Zully Pedrozo

Cardiovascular disease (CVD) is the leading cause of morbidity and mortality worldwide. Although treatments have improved, development of novel therapies for patients with CVD remains a major research goal. Apoptosis, necrosis, and autophagy occur in cardiac myocytes, and both gradual and acute cell death are hallmarks of cardiac pathology, including heart failure, myocardial infarction, and ischemia/reperfusion. Pharmacological and genetic inhibition of autophagy, apoptosis, or necrosis diminishes infarct size and improves cardiac function in these disorders. Here, we review recent progress in the fields of autophagy, apoptosis, and necrosis. In addition, we highlight the involvement of these mechanisms in cardiac pathology and discuss potential translational implications.

The hexosamine biosynthetic pathway (HBP) generates uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) for glycan synthesis and O-linked GlcNAc (O-GlcNAc) protein modifications. Despite the established role of the HBP in metabolism and multiple diseases, regulation of the HBP remains largely undefined. Here, we show that spliced X-box binding protein 1 (Xbp1s), the most conserved signal transducer of the unfolded protein response (UPR), is a direct transcriptional activator of the HBP. We demonstrate that the UPR triggers HBP activation via Xbp1s-dependent transcription of genes coding for key, rate-limiting enzymes. We further establish that this previously unrecognized UPR-HBP axis is triggered in a variety of stress conditions. Finally, we demonstrate a physiologic role for the UPR-HBP axis by showing that acute stimulation of Xbp1s in heart by ischemia/reperfusion confers robust cardioprotection in part through induction of the HBP. Collectively, these studies reveal that Xbp1s couples the UPR to the HBP to protect cells under stress.

Reperfusion accounts for a substantial fraction of the myocardial injury occurring with ischemic heart disease. Yet, no standard therapies are available targeting reperfusion injury. Here, we tested the hypothesis that suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor approved for cancer treatment by the US Food and Drug Administration, will blunt reperfusion injury.Twenty-one rabbits were randomly assigned to 3 groups: (1) vehicle control, (2) SAHA pretreatment (1 day before and at surgery), and (3) SAHA treatment at the time of reperfusion only. Each arm was subjected to ischemia/reperfusion surgery (30 minutes coronary ligation, 24 hours reperfusion). In addition, cultured neonatal and adult rat ventricular cardiomyocytes were subjected to simulated ischemia/reperfusion to probe mechanism. SAHA reduced infarct size and partially rescued systolic function when administered either before surgery (pretreatment) or solely at the time of reperfusion. SAHA plasma concentrations were similar to those achieved in patients with cancer. In the infarct border zone, SAHA increased autophagic flux, assayed in both rabbit myocardium and in mice harboring an RFP-GFP-LC3 transgene. In cultured myocytes subjected to simulated ischemia/reperfusion, SAHA pretreatment reduced cell death by 40%. This reduction in cell death correlated with increased autophagic activity in SAHA-treated cells. RNAi-mediated knockdown of ATG7 and ATG5, essential autophagy proteins, abolished SAHA's cardioprotective effects.The US Food and Drug Administration-approved anticancer histone deacetylase inhibitor, SAHA, reduces myocardial infarct size in a large animal model, even when delivered in the clinically relevant context of reperfusion. The cardioprotective effects of SAHA during ischemia/reperfusion occur, at least in part, through the induction of autophagic flux.

Endoplasmic reticulum (ER) stress activates an adaptive unfolded protein response (UPR) that facilitates cellular repair, however, under prolonged ER stress, the UPR can ultimately trigger apoptosis thereby terminating damaged cells.The molecular mechanisms responsible for execution of the cell death program are relatively well characterized, but the metabolic events taking place during the adaptive phase of ER stress remain largely undefined.Here we discuss emerging evidence regarding the metabolic changes that occur during the onset of ER stress and how ER influences mitochondrial function through mechanisms involving calcium transfer, thereby facilitating cellular adaptation.Finally, we highlight how dysregulation of ER-mitochondrial calcium homeostasis during prolonged ER stress is emerging as a novel mechanism implicated in the onset of metabolic disorders.

