments employing neonatal rat ventricular myocytes (NRMVs) confirmed that NF2 regulated expression of ERRβ and ERRγ, and DNA pulldown assays demonstrated NF2 association with ERRβ and ERRγ proximal promoters. Luciferase reporter assays demonstrated that NF2 modulates ERR function in NRVMs. Based on these findings, we propose that transient upregulation of myocardial NF2 expression during PO stress is compensatory and regulates metabolic gene expression according to energy demand.

Cardiovascular disease is the leading cause of death in the US and was responsible for nearly 1 of every 3 deaths nationwide in 2010 [ [1] Go A.S. Mozaffarian D. Roger V.L. Benjamin E.J. Berry J.D. Blaha M.J. et al. Heart disease and stroke statistics—2014 update: a report from the American Heart Association. Circulation. 2014; 129: e28-e292 Crossref PubMed Scopus (4449) Google Scholar ]. In addition to hypertension, age, obesity and tobacco use among others, cardiac hypertrophy is also recognized as an independent risk factor for cardiovascular morbidity and mortality [ [2] Hill J.A. Olson E.N. Cardiac plasticity. N Engl J Med. 2008; 358: 1370-1380 Crossref PubMed Scopus (892) Google Scholar ]. While initially thought to be a compensatory response required to maintain heart function in the face of added workload or pathologic insult, decompensation and remodeling eventually occur, concomitant with a decline in output, ultimately leading to failure. Depending on the type of stress, the myocardium can respond by enlarging in two distinct ways [ [3] Grossman W. Jones D. McLaurin L.P. Wall stress and patterns of hypertrophy in the human left ventricle. J Clin Invest. 1975; 56: 56-64 Crossref PubMed Scopus (1870) Google Scholar ]. Concentric hypertrophy is defined by increased ventricular wall thickness and typically involves adding sarcomeres in parallel (i.e. widening of individual cardiomyocytes). This mode is often associated with somewhat improved or maintained systolic cardiac performance and reduced diastolic function without chamber dilation. In contrast, eccentric hypertrophy occurs through the addition of sarcomeres in serial (i.e. lengthening of cardiomyocytes) and is typically characterized by chamber dilatation and decreased systolic function. Further elucidation of the underlying mechanisms responsible for directing the heart toward one mode relative to the other could have great therapeutic potential and remains as an active focus of cardiovascular biology.

Noonan syndrome is a relatively common autosomal dominant disorder, affecting every 1:1000–2500 births. It is named after Dr. Jacqueline Noonan, as she was the first to describe the characteristic phenotype consisting of distinct facial features, cranial abnormalities, short stature, chest deformities, and congenital heart disease [ 1 Romano A.A. Allanson J.E. Dahlgren J. Gelb B.D. Hall B. Pierpont M.E. et al. Noonan syndrome: clinical features, diagnosis, and management guidelines. Pediatrics. Oct 2010; 126: 746-759 Crossref PubMed Scopus (383) Google Scholar , 2 Aoki Y. Niihori T. Narumi Y. Kure S. Matsubara Y. The RAS/MAPK syndromes: novel roles of the RAS pathway in human genetic disorders. Hum Mutat. Aug 2008; 29: 992-1006 Crossref PubMed Scopus (275) Google Scholar ]. LEOPARD syndrome (multiple lentigines, electrocardiographic conduction abnormalities, ocular hypertelorism, pulmonary stenosis, abnormal genitalia, retardation of growth and sensorineural deafness) is a closely related genetic disorder that presents many of the same features as Noonan, but occurs less frequently [ [3] Tidyman W.E. Rauen K.A. The RASopathies: developmental syndromes of Ras/MAPK pathway dysregulation. Curr Opin Genet Dev. Jun 2009; 19: 230-236 Crossref PubMed Scopus (539) Google Scholar ]. These two syndromes are part of a larger class of related, yet distinct, developmental disorders, which also includes cardio-facio-cutaneous (CFC) syndrome, Costello syndrome and neurofibromatosis type 1 (NF1). To date, the observed germline mutations linked to these disorders have been confined to established Ras signaling components, and typically result in gain-of-function signaling outcomes that are thought to mediate the disease state. For this reason, they are collectively referred to as RASopathies. Interestingly, although overall phenotypes and associated mutations differ between these disorders, one commonality is that all are known to exhibit cardiac abnormalities. However, the nature of the cardiac defects is divergent among various mutations and the cause of such diversity is not well understood.