Mitochondria are key organelles for ATP production in cardiomyocytes, which is regulated by processes of fission and fusion. We hypothesized that the mitochondria fusion protein dynamin-related protein 1 (Drp1) inhibition, attenuates ischemia-reperfusion (I/R) injury through modifications in mitochondrial metabolism. Rats were subjected to I/R through coronary artery ligation, and isolated cardiomyocytes were treated with an ischemia-mimicking solution. In vivo, cardiac function, myocardial infarction area, and mitochondrial morphology were determined, whereas in vitro, viability, mitochondrial membrane potential, intracellular ATP levels, and oxygen consumption rate (OCR) were assessed. In both models, an adenovirus expressing Drp1 dominant-negative K38A (Drp1K38A) was used to induce Drp1 loss-of-function. Our results showed that I/R stimulated mitochondrial fission. Myocardial infarction size and cell death induced by I/R were significantly reduced, whereas cardiac function after I/R was improved in Drp1K38A-treated rats compared with controls. Drp1K38A-transduced cardiomyocytes showed lower OCR with no decrease in intracellular ATP levels, and on I/R, a larger decrease in OCR with a smaller reduction in intracellular ATP level was observed. However, proton leak-associated oxygen consumption was comparatively higher in Drp1K38A-treated cardiomyocytes, suggesting a protective mitochondrial uncoupling effect against I/R. Collectively, our results show that Drp1 inhibition triggers cardioprotection by reducing mitochondrial metabolism during I/R.

We have shown previously that electrically induced tachycardia effectively produces myocardial preconditioning. Among other effects, tachycardia increases calcium release rates in microsomal fractions enriched in sarcoplasmic reticulum (SR) isolated from dog cardiac ventricular muscle. Here, we report that preconditioning tachycardia increased twofold the NADPH oxidase activity of isolated SR-enriched microsomal fractions, measured as NADPH-dependent generation of superoxide anion and hydrogen peroxide. Tachycardia also augmented the association of rac1 and the NADPH oxidase cytosolic subunit p47(phox) to the microsomal fraction, without modifying the content of the membrane integral subunit gp91(phox). Microsomes from control animals displayed endogenous S-glutathionylation of cardiac ryanodine receptors (RyR2); in microsomal fractions isolated after tachycardia RyR2 S-glutathionylation levels were 1.7-fold higher than in controls. Parallel in vitro experiments showed that NADPH produced a transient increase in calcium release rates and enhanced 1.6-fold RyR2 S-glutathionylation in control microsomes but had marginal or no effects on microsomes isolated after tachycardia. Catalase plus superoxide dismutase, and the NADPH oxidase inhibitors apocynin and diphenyleneiodonium prevented the in vitro stimulation of calcium release rates and RyR2 S-glutathionylation induced by NADPH, suggesting NADPH oxidase involvement. Conversely, addition of reducing agents to vesicles incubated with NADPH markedly inhibited calcium release and prevented RyR2 S-glutathionylation. We propose that tachycardia stimulates NADPH oxidase activity, which by enhancing RyR2 redox modifications such as S-glutathionylation, would contribute to sustain faster calcium release rates during conditions of increased cardiac activity. This response may be an important component of tachycardia-induced preconditioning.

Aggresomes are dynamic structures formed when the ubiquitin-proteasome system is overwhelmed with aggregation-prone proteins. In this process, small protein aggregates are actively transported towards the microtubule-organizing center. A functional role for autophagy in the clearance of aggresomes has also been proposed. In the present work we investigated the molecular mechanisms involved on aggresome formation in cultured rat cardiac myocytes exposed to glucose deprivation. Confocal microscopy showed that small aggregates of polyubiquitinated proteins were formed in cells exposed to glucose deprivation for 6 h. However, at longer times (18 h), aggregates formed large perinuclear inclusions (aggresomes) which colocalized with gamma-tubulin (a microtubule-organizing center marker) and Hsp70. The microtubule disrupting agent vinblastine prevented the formation of these inclusions. Both small aggregates and aggresomes colocalized with autophagy markers such as GFP-LC3 and Rab24. Glucose deprivation stimulates reactive oxygen species (ROS) production and decreases intracellular glutathione levels. ROS inhibition by N-acetylcysteine or by the adenoviral overexpression of catalase or superoxide dismutase disrupted aggresome formation and autophagy induced by glucose deprivation. In conclusion, glucose deprivation induces oxidative stress which is associated with aggresome formation and activation of autophagy in cultured cardiac myocytes.

Our objective was to investigate in cardiac muscle the contribution of NADPH oxidase to (a) ryanodine receptor-2 (RyR2) S-glutathionylation and (b) the preconditioning effects of exercise and tachycardia on infarct size following coronary artery occlusion.We measured NADPH oxidase activity, RyR2 S-glutathionylation, and calcium release kinetics in sarcoplasmic reticulum (SR) vesicles isolated from dog ventricular muscle after exercise and tachycardia, plus or minus prior administration of the NADPH oxidase inhibitor apocynin. In ventricular muscle sections, we studied the colocalization of NADPH oxidase and RyR2 by confocal microscopy using fluorescent antibodies. We determined the effect of apocynin on the infarct size produced by occlusion of the descendent anterior coronary artery in animals preconditioned by exercise or tachycardia. Exercise and tachycardia increased NADPH oxidase activity, RyR2 S-glutathionylation, and calcium release rates in isolated SR vesicles. Cardiac muscle sections displayed significant colocalization of NADPH oxidase and RyR2, suggesting direct and specific effects of reactive oxygen species (ROS) produced by NADPH oxidase on RyR2 activation. The NADPH oxidase inhibitor apocynin prevented the increase in RyR2 S-glutathionylation, reduced calcium release activity, and completely prevented the protective effects of exercise and tachycardia on infarct size.The loss of cardioprotection induced by the NADPH oxidase inhibitor suggests that ROS generated by this enzyme are important mediators of the preconditioning response, which presumably involves NADPH oxidase-induced RyR2 S-glutathionylation.

Pharmacological preconditioning limits myocardial infarct size after ischemia/reperfusion. Dexmedetomidine is an α2-adrenergic receptor agonist used in anesthesia that may have cardioprotective properties against ischemia/reperfusion injury. We investigate whether dexmedetomidine administration activates cardiac survival kinases and induces cardioprotection against regional ischemia/reperfusion injury. In in vivo and ex vivo models, rat hearts were subjected to 30 min of regional ischemia followed by 120 min of reperfusion with dexmedetomidine before ischemia. The α2-adrenergic receptor antagonist yohimbine was also given before ischemia, alone or with dexmedetomidine. Erk1/2, Akt and eNOS phosphorylations were determined before ischemia/reperfusion. Cardioprotection after regional ischemia/reperfusion was assessed from infarct size measurement and ventricular function recovery. Localization of α2-adrenergic receptors in cardiac tissue was also assessed. Dexmedetomidine preconditioning increased levels of phosphorylated Erk1/2, Akt and eNOS forms before ischemia/reperfusion; being significantly reversed by yohimbine in both models. Dexmedetomidine preconditioning (in vivo model) and peri-insult protection (ex vivo model) significantly reduced myocardial infarction size, improved functional recovery and yohimbine abolished dexmedetomidine-induced cardioprotection in both models. The phosphatidylinositol 3-kinase inhibitor LY-294002 reversed myocardial infarction size reduction induced by dexmedetomidine preconditioning. The three isotypes of α2-adrenergic receptors were detected in the whole cardiac tissue whereas only the subtypes 2A and 2C were observed in isolated rat adult cardiomyocytes. These results show that dexmedetomidine preconditioning and dexmedetomidine peri-insult administration produce cardioprotection against regional ischemia/reperfusion injury, which is mediated by the activation of pro-survival kinases after cardiac α2-adrenergic receptor stimulation.

Background— L-type calcium channel activity is critical to afterload-induced hypertrophic growth of the heart. However, the mechanisms governing mechanical stress–induced activation of L-type calcium channel activity are obscure. Polycystin-1 (PC-1) is a G protein–coupled receptor–like protein that functions as a mechanosensor in a variety of cell types and is present in cardiomyocytes. Methods and Results— We subjected neonatal rat ventricular myocytes to mechanical stretch by exposing them to hypo-osmotic medium or cyclic mechanical stretch, triggering cell growth in a manner dependent on L-type calcium channel activity. RNAi-dependent knockdown of PC-1 blocked this hypertrophy. Overexpression of a C-terminal fragment of PC-1 was sufficient to trigger neonatal rat ventricular myocyte hypertrophy. Exposing neonatal rat ventricular myocytes to hypo-osmotic medium resulted in an increase in α1C protein levels, a response that was prevented by PC-1 knockdown. MG132, a proteasomal inhibitor, rescued PC-1 knockdown–dependent declines in α1C protein. To test this in vivo, we engineered mice harboring conditional silencing of PC-1 selectively in cardiomyocytes (PC-1 knockout) and subjected them to mechanical stress in vivo (transverse aortic constriction). At baseline, PC-1 knockout mice manifested decreased cardiac function relative to littermate controls, and α1C L-type calcium channel protein levels were significantly lower in PC-1 knockout hearts. Whereas control mice manifested robust transverse aortic constriction–induced increases in cardiac mass, PC-1 knockout mice showed no significant growth. Likewise, transverse aortic constriction–elicited increases in hypertrophic markers and interstitial fibrosis were blunted in the knockout animals Conclusion— PC-1 is a cardiomyocyte mechanosensor that is required for cardiac hypertrophy through a mechanism that involves stabilization of α1C protein.

HDAC inhibitors prevent pathological cardiac hypertrophy by suppressing a growth-promoting kinase.