After publication of this review [1] it emerged that the references in the Introduction section were incorrect. Several references were omitted and other references were numbered incorrectly. These errors were introduced during the final production process by the publisher and SpringerOpen apologises for any inconvenience caused. The relevant Introduction and references have been replaced with: Myocardial infarction (MI), or insufficient blood flow to the heart muscle, promotes the death and loss of cardiomyocytes resulting in heart damage and impaired cardiac function. While patient survival following MI has improved, the prognosis is typically poor and can eventually progress to heart failure, a leading cause of morbidity and mortality (one). Because mature cardiomyocytes have a limited capacity to re-enter the cell cycle and proliferate [116, 117], the ability of the adult heart to regenerate is similarly restricted and cannot adequately replace lost cardiomyocytes. The Hippo signaling pathway is evolutionarily conserved from flies to mammals and has emerged as an important regulator of both cell survival and proliferation (four) [20]. Importantly, this cascade also appears critical for proper mammalian heart development and the post-natal response to cardiac stress and injury [60, 123, 124]. It is therefore plausible to hypothesize that Hippo signaling could be targeted to promote heart regeneration after MI and heart injury. This review will provide an overview of the Hippo pathway and examine its role in cardiac development, disease and regeneration. The omitted references can be found below: Reference one Go, A. S., Mozaffarian, D., Roger, V. L., Benjamin, E. J., Berry, J. D., Blaha, M. J., Dai, S., Ford, E. S., Fox, C. S., Franco, S., Fullerton, H. J., Gillespie, C., Hailpern, S. M., Heit, J. A., Howard, V. J., Huffman, M. D., Judd, S. E., Kissela, B. M., Kittner, S. J., Lackland, D. T., Lichtman, J. H., Lisabeth, L. D., Mackey, R. H., Magid, D. J., Marcus, G. M., Marelli, A., Matchar, D. B., McGuire, D. K., Mohler, E. R., 3rd, Moy, C. S., Mussolino, M. E., Neumar, R. W., Nichol, G., Pandey, D. K., Paynter, N. P., Reeves, M. J., Sorlie, P. D., Stein, J., Towfighi, A., Turan, T. N., Virani, S. S., Wong, N. D., Woo, D., Turner, M. B., American Heart Association Statistics, C. and Stroke Statistics, S. (2014) Heart disease and stroke statistics--2014 update: a report from the American Heart Association. Circulation. 129, e28-e292 Reference four Staley, B. K. and Irvine, K. D. (2012) Hippo signaling in Drosophila: recent advances and insights. Developmental dynamics : an official publication of the American Association of Anatomists. 241, 3–15

Abstract Introduction: Neurofibromin 2 (NF2) is a tumor suppressor that can engage the Hippo signaling pathway and modulate cell proliferation and survival. We previously demonstrated that NF2 mediates cardiomyocyte apoptosis and injury caused by acute myocardial infarction (MI) through inhibition of the Hippo target YAP. However, the function of NF2 in the heart remains largely uncharacterized. Our current study sought to determine whether NF2 contributes to cardiac remodeling, dysfunction, and heart failure due to chronic stress. Methods and Results: We used a transverse aortic constriction (TAC) model in WT C57BL/6J mice to generate chronic pressure overload (PO) stress, which elicits cardiac hypertrophy, remodeling, and eventually heart failure. We found that NF2 is transiently upregulated in mouse myocardium in response to PO stress. To investigate if increased NF2 contributes to pathology, we used cardiomyocyte-specific NF2 knockout (cKO) mice. At baseline, adult NF2 cKO mice had normal cardiac morphology and function. However, following TAC, NF2 cKO hearts unexpectedly showed worsened cardiac function, assessed by echocardiography and hemodynamic analysis, compared to littermate controls. Interestingly, no differences in the extent of TAC-induced cardiac hypertrophy or cardiomyocyte apoptosis were observed between NF2 cKO and control mice. To investigate the mechanism underlying the NF2 cKO phenotype, we performed RNAseq. Our analysis indicated downregulation of several metabolic pathways including oxidative phosphorylation (OXPHOS) and fatty acid oxidation (FAO) in unstressed NF2 cKO hearts, which was confirmed by qPCR. We also determined that ATP content in NF2 cKO hearts was significantly reduced at baseline compared to control mice. Estrogen-related receptors (ERRs) are important regulators of mitochondrial biogenesis and metabolic function. RNAseq revealed the downregulation of ERRβ and ERRγ in NF2 cKO hearts, which was confirmed by qPCR. Experiments in neonatal cardiomyocytes confirmed that regulation of ERRβ and ERRγ expression by NF2 is cell autonomous. Luciferase reporter assays demonstrated that modulation of NF2 regulates ERR function in cardiomyocytes. We also found that YAP activation is enhanced in NF2 cKO hearts and cardiomyocytes deficient for NF2. Co-IP analysis indicated that YAP associates with components of the nucleosome remodeling and deacetylase (NuRD) complex in cardiomyocytes, which may mediate targeted gene suppression. Conclusion: Myocardial NF2 expression is upregulated and may be compensatory during PO stress through preservation of metabolic gene expression and energy content in the heart. Citation Format: Yu Zhang, Wataru Mizushima, Shinichi Oka, Peiyong Zhai, Dominic Del Re. Neurofibromin 2 regulates metabolism in the heart [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr B19.

Cardiac remodeling promotes heart failure (HF). Cardiomyocyte (CM) death is one of the mechanisms to develop cardiac remodeling. We recently reported that Mst1 phosphorylates Bcl-xL at Ser14, which promotes apoptosis by inducing dissociation of Bcl-xL from Bax and consequent activation of Bax in CMs. Its phosphorylation is increased in response to ischemia-reperfusion (IR) in an Mst1-dependent manner. However, the functional significance of endogenous Bcl-xL phosphorylation remains unclear in vivo. To address this question, knock-in (KI) mice with alanine mutation at Ser14 in Bcl-x were generated. At baseline, cardiac function was similar between wild-type (WT) and heterozygous KI (HKI) mice (EF 76% and 79%, respectively). HKI mice exhibited smaller % infarct area (30%) than WT (43%) (p=0.016) upon IR, suggesting that phosphorylation of endogenous Bcl-xL at Ser14 plays an essential role in mediating IR injury. In order to test the role of Bcl-xL phosphorylation in the development of HF, HKI and WT mice were subjected to permanent ligation of LAD for 4 weeks. During progression of cardiac remodeling, Mst1 was activated in both WT and HKI mice. Phosphorylation of Bcl-xL and Bcl-xS, an alternative transcriptional variant of Bcl-x, both at Ser14, were increased in WT mice, which were abrogated in HKI mice. The infarct area evaluated with TTC staining at Day 1 was similar in WT and HKI mice (59.1% and 61.2%, p=0.65). Four weeks after myocardial infarction (MI), WT mice exhibited lower cardiac contraction (EF 46.5%) and higher LVEDP (10.8mmHg) than those in HKI mice (EF 68.9% and LVEDP 7.0mmHg) (both p&lt;0.05). Scar area and TUNEL-positive CMs were greater in WT (49.0% and 1.6%, respectively) than in HKI mice (29.2% and 0.4%, respectively) (both p&lt;0.05). Cleaved caspase 3 and 9 were significantly increased (3.2- and 5.7-fold, respectively) in WT but not in HKI mice. In vitro experiments with overexpression of phospho-mimicking mutant (Bcl-xS-S14D) showed 13% reduction in cell viability compared with that of phospho-resistant mutant (Bcl-xS-S14A) (p=0.01%). Our results suggest that phosphorylation of Bcl-xL and Bcl-xS at Ser14 contributes to CM death in response to IR and chronic MI in vivo, thereby promoting cardiac remodeling and HF.