The alpha2-adrenergic receptor agonist Dexmedetomidine (Dex) is a sedative medication used by anesthesiologists. Dex protects the heart against ischemia-reperfusion (IR) and can also act as a preconditioning mimetic. The mechanisms involved in Dex-dependent cardiac preconditioning, and whether this action occurs directly or indirectly on cardiomyocytes, still remain unclear. The endothelial nitric oxide synthase (eNOS)/nitric oxide (NO) signaling pathway and endothelial cells are known to play key roles in cardioprotection against IR injury. Therefore, the aims of this work were to evaluate whether the eNOS/NO pathway mediates the pharmacological cardiac effect of Dex, and whether endothelial cells are required in this cardioprotective action. Isolated adult rat hearts were treated with Dex (10nM) for 25min and the dimerization of eNOS and production of NO were measured. Hearts were then subjected to global IR (30/120min) and the role of the eNOS/NO pathway was evaluated. Dex promoted the activation of eNOS and production of NO. Dex reduced the infarct size and improved the left ventricle function recovery, but this effect was reversed when Dex was co-administered with inhibitors of the eNOS/NO/PKG pathway. In addition, Dex was unable to reduce cell death in isolated adult rat cardiomyocytes subjected to simulated IR. Cardiomyocyte death was attenuated by co-culturing them with endothelial cells pre-treated with Dex. In summary, our results show that Dex triggers cardiac protection by activating the eNOS/NO signaling pathway. This pharmacological effect of Dex requires its interaction with the endothelium.

Autophagy is an evolutionarily ancient process of intracellular protein and organelle recycling required to maintain cellular homeostasis in the face of a wide variety of stresses. Dysregulation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) leads to oxidative damage. Both autophagy and ROS/RNS serve pathological or adaptive roles within cardiomyocytes, depending on the context.ROS/RNS and autophagy communicate with each other via both transcriptional and post-translational events. This cross talk, in turn, regulates the structural integrity of cardiomyocytes, promotes proteostasis, and reduces inflammation, events critical to disease pathogenesis.Dysregulation of either autophagy or redox state has been implicated in many cardiovascular diseases. Cardiomyocytes are rich in mitochondria, which make them particularly sensitive to oxidative damage. Maintenance of mitochondrial homeostasis and elimination of defective mitochondria are each critical to the maintenance of redox homeostasis.The complex interplay between autophagy and oxidative stress underlies a wide range of physiological and pathological events and its elucidation holds promise of potential clinical applicability.

The regulator of calcineurin 1 (RCAN1) inhibits CN (calcineurin), a Ca2+-activated protein phosphatase important in cardiac remodeling. In humans, RCAN1 is located on chromosome 21 in proximity to the Down syndrome critical region. The hearts and brains of Rcan1 KO mice are more susceptible to damage from ischemia/reperfusion (I/R); however, the underlying cause is not known.Mitochondria are key mediators of I/R damage. The goal of these studies was to determine the impact of RCAN1 on mitochondrial dynamics and function.Using both neonatal and isolated adult cardiomyocytes, we show that, when RCAN1 is depleted, the mitochondrial network is more fragmented because of increased CN-dependent activation of the fission protein, DRP1 (dynamin-1-like). Mitochondria in RCAN1-depleted cardiomyocytes have reduced membrane potential, O2 consumption, and generation of reactive oxygen species, as well as a reduced capacity for mitochondrial Ca2+ uptake. RCAN1-depleted cardiomyocytes were more sensitive to I/R; however, pharmacological inhibition of CN, DRP1, or CAPN (calpains; Ca2+-activated proteases) restored protection, suggesting that in the absence of RCAN1, CAPN-mediated damage after I/R is greater because of a decrease in the capacity of mitochondria to buffer cytoplasmic Ca2+. Increasing RCAN1 levels by adenoviral infection was sufficient to enhance fusion and confer protection from I/R. To examine the impact of more modest, and biologically relevant, increases in RCAN1, we compared the mitochondrial network in induced pluripotent stem cells derived from individuals with Down syndrome to that of isogenic, disomic controls. Mitochondria were more fused, and O2 consumption was greater in the trisomic induced pluripotent stem cells; however, coupling efficiency and metabolic flexibility were compromised compared with disomic induced pluripotent stem cells. Depletion of RCAN1 from trisomic induced pluripotent stem cells was sufficient to normalize mitochondrial dynamics and function.RCAN1 helps maintain a more interconnected mitochondrial network, and maintaining appropriate RCAN1 levels is important to human health and disease.

Type-2 ryanodine receptors (RyR2) – the calcium release channels of cardiac sarcoplasmic reticulum – have a central role in cardiac excitation–contraction coupling. In the heart, ischemia/reperfusion causes a rapid and significant decrease in RyR2 content but the mechanisms responsible for this effect are not fully understood. We have studied the involvement of three proteolytic systems – calpains, the proteasome and autophagy – on the degradation of RyR2 in rat neonatal cardiomyocyte cultures subjected to simulated ischemia/reperfusion (sI/R). We found that 8 h of ischemia followed by 16 h of reperfusion decreased RyR2 content by 50% without any changes in RyR2 mRNA. Specific inhibitors of calpains and the proteasome prevented the decrease of RyR2 caused by sI/R, implicating both pathways in its degradation. Proteasome inhibitors also prevented the degradation of calpastatin, the endogenous calpain inhibitor, hindering the activation of calpain induced by calpastatin degradation. Autophagy was activated during sI/R as evidenced by the increase in LC3-II and beclin-1, two proteins involved in autophagosome generation, and in the emergence of GFP-LC3 containing vacuoles in adenovirus GFP-LC3 transduced cardiomyocytes. Selective autophagy inhibition, however, induced even further RyR2 degradation, making unlikely the participation of autophagy in sI/R-induced RyR2 degradation. Our results suggest that calpain activation as a result of proteasome-induced degradation of calpastatin initiates RyR2 proteolysis, which is followed by proteasome-dependent degradation of the resulting RyR2 fragments. The decrease in RyR2 content during ischemia/reperfusion may be relevant to the decrease of heart contractility after ischemia.

Chaperone-mediated autophagy (CMA) is a selective mechanism for the degradation of soluble cytosolic proteins bearing the sequence KFERQ. These proteins are targeted by chaperones and delivered to lysosomes where they are translocated into the lysosomal lumen and degraded via the lysosome-associated membrane protein type 2A (LAMP-2A). Mutations in LAMP2 that inhibit autophagy result in Danon disease characterized by hypertrophic cardiomyopathy. The ryanodine receptor type 2 (RyR2) plays a key role in cardiomyocyte excitation-contraction and its dysfunction can lead to cardiac failure. Whether RyR2 is degraded by CMA is unknown.To induce CMA, cultured neonatal rat cardiomyocytes were treated with geldanamycin (GA) to promote protein degradation through this pathway. GA increased LAMP-2A levels together with its redistribution and colocalization with Hsc70 in the perinuclear region, changes indicative of CMA activation. The inhibition of lysosomes but not proteasomes prevented the loss of RyR2. The recovery of RyR2 content after incubation with GA by siRNA targeting LAMP-2A suggests that RyR2 is degraded via CMA. In silico analysis also revealed that the RyR2 sequence harbours six KFERQ motifs which are required for the recognition Hsc70 and its degradation via CMA. Our data suggest that presenilins are involved in RyR2 degradation by CMA.These findings are consistent with a model in which oxidative damage of the RyR2 targets it for turnover by presenilins and CMA, which could lead to removal of damaged or leaky RyR2 channels.

Many important components of the cardiovascular system display circadian rhythmicity. In both humans and mice, cardiac damage from ischemia/reperfusion (I/R) is greatest at the transition from sleep to activity. The causes of this window of susceptibility are not fully understood. In the murine heart we have reported high amplitude circadian oscillations in the expression of the cardioprotective protein regulator of calcineurin 1 (Rcan1). This study was designed to test whether Rcan1 contributes to the circadian rhythm in cardiac protection from I/R damage. Wild type (WT), Rcan1 KO, and Rcan1-Tg mice, with cardiomyocyte-specific overexpression of Rcan1, were subjected to 45min of myocardial ischemia followed by 24h of reperfusion. Surgeries were performed either during the first 2h (AM) or during the last 2h (PM) of the animal's light phase. The area at risk was the same for all genotypes at either time point; however, in WT mice, PM-generated infarcts were 78% larger than AM-generated infarcts. Plasma cardiac troponin I levels were likewise greater in PM-operated animals. In Rcan1 KO mice there was no significant difference between the AM- and PM-operated hearts, which displayed greater indices of damage similar to that of PM-operated WT animals. Mice with cardiomyocyte-specific overexpression of human RCAN1, likewise, showed no time-of-day difference, but had smaller infarcts comparable to those of AM-operated WT mice. In vitro, cardiomyocytes depleted of RCAN1 were more sensitive to simulated I/R and the calcineurin inhibitor, FK506, restored protection. FK506 also conferred protection to PM-infarcted WT animals. Importantly, transcription of core circadian clock genes was not altered in Rcan1 KO hearts. These studies identify the calcineurin/Rcan1-signaling cascade as a potential therapeutic target through which to benefit from innate circadian changes in cardiac protection without disrupting core circadian oscillations that are essential to cardiovascular, metabolic, and mental health.