Introduction: Expression of miR-206 is upregulated by YAP, a key transcription co-factor controlled by the Hippo signaling pathway, and mediates YAP-induced hypertrophy and survival of cardiomyocytes. Although miR-206 is known to promote hypertrophy of skeletal muscle, the role of miR-206 in the heart under clinically relevant conditions in vivo remains unknown. We investigated the role of miR-206 in mediating cardiac hypertrophy in response to pressure overload (PO). Results: The level of miR-206 in the mouse heart, as evaluated by qRT-PCR, was upregulated 2.9 fold (p&lt;0.05) 7 days after transverse aortic constriction (TAC) compared to sham operation. In order to evaluate the involvement of miR-206 in cardiac hypertrophy, wild-type C57B/6J mice were administered LNA inhibitor designed to selectively inhibit miR-206, or control scrambled LNA, by tail vein injection. Specificity of the LNA inhibitor was confirmed by qRT-PCR analysis of miRNA expression 48 hours after treatment. Notably, the LNA inhibitor did not affect the level of miR-1, which has a sequence similarity with miR-206. After 48 hours, mice from both treatment groups were subjected to sham operation or TAC. After 7 days of TAC, echocardiography was performed and mice were sacrificed. Upregulation of myocardial miR-206 expression levels after 7 days TAC observed in LNA control-treated mice was completely abolished in LNA-anti-206 -treated mice. A significant increase in left ventricular weight/tibial length (mg/mm) in LNA control-treated mice following TAC was observed (sham vs TAC: 3.7, 4.8, p&lt;0.05); however, no increase was observed in LNA-anti-206 -treated mice (3.8, 3.8). We also noted significant differences in chamber wall thickness (mm) between the LNA-control and LNA-anti-206-treated TAC groups (diastolic posterior wall 0.91, 0.61, p&lt;0.05). Additionally, cardiomyocyte cross sectional area (1.23, 0.9, p&lt;0.05) and ANF expression (2.5, 1.3, P&lt;0.05) were significantly increased in the LNA control-treated TAC group, and these responses were attenuated in the LNA-anti-206-treated mice. Conclusions: These data demonstrate that inhibition of miR-206 impairs PO-induced hypertrophy and indicates that miR-206 is an important endogenous mediator of heart growth in response to PO.

Yes-Associated Protein (YAP), a downstream effector of the Hippo pathway, plays an important role in regulating cell proliferation and survival in mammalian cells. We have shown that cardiac-specific loss of YAP leads to increased cardiomyocyte (CM) apoptosis and impaired hypertrophy during chronic myocardial infarction in the mouse heart. However, it remains unclear whether YAP mediates hypertrophy of individual CMs under stress conditions in vivo. We hypothesized that endogenous YAP plays an essential role in mediating hypertrophy and survival of CMs in response to pressure overload (PO). Three-month-old YAP+/fl;α-MHC-Cre (YAP-cKO) and YAP+/fl (control) mice were subjected to transverse aortic constriction (TAC). Two weeks later, YAP-cKO and control mice developed similar levels of cardiac hypertrophy (left ventricular (LV) weight/tibia length: 7.27±0.38, 6.93±0.29) compared to sham (5.08±0.14, 4.07±0.33). LV CM cross sectional area was similarly increased by TAC in YAP-cKO and control mice compared to their respective shams. Induction of fetal-type genes, such as Anf and Myh7, was also similar in YAP-cKO and control mice. YAP-cKO and control mice exhibited similar baseline LV systolic function (ejection fraction (EF): 75, 76%). YAP-cKO mice had significantly decreased LV function after TAC compared to Sham-control mice (EF: 51%, 76%, p&lt;0.05) and TAC-control mice (75%, p&lt;0.05). LV end diastolic pressure (LVEDP, mmHg) was significantly increased (19.3 ±3.2, 9.8±1.6, p&lt;0.05), and LV +dP/dt (mmHg/s, 7250±588, 9500±453, p&lt;0.01) and -dP/dt (mmHg/s, 6000±433, 7781± 314, p&lt;0.05) were significantly decreased in YAP-cKO compared to in control mice after TAC. LV end diastolic diameter (mm) was significantly greater in YAP-cKO than in control mice after TAC (3.95±0.11, 3.35±0.15, p&lt;0.05), whereas LV pressure was similar, suggesting that LV wall stress was elevated in YAP-cKO compared to in control mice. Since cardiac hypertrophy in YAP-cKO mice is similar to that in control mice despite elevated wall stress, the lack of YAP appears to limit the extent of cardiac hypertrophy in response to increased wall stress. These data suggest that endogenous YAP plays an important role in mediating adaptive hypertrophy and protecting the heart against PO.