Considerable evidence points to critical roles of intracellular Ca2+ homeostasis in the modulation and control of autophagic activity. Yet, underlying molecular mechanisms remain unknown. Mutations in the gene (pkd2) encoding polycystin-2 (PC2) are associated with autosomal dominant polycystic kidney disease (ADPKD), the most common inherited nephropathy. PC2 has been associated with impaired Ca2+ handling in cardiomyocytes and indirect evidence suggests that this protein may be involved in autophagic control. Here, we investigated the role for PC2 as an essential regulator of Ca2+ homeostasis and autophagy.Activation of autophagic flux triggered by mTOR inhibition either pharmacologically (rapamycin) or by means of nutrient depletion was suppressed in cells depleted of PC2. Moreover, cardiomyocyte-specific PC2 knockout mice (αMhc-cre;Pkd2F/F mice) manifested impaired autophagic flux in the setting of nutrient deprivation. Stress-induced autophagy was blunted by intracellular Ca2+ chelation using BAPTA-AM, whereas removal of extracellular Ca2+ had no effect, pointing to a role of intracellular Ca2+ homeostasis in stress-induced cardiomyocyte autophagy. To determine the link between stress-induced autophagy and PC2-induced Ca2+ mobilization, we over-expressed either wild-type PC2 (WT) or a Ca2+-channel deficient PC2 mutant (PC2-D509V). PC2 over-expression increased autophagic flux, whereas PC2-D509V expression did not. Importantly, autophagy induction triggered by PC2 over-expression was attenuated by BAPTA-AM, supporting a model of PC2-dependent control of autophagy through intracellular Ca2+. Furthermore, PC2 ablation was associated with impaired Ca2+ handling in cardiomyocytes marked by partial depletion of sarcoplasmic reticulum Ca2+ stores. Finally, we provide evidence that Ca2+-mediated autophagy elicited by PC2 is a mechanism conserved across multiple cell types.Together, this study unveils PC2 as a novel regulator of autophagy acting through control of intracellular Ca2+ homeostasis.

We investigate here the role of reactive oxygen species and nitric oxide in iron-induced cardiomyocyte hypertrophy or cell death. Cultured rat cardiomyocytes incubated with 20 microM iron (added as FeCl(3)-Na nitrilotriacetate, Fe-NTA) displayed hypertrophy features that included increased protein synthesis and cell size, plus realignment of F-actin filaments along with sarcomeres and activation of the atrial natriuretic factor gene promoter. Incubation with higher Fe-NTA concentrations (100 microM) produced cardiomyocyte death by necrosis. Incubation for 24 h with Fe-NTA (20-40 microM) or the nitric oxide donor Delta-nonoate increased iNOS mRNA but decreased iNOS protein levels; under these conditions, iron stimulated the activity and the dimerization of iNOS. Fe-NTA (20 microM) promoted short- and long-term generation of reactive oxygen species, whereas preincubation with l-arginine suppressed this response. Preincubation with 20 microM Fe-NTA also attenuated the necrotic cell death triggered by 100 microM Fe-NTA, suggesting that these preincubation conditions have cardioprotective effects. Inhibition of iNOS activity with 1400 W enhanced iron-induced ROS generation and prevented both iron-dependent cardiomyocyte hypertrophy and cardioprotection. In conclusion, we propose that Fe-NTA (20 microM) stimulates iNOS activity and that the enhanced NO production, by promoting hypertrophy and enhancing survival mechanisms through ROS reduction, is beneficial to cardiomyocytes. At higher concentrations, however, iron triggers cardiomyocyte death by necrosis.

Herp is an endoplasmic reticulum (ER) stress inducible protein that participates in the ER-associated protein degradation (ERAD) pathway. However, the contribution of Herp to other protein degradation pathways like autophagy and its connection to other types of stress responses remain unknown. Here we report that Herp regulates autophagy to clear poly-ubiquitin (poly-Ub) protein aggregates. Proteasome inhibition and glucose starvation (GS) led to a high level of poly-Ub protein aggregation that was drastically reduced by stably knocking down Herp (shHerp cells). The enhanced removal of poly-Ub inclusions protected cells from death caused by glucose starvation. Under basal conditions and increasingly after stress, higher LC3-II levels and GFP-LC3 puncta were observed in shHerp cells compared to control cells. Herp knockout cells displayed basal up-regulation of two essential autophagy regulators-Atg5 and Beclin-1, leading to increased autophagic flux. Beclin-1 up-regulation was due to a reduction in Hrd1 dependent proteasomal degradation, and not at transcriptional level. The consequent higher autophagic flux was necessary for the clearance of aggregates and for cell survival. We conclude that Herp operates as a relevant factor in the defense against glucose starvation by modulating autophagy levels. These data may have important implications due to the known up-regulation of Herp in pathological states such as brain and heart ischemia, both conditions associated to acute nutritional stress.

Ventricular arrhythmias are a common cause of sudden cardiac death, and their occurrence is higher in obese subjects. Abnormal gating of ryanodine receptors (RyR2), the calcium release channels of the sarcoplasmic reticulum, can produce ventricular arrhythmias. Since obesity promotes oxidative stress and RyR2 are redox-sensitive channels, we investigated whether the RyR2 activity was altered in obese mice. Mice fed a high fat diet (HFD) became obese after eight weeks and exhibited a significant increase in the occurrence of ventricular arrhythmias. Single RyR2 channels isolated from the hearts of obese mice were more active in planar bilayers than those isolated from the hearts of the control mice. At the molecular level, RyR2 channels from HFD-fed mice had substantially fewer free thiol residues, suggesting that redox modifications were responsible for the higher activity. Apocynin, provided in the drinking water, completely prevented the appearance of ventricular arrhythmias in HFD-fed mice, and normalized the activity and content of the free thiol residues of the protein. HFD increased the expression of NOX4, an isoform of NADPH oxidase, in the heart. Our results suggest that HFD increases the activity of RyR2 channels via a redox-dependent mechanism, favoring the appearance of ventricular arrhythmias.

Cardiac fibroblasts (CFs) activation is a common response to most pathological conditions affecting the heart, characterized by increased cellular secretory capacity and increased expression of fibrotic markers, such as collagen I and smooth muscle actin type alpha (α-SMA). Fibrotic activation of CFs induces the increase in tissue protein content, with the consequent tissue stiffness, diastolic dysfunction, and heart failure. Therefore, the search for new mechanisms of CFs activation is important to find novel treatments for cardiac diseases characterized by fibrosis. In this regard, TGF-β1, a cytokine with proinflammatory and fibrotic properties, is crucial in the CFs activation and the development of fibrotic diseases, whereas its molecular targets are not completely known. Serum and glucocorticoid-regulated kinase (SGK1) is a protein involved in various pathophysiological phenomena, especially cardiac and renal diseases that curse with fibrosis. Additionally, SGK1 phosphorylates and regulates the activity and expression of several targets, highlighting FoxO3a for its role in the regulation of oxidative stress and CFs activation induced by TGF-β1. However, the regulation of SGK1 by TGF-β1 and its role in CFs activation have not been studied. In this work, we evaluate the role of SGK1 in CFs isolated from neonatal Sprague-Dawley rats. The participation of SGK1 in the fibrotic activation of CFs induced by TGF-β1 was analyzed, using an inhibitor or siRNA of SGK1. In addition, the role of SGK1 on the regulation of FoxO3a and oxidative stress induced by TGF-β1 was analyzed. Our results indicate that TGF-β1 increased both the activity and expression of SGK1 in CFs, requiring the activation of MAPKs, ERK1/2, p38 and JNK, while inhibition and silencing of SGK1 prevented TGF-β1-induced fibrotic activation of CFs. In addition, SGK1 inhibition prevented FoxO3a inactivation and expression reduction, catalase and SOD2 expression decrease, and the increase of oxidative stress induced by TGF-β1. Taken together, our results position SGK1 as an important regulator of CFs activation driven by TGF-β1, at least in part, through the regulation of FoxO3a and oxidative stress.

FK866 is an inhibitor of the NAD+ synthesis rate-limiting enzyme nicotinamide phosphoribosyltransferase (NAMPT). Using FK866 to target NAD+ synthesis has been proposed as a treatment for inflammatory diseases and cancer. However, use of FK866 may pose cardiovascular risks, as NAMPT expression is decreased in various cardiomyopathies, with low NAD+ levels playing an important role in cardiovascular disease progression. In addition, low NAD+ levels are associated with cardiovascular risk conditions such as aging, dyslipidemia, and type II diabetes mellitus. The aim of this work was to study the effects of FK866-induced NAD+ depletion on mitochondrial metabolism and adaptive stress responses in cardiomyocytes. FK866 was used to deplete NAD+ levels in cultured rat cardiomyocytes. Cell viability, mitochondrial metabolism, and adaptive responses to insulin, norepinephrine, and H2O2 were assessed in cardiomyocytes. The drop in NAD+ induced by FK866 decreased mitochondrial metabolism without changing cell viability. Insulin-stimulated Akt phosphorylation, glucose uptake, and H2O2-survival were compromised by FK866. Glycolytic gene transcription was increased, whereas cardiomyocyte hypertrophy induced by norepinephrine was prevented. Restoring NAD+ levels via nicotinamide mononucleotide administration reestablished mitochondrial metabolism and adaptive stress responses. This work shows that FK866 compromises mitochondrial metabolism and the adaptive response of cardiomyocytes to norepinephrine, H2O2, and insulin.