Neurofibromin 2 (NF2)/merlin is a tumor suppressor that can regulate activation of the hippo pathway in Drosophila and the mammalian liver, thereby inhibiting cell growth and promoting apoptosis. We have shown previously that the mammalian homologue of hippo, Mst1, has a similar function in the heart. Therefore, we sought to determine whether NF2 can mediate activation of Mst1 in the heart and modulate cardiac ischemia/reperfusion (I/R) injury. Both NF2 and Mst1 are activated by oxidative stress in isolated cardiomyocytes in vitro and in the heart following I/R. Increased expression of NF2 in cardiomyocytes increased Mst1 phosphorylation (2.9±0.2-fold vs LacZ, p&lt;0.05) and elicited apoptosis (4.8±1.7-fold vs LacZ, p&lt;0.05) that was prevented by co-expression of kinase inactive Mst1 (61±12% decrease vs. NF2, p&lt;0.05). To examine the importance of endogenous NF2 in I/R injury, we employed two different genetic models - systemic heterozygous nf2 deletion ( nf2 +/- ) and cardiomyocyte-specific disruption using nf2 floxed mice crossed with αMHC-Cre transgenic mice ( nf2 +/flox Cre). We performed global I/R (30/60min) injury on isolated perfused hearts using the Langendorff method. Functional parameters and infarct size were determined following the reperfusion phase. While nf2 +/- hearts showed no difference in function and infarct size (45±5% vs 48±7%, NS) compared to WT, nf2 +/flox Cre hearts had significantly smaller infarcts (22±5% vs 53±8%, p&lt;0.05) and improved function versus nf2 +/flox controls. nf2 +/- hearts also failed to protect against I/R injury in vivo as Infarct/AAR (34±3% vs 32±2%, NS) and myocyte apoptosis (1.5±0.2% vs 1.7±0.3%, NS) were unchanged despite significant attenuation of Mst1 activation in ventricular homogenates (71±8% reduced vs WT, p&lt;0.05). Preliminary experiments to determine the effect of cardiomyocyte nf2 deletion show a trend toward protection (Infarct/AAR, 23±6% vs 33±6%, n=4) in nf2 +/flox Cre versus nf2 +/flox control mice. Based on these data we conclude that nf2 disruption in cardiomyocytes is protective against I/R injury through inhibition of Mst1 activity. However, NF2 signaling in residential non-myocytes appears beneficial for the heart and obscures the detrimental function of NF2 in cardiomyocytes during I/R.

Our previous work demonstrated Rassf1A to be a critical mediator of Mst1 activation, the chief component of the mammalian Hippo pathway, in heart failure. In the setting of ischemia/reperfusion (I/R) injury, Mst1 is robustly activated and promotes injury in the heart; however, its regulation remains unclear. Using genetically modified mice in which expression of Rassf1A is altered in a cell type-specific manner, we demonstrate that systemic deletion of rassf1a -/- (KO) does not alter infarct size (36±4% vs 33±3%) or cardiac myocyte apoptosis (1.7±0.2% vs 2.1±0.4%) following I/R versus WT. Conversely, mice harboring deletion of rassf1a in cardiac myocytes ( rassf1a flox/flox αMHC-Cre, CKO) have smaller infarcts (23±2 vs 38±3%, p&lt;0.05) and less apoptosis (0.9±0.2 vs 2.0±0.2%, p&lt;0.05) after I/R. Importantly, attenuation of Mst1 activation in ventricular homogenates was observed in both deletion models, implicating Rassf1A as a positive regulator of Mst1 during I/R. Langendorff global I/R injury yielded similar results - no protection in KO, yet significant protection in CKO hearts versus control mice, suggesting that native cardiac cells are sufficient to mediate this response. Using isolated cardiac fibroblast cultures we determined that Rassf1A negatively regulates activation of Akt in response to oxidative stress. Further, knockdown of endogenous Rassf1A causes increased NF-κB activity, a response mediated by Akt. A candidate molecule screen found exaggerated TNF-α expression in non-myocytes of KO hearts compared to WT (4.8±0.7fold, p&lt;0.05), whereas TNF-α expression was attenuated in CKO hearts (reduced 65±8% vs control, p&lt;0.05). Finally, WT and KO mice were administered TNF-α neutralizing antibody or control IgG and subjected to I/R. KO hearts treated with TNF-α Ab, but not IgG, had reduced infarcts (19±4% vs 34±5%, p&lt;0.05), but no significant reduction in infarct size was observed in WT mice given TNF-α Ab. Taken together these data suggest that TNF-α blockade prevents the deleterious consequences of Rassf1A deletion in non-myocytes while unmasking the protective effect of Rassf1A deletion in cardiac myocytes following I/R. Further, these results demonstrate the importance of non-myocytes in modulating cardiac myocyte survival and I/R injury

Mammalian sterile 20-like kinases 1 and 2 (Mst1/2) are highly conserved and are thought to have similar pro-apoptotic and anti-cell growth properties. Previous work has demonstrated that increased expression of Mst1 causes robust cardiomyocyte apoptosis, heart failure and premature death in mice, yet the function of endogenous Mst1/2 has not been investigated. The goal of this study was to determine the role of Mst1 and Mst2 in the development of heart failure using genetically altered Mst isoform-specific mice. Systemic deletion of both Mst isoforms is embryonic lethal; therefore we utilized mice harboring floxed Mst1 alleles on an Mst2-/- background and bred them with α-MHC Cre mice to disrupt both isoforms in cardiomyocytes (Mst DKO). Animals were subjected to transverse aortic constriction for 4 weeks or sham. Echocardiography was performed prior to intervention and immediately before sacrifice. At 8 weeks of age, no significant differences in the cardiac phenotype were observed between control, Mst1-/-, Mst2-/- or Mst DKO mice. In response to pressure overload (PO), control mice had increased heart weight/tibia length (HW/TL), left ventricle weight/tibia length (LVW/TL) and cardiomyocyte cross-sectional area as expected. Surprisingly, this hypertrophic response was significantly attenuated in Mst1-/- and Mst2-/- mice. Interestingly, Mst DKO mice hypertrophied similar to controls. Apoptosis and fibrosis were increased, and LV ejection fraction (LVEF) was decreased after PO in control mice. Mst1-/- and Mst2-/- mice had attenuated apoptosis and fibrosis; however, LVEF was decreased to levels similar to controls. Interestingly, Mst DKO mice also showed attenuated apoptosis and fibrosis but had significantly worse LVEF compared to control mice. Studies are ongoing to address the mechanism responsible for worsened cardiac function observed in Mst DKO mice despite suppression of fibrosis and apoptosis after PO.

Our previous work demonstrated Rassf1A to be a critical mediator of Mst1 activation, the chief component of the mammalian Hippo pathway, in heart failure. In the setting of ischemia/reperfusion (I/R) injury, Mst1 is robustly activated in the heart; however, its regulation remains unclear. Further, the role of Rassf1A in I/R injury has not been investigated. Using genetically modified mice in which expression of Rassf1A is altered in a cell type-specific manner, we demonstrate that systemic deletion of rassf1a (KO) does not alter infarct size (36±4% vs 33±3%) or cardiac myocyte apoptosis (1.7±0.2% vs 2.1±0.4%) following I/R versus WT. Conversely, mice harboring deletion of rassf1a in cardiac myocytes (CKO) have smaller infarcts (38±3% vs 23±2%, p&lt;0.05) and less apoptosis (2.0±0.2% vs 0.9±0.2%) after I/R. Importantly, attenuation of Mst1 activation in ventricular homogenates was observed in both deletion models, implicating Rassf1A as a positive regulator of Mst1 during I/R. Langendorff global I/R injury yielded similar results - no protection in KO, yet significant protection in CKO hearts versus control mice, suggesting that native cardiac cells are sufficient to mediate this response. A candidate molecule screen found exaggerated TNF-α expression in KO hearts compared to WT (4.8±0.7% vs 1.0±0.3%), whereas TNF-α expression was attenuated in CKO hearts (0.9±0.2% vs 0.3±0.1%). Finally, WT and KO mice were administered TNF-α neutralizing or control IgG and subjected to I/R. KO hearts treated with TNF-α Ab, but not IgG, had reduced infarcts (19±4% vs 34±5%), but no significant reduction in infarct size was observed in WT mice given TNF-α Ab. Taken together these data suggest that TNF-α blockade prevents the deleterious consequences of Rassf1A deletion in non-myocytes while unmasking the protective effect of Rassf1A deletion in cardiac myocytes following I/R. Further, these results demonstrate the importance of non-myocytes in modulating cardiac myocyte survival and I/R injury.

Acute myocardial ischemia and reperfusion (I/R) cause damage to the heart, thereby impairing its ability to function effectively. Therapies aimed at preventing I/R injury are limited and a better understanding of the mechanisms involved remains critical. In hearts of mice subjected to I/R we found differing kinetics of H- and K-Ras activation, and only K-Ras modification following oxidative stress was observed. Expression of active H-Ras12V caused increased phosphorylation of AKT (1.8±0.3-fold vs LacZ) and ERK1/2 (3.3±0.3-fold vs LacZ) and protected cardiomyocytes against apoptosis, whereas K-Ras12V activated mammalian sterile 20-like kinase 1 (Mst1)(2.1±0.1-fold vs LacZ), which led to myocyte death. Endogenous K-Ras, but not H-Ras, was detected at mitochondria and associated with Ras association domain family 1 isoform A (RASSF1A) in both cardiomyocytes and mouse hearts. Genetic disruption of RASSF1A prevented K-Ras-induced Mst1 activation and inhibited Mst1 translocation to mitochondria in response to oxidative stress. Mass spectrometry analysis revealed that active Mst1 phosphorylates Bcl-xL at Ser14, a previously unreported residue, in cardiomyocytes and mouse heart, and mediates Bcl-xL phosphorylation in response to I/R in vivo. Ser14 phosphorylation attenuated Bcl-xL-Bax binding and the phosphomimetic Bcl-xL S14D mutant showed a weakened protective capacity against apoptotic insult (cell viability; LacZ + H2O2 45±5%, XL WT + H2O2 76±4%, XL SD + H2O2 51±5%, p&lt;0.05). Following in vivo I/R, Kras+/- mice had significantly smaller myocardial infarct size vs WT mice (22±4% vs 39±2%, p&lt;0.05), while no significant difference in infarct size was observed in Hras+/- mice (37±3% vs 38±3%). Conversely, cardiac-specific K-Ras12V TG mice had increased Mst1 activation and significantly larger infarcts vs NTG mice (67±10% vs 36±2%, p&lt;0.05). This myocardial damage was rescued in bigenic mice expressing both K-Ras12V and Dn-Mst1 (41±7%, p&lt;0.05), implicating Mst1 as a critical mediator of K-Ras in vivo. Our work demonstrates a distinct role for K-Ras as an endogenous promoter of mammalian Hippo signaling, and identifies a novel mechanism involving phosphorylation of Bcl-xL by which Mst1 mediates cardiomyocyte apoptosis and I/R injury in the heart.