Cardiomyocyte loss is the main cause of myocardial dysfunction following an ischemia-reperfusion (IR) injury. Mitochondrial dysfunction and altered mitochondrial network dynamics play central roles in cardiomyocyte death. Proteasome inhibition is cardioprotective in the setting of IR; however, the mechanisms underlying this protection are not well-understood. Several proteins that regulate mitochondrial dynamics and energy metabolism, including Mitofusin-2 (Mfn2), are degraded by the proteasome. The aim of this study was to evaluate whether proteasome inhibition can protect cardiomyocytes from IR damage by maintaining Mfn2 levels and preserving mitochondrial network integrity. Using ex vivo Langendorff-perfused rat hearts and in vitro neonatal rat ventricular myocytes, we showed that the proteasome inhibitor MG132 reduced IR-induced cardiomyocyte death. Moreover, MG132 preserved mitochondrial mass, prevented mitochondrial network fragmentation, and abolished IR-induced reductions in Mfn2 levels in heart tissue and cultured cardiomyocytes. Interestingly, Mfn2 overexpression also prevented cardiomyocyte death. This effect was apparently specific to Mfn2, as overexpression of Miro1, another protein implicated in mitochondrial dynamics, did not confer the same protection. Our results suggest that proteasome inhibition protects cardiomyocytes from IR damage. This effect could be partly mediated by preservation of Mfn2 and therefore mitochondrial integrity.

Down syndrome (DS) is a genetic disorder caused by a trisomy of the human chromosome 21 (Hsa21). Overexpression of Hsa21 genes that encode proteins and non-coding RNAs (ncRNAs) can disrupt several cellular functions and biological processes, especially in the heart. Congenital heart defects (CHDs) are present in 45-50% of individuals with DS. Here, we describe the genetic background of this condition (Hsa21 and non-Hsa21 genes), including the role of ncRNAs, and the relevance of these new players in the study of the pathophysiology of DS heart diseases. Additionally, we discuss several distinct pathways in cardiomyocytes which help maintain a functional heart, but that might trigger hypertrophy and oxidative stress when altered. Moreover, we highlight the importance of investigating how mitochondrial and lysosomal dysfunction could eventually contribute to understanding impaired heart function and development in subjects with the Hsa21 trisomy. Altogether, this review focuses on the newest insights about the gene expression, molecular pathways, and organelle alterations involved in the cardiac phenotype of DS.

Acute myocardial infarction is one of the leading causes of death worldwide and thus, an extensively studied disease. Nonetheless, the effects of ischemia/reperfusion injury elicited by oxidative stress on cardiac fibroblast function associated with tissue repair are not completely understood. Ascorbic acid, deferoxamine, and N-acetylcysteine (A/D/N) are antioxidants with known cardioprotective effects, but the potential beneficial effects of combining these antioxidants in the tissue repair properties of cardiac fibroblasts remain unknown. Thus, the aim of this study was to evaluate whether the pharmacological association of these antioxidants, at low concentrations, could confer protection to cardiac fibroblasts against simulated ischemia/reperfusion injury. To test this, neonatal rat cardiac fibroblasts were subjected to simulated ischemia/reperfusion in the presence or absence of A/D/N treatment added at the beginning of simulated reperfusion. Cell viability was assessed using trypan blue staining, and intracellular reactive oxygen species (ROS) production was assessed using a 2′,7′-dichlorofluorescin diacetate probe. Cell death was measured by flow cytometry using propidium iodide. Cell signaling mechanisms, differentiation into myofibroblasts and pro-collagen I production were determined by Western blot, whereas migration was evaluated using the wound healing assay. Our results show that A/D/N association using a low concentration of each antioxidant increased cardiac fibroblast viability, but that their separate administration did not provide protection. In addition, A/D/N association attenuated oxidative stress triggered by simulated ischemia/reperfusion, induced phosphorylation of pro-survival extracellular-signal-regulated kinases 1/2 (ERK1/2) and PKB (protein kinase B)/Akt, and decreased phosphorylation of the pro-apoptotic proteins p38- mitogen-activated protein kinase (p38-MAPK) and c-Jun-N-terminal kinase (JNK). Moreover, treatment with A/D/N also reduced reperfusion-induced apoptosis, evidenced by a decrease in the sub-G1 population, lower fragmentation of pro-caspases 9 and 3, as well as increased B-cell lymphoma-extra large protein (Bcl-xL)/Bcl-2-associated X protein (Bax) ratio. Furthermore, simulated ischemia/reperfusion abolished serum-induced migration, TGF-β1 (transforming growth factor beta 1)-mediated cardiac fibroblast-to-cardiac myofibroblast differentiation, and angiotensin II-induced pro-collagen I synthesis, but these effects were prevented by treatment with A/D/N. In conclusion, this is the first study where a pharmacological combination of A/D/N, at low concentrations, protected cardiac fibroblast viability and function after simulated ischemia