The small GTPase RhoA has established roles in regulating cell growth and cytoskeletal dynamics but the role of RhoA in modulating cardiac hypertrophy and cell survival remains elusive. Inducible cardiac‐specific RhoA mice (RhoA DTG) expressing 2–5 fold increase in active RhoA showed no evident basal phenotype. Remarkably, isolated RhoA DTG hearts subjected to 45min global ischemia followed by 2h reperfusion displayed a 60% reduction in infarct size, decreased lactate dehydrogenase release and significantly improved post‐ischemic cardiac function compared to the wild‐type hearts. RhoA DTG hearts subjected to 30min coronary occlusion followed by 24h reperfusion in vivo showed even greater protection with infarct size reduced by more than 70%. The Rho Kinase (ROCK) inhibitor Y‐27632 enhanced rather than blocking the protection in RhoA DTGs, suggesting that ROCK is not downstream of RhoA‐mediated ischemia/reperfusion (I/R) protection. Activated protein kinase C epsilon and protein kinase D were significantly increased in the RhoA DTG heart, and their roles in the protective effect of RhoA are currently under investigation. In conclusion, inducible and cardiac‐specific RhoA‐expression provides strong protection against I/R injury in the mouse heart both ex vivo and in vivo, contrasting with the conventional notion that RhoA/ROCK signaling in the heart is maladaptive. (HL28143)

Isoform specific functions of Ras proteins exist, yet the role of endogenous Ras isoforms in the heart remains unknown. The goal of this study was to determine the role of H- and K-Ras in the devel...

Yes-Associated Protein (YAP), a downstream effector of the Hippo pathway, regulates cell proliferation and survival. Cardiac-specific loss of YAP leads to increased cardiomyocyte (CM) apoptosis and...

Inflammation is an integral component of cardiovascular disease and is thought to contribute to cardiac dysfunction in ischemic and non-ischemic models of heart failure. While ischemia-induced inflammation has been extensively studied in the heart, relatively less is known regarding cardiac inflammation during non-ischemic stress. Recent work has implicated a role for Yes-associated protein (YAP), a transcription co-factor, in modulating cardiac inflammation in response to ischemic injury; however, whether YAP influences inflammation in the heart during non-ischemic stress is not described. We hypothesized that YAP mediates a pro-inflammatory response during pressure overload (PO)-induced non-ischemic injury, and that targeted YAP inhibition in the myeloid compartment is cardioprotective. In mice, PO elicits an immune response characterized by infiltration of myeloid cells that precedes cardiac dysfunction. Myeloid cells isolated from the heart after 7 days PO showed evidence of increased YAP activity. Myeloid-specific YAP knockout mice (YAPF/F ;LysMCre ) were subjected to PO stress. After 4 weeks, cardiac hypertrophy was similar between myeloid YAP KO mice and controls. However, systolic dysfunction, cardiac fibrosis, apoptosis, "fetal gene" induction, and additional indicators of pathological remodeling were attenuated in myeloid YAP KO mice compared to controls. Additionally, inflammatory gene expression and CCR2+ macrophage infiltration in the myocardium were significantly attenuated in myeloid YAP KO mice after PO, indicating reduced inflammation compared to controls. Conversely, angiogenic indicators and pro-resolving genes were enhanced in myeloid YAP KO hearts. Experiments using primary bone marrow-derived macrophages (BMDMs) from YAP KO and control mice demonstrated that YAP suppression attenuated "M1-like" inflammatory gene expression, while "M2-like" resolving genes were augmented following stimulation. The inflammasome is a multiprotein complex and important facilitator of cytokine processing that mediates inflammation in the injured heart. We observed attenuated inflammasome priming and function in YAP deficient BMDMs, as well as in myeloid YAP KO hearts following PO, indicating disruption of inflammasome induction. Finally, we leveraged nanoparticle-mediated delivery of the YAP inhibitor verteporfin to test the translational potential of targeting YAP to prevent heart failure. An alternate-day dosing regimen of drug-loaded nanoparticles attenuated inflammation and fibrosis, and improved myocardial capillary density after 2 weeks PO compared to DMSO nanoparticle control treatment. Together these data implicate myeloid YAP as an important molecular nodal point that facilitates cardiac inflammation and pathology during PO stress and suggest that selective inhibition of YAP may prove a novel therapeutic target in non-ischemic heart disease.

The small G‐protein RhoA is a transducer of signals from both GPCRs and the extracellular matrix, influencing cell morphology as well as gene expression. A role for RhoA in cultured cardiomyocyte hypertrophy has been established, however the possibility that RhoA can influence myocyte survival has not been examined. We find that adenoviral expression of activated RhoA protects neonatal rat cardiomyocytes from apoptotic insults induced by peroxide or glucose deprivation. Interestingly, expression of RhoA induces PI3K‐dependent activation of the cardioprotective kinase Akt. It has been reported that RhoA mediates FAK activation and that FAK can directly interact with the p85 regulatory subunit of PI3K. Accordingly, we examined the possible role of FAK in RhoA signaling to Akt. RhoA expression increased FAK phosphorylation and focal adhesion formation in cardiomyocytes in a Rho kinase‐dependent manner. Further, RhoA‐induced Akt activation is prevented by co‐expression of FRNK, a negative regulator of FAK, suggesting that FAK is not only activated but mediates this response. Expression of FRNK also blocked the protective effect afforded by RhoA further implicating FAK in this signaling pathway. The finding that RhoA activation protects cardiomyocytes through FAK‐dependent Akt activation suggests that activation of similar pathways by mechanical stretch could regulate cardiomyocyte survival. NHLBI R01 HL‐28143

Inflammation is an integral component of cardiovascular disease and is thought to contribute to cardiac dysfunction in ischemic and non-ischemic models of heart failure. While ischemia-induced inflammation has been extensively studied in the heart, relatively less is known regarding cardiac inflammation during non-ischemic stress. Recent work has implicated a role for Yes-associated protein (YAP), a transcription co-factor, in modulating cardiac inflammation in response to ischemic injury; however, whether YAP influences inflammation in the heart during non-ischemic stress is not described. We hypothesized that YAP mediates a pro-inflammatory response during pressure overload (PO)-induced non-ischemic injury, and that targeted YAP inhibition in the myeloid compartment is cardioprotective. In mice, PO elicits an immune response characterized by infiltration of myeloid cells that precedes cardiac dysfunction. Myeloid cells isolated from the heart after 7 days PO showed evidence of increased YAP activity. Myeloid-specific YAP knockout mice (YAP F/F ;LysM Cre ) were subjected to PO stress. After 4 weeks cardiac hypertrophy was similar between myeloid YAP KO mice and controls. However, systolic dysfunction, cardiac fibrosis, apoptosis, “fetal gene” induction, and additional indicators of pathological remodeling were attenuated in myeloid YAP KO mice compared to controls. Additionally, inflammatory gene expression and macrophage infiltration in the myocardium were significantly attenuated in myeloid YAP KO mice after PO indicating reduced inflammation compared to controls. Conversely, angiogenic indicators and pro-resolving genes were enhanced in myeloid YAP KO hearts. Experiments using primary bone marrow-derived macrophages (BMDMs) from YAP KO and control mice demonstrated that YAP suppression attenuated “M1-like” inflammatory gene expression, while “M2-like” resolving genes were augmented following stimulation. The inflammasome is a multiprotein complex and important facilitator of cytokine processing that mediates inflammation in the injured heart. We observed attenuated inflammasome priming and function in YAP deficient BMDMs, as well as in myeloid YAP KO hearts following PO, indicating disruption of inflammasome induction. Finally, we leveraged nanoparticle-mediated delivery of the YAP inhibitor verteporfin to test the translational potential of targeting YAP to prevent heart failure. An alternate-day dosing regimen of drug-loaded nanoparticles attenuated inflammation and fibrosis, and improved myocardial capillary density after PO compared to DMSO nanoparticle control treatment. Together these data implicate myeloid YAP as an important molecular nodal point that facilitates cardiac inflammation and pathology during PO stress and suggest that selective inhibition of YAP may prove a novel therapeutic target in non-ischemic heart disease.

Cardiac hypertrophy is an adaptive response at least initially since it reduces wall stress. Phosphorylation of Bcl-xL at Serine (Ser) 14, which dissociates Bcl-xL from Bax and promotes apoptosis, is increased in the heart within one hour after transverse aortic constriction (TAC)-induced pressure overload (PO). Here, we investigated how the increased Ser14-phosphorylation affects hypertrophy during PO. The Bcl-xL knock-in (KI) mice, in which Ser14 was replaced with Ala (Ser14Ala), exhibited a significantly greater mortality than wild-type (WT) mice (p=0.001) after TAC, with elevated end diastolic pressure (LVEDP, 34.6 vs 16.5 mmHg, p&lt;0.05), impaired systolic function (EF, 38.2% vs 67.5%, p&lt;0.001), and increased fibrosis (1.6-fold, p&lt;0.001). The level of apoptosis was similar between the KI and WT mice one week after TAC, as assessed by TUNEL staining. The KI mice showed less cardiomyocyte and cardiac hypertrophy (cardiomyocyte size, 0.71-fold; heart weight/tibia length, 0.88-fold, both P&lt;0.001). Adult cardiomyocytes isolated from the KI mice two days after TAC showed significantly lower contractility compared to those isolated from WT mice (0.32-fold, p&lt;0.001). Mechanistically, gene set enrichment analysis using the RNA-seq data obtained from one-day TAC hearts showed that ion channel activity-related gene sets enriched in WT mice are downregulated in the KI mice. In line with this result, angiotensin II (Ang II) increased Ser14-phosphorylation and cytosolic Ca 2+ level in WT-MEFs in vitro , whereas MEFs isolated from the KI mice showed a significantly lower elevation of cytosolic Ca 2+ against Ang II. Proteomics analysis showed that Ankyrin, an anchoring protein that targets and stabilizes ion channels on the membrane, interacts with endogenous Bcl-xL in the heart. Taken together, these data suggest that phosphorylation of Bcl-xL at Ser14 is critical for augmenting Ca 2+ release from the sarcoplasmic reticulum by modulating the ion channel activity in part via Ankyrin against PO or AngII, thereby developing compensatory hypertrophy and maintaining contractile function. Our findings indicate that increasing the Bcl-xL-Ser14 phosphorylation during acute phase PO could be a potential therapeutic strategy for maintaining cardiac function.

Background

Introduction: Neurofibromin 2 (NF2) is a tumor suppressor that modulates cell proliferation and survival. We previously demonstrated that NF2 mediates cardiomyocyte apoptosis and injury caused by a...

The small G-protein RhoA is a transducer of signals from both GPCRs and the extracellular matrix, influencing cell morphology as well as gene expression. A role for RhoA has been firmly established in cultured cardiomyocyte hypertrophy, however the possibility that RhoA can influence myocyte survival or apoptosis has not been examined. Previous work from our lab employing cardiac specific RhoA transgenic mice demonstrated that RhoA overexpression leads to premature death, apparently secondary to the development of heart failure. We have now determined that adenoviral overexpression of activated RhoA in neonatal rat cardiomyocytes induces early changes indicative of hypertrophy which transitions to apoptosis, with the accompanying hallmarks of caspase activation and nucleosomal DNA fragmentation. To delineate the mechanism of RhoA-mediated apoptosis we tested the effects of the Rho kinase inhibitors Y-27632 and HA-1077. Treatment with Y-27632 or HA-1077 prevented nucleosome fragmentation and caspase-3 cleavage. Activated RhoA increases caspase-9 cleavage but not that of caspase-8, and a caspase-9 selective inhibitor prevented DNA fragmentation whereas inhibition of caspase-8 did not, consistent with involvement of a mitochondrial death pathway. Further, RhoA induces a 3-4 fold upregulation in protein levels of the proapoptotic Bcl family protein Bax, which is sensitive to Rho kinase inhibition. Bax involvement in this apoptotic effect was tested by treatment with a Bax inhibitory peptide, which markedly attenuated RhoA-induced DNA fragmentation and caspase-9 and -3 activation. The mechanism of Bax upregulation by RhoA is currently under investigation.

Background: Neurofibromin 2 (NF2) is a tumor suppressor and signaling platform that can engage the Hippo pathway to modulate cell proliferation and survival. We previously demonstrated that NF2 mediates injury caused by acute myocardial infarction (MI) through inhibition of the Hippo target YAP. However, NF2 function in the heart remains largely uncharacterized. This study sought to determine whether NF2 contributes to cardiac remodeling, dysfunction, and heart failure resulting from chronic stress. Methods and Results: We used a transverse aortic constriction (TAC) model to generate pressure overload (PO), which elicits cardiac hypertrophy, remodeling and heart failure. We found that NF2 is transiently upregulated in WT C57Bl/6J myocardium in response to PO. To investigate if increased NF2 contributes to pathology, we used cardiomyocyte-specific (αMHC-Cre) NF2 knockout (CKO) mice. At baseline, adult NF2 CKO mice had normal cardiac morphology and function. However, following TAC, NF2 CKO hearts unexpectedly showed worsened cardiac function compared to littermate controls. No differences in the extent of TAC-induced hypertrophy or apoptosis were observed between genotypes. RNAseq analysis indicated downregulation of several metabolic pathways including oxidative phosphorylation (OXPHOS) and fatty acid oxidation (FAO) in unstressed NF2 CKO hearts. RNAseq identified, and qPCR confirmed, downregulation of ERRβ and ERRγ expression and function in NF2 CKO hearts, as well as ATP content. Critical ETC genes were also suppressed in NF2 CKO hearts after 3 days PO. Experiments in neonatal rat cardiomyocytes (NRCM) confirmed that altered ERRβ and ERRγ expression and function, as well as ATP content, by NF2 is cell autonomous. Mechanistically, we discovered that nuclear NF2 is enriched following TAC, and that NF2 associates with the proximal promoter regions of ERRβ and ERRγ using DNA pulldown assay. We also found that NF2 activates transcription in NRCMs using the Gal4-UAS system. Future studies will identify transcription factors important for mediating this response. Conclusion: Myocardial NF2 is transiently upregulated and may be compensatory during PO through preservation of metabolic gene expression and energy content in the heart.

Inflammation is a component of cardiovascular disease and is thought to contribute to cardiac dysfunction in ischemic and non-ischemic models of heart failure. While ischemia-induced inflammation has been extensively studied in the heart, relatively less is known regarding cardiac inflammation during non-ischemic stress. Recent work has implicated a role for Yes-associated protein (YAP), a transcriptional co-factor, in modulating cardiac inflammation and remodeling after myocardial infarction. We hypothesized that YAP mediates a pro-inflammatory response during pressure overload (PO)-induced non-ischemic injury, and that targeted YAP inhibition is cardioprotective. PO in mice elicits an immune response characterized by infiltration of myeloid cells that precedes cardiac dysfunction. Myeloid cells isolated from the heart after 7d PO showed evidence of increased YAP activity. Myeloid-specific YAP knockout mice (YAP F/F ;LysM Cre ) were subjected to PO stress. After 4 weeks, cardiac hypertrophy was similar between YAP KO mice and controls. However, systolic dysfunction, cardiac fibrosis, and indicators of pathological remodeling were all attenuated in YAP KO mice compared to controls. Additionally, inflammatory gene expression and macrophage infiltration to the myocardium were significantly attenuated in YAP KO mice after PO, indicating reduced inflammation compared to controls. Experiments using RAW264.7 macrophages and primary bone marrow-derived macrophages (BMDMs) from YAP KO and control mice demonstrated that increased YAP expression enhanced, while YAP suppression attenuated, inflammatory gene expression. The inflammasome is a multiprotein complex and important facilitator of cytokine processing that mediates inflammation in the PO heart. We observed attenuated inflammasome priming and function in YAP deficient BMDMs, as well as in YAP KO hearts following PO, indicating disruption of inflammasome induction. Together these data implicate YAP as an important mediator of inflammasome function and cardiac inflammation during PO stress and suggest that selective inhibition of YAP in the myeloid compartment may prove a novel therapeutic target in non-ischemic heart disease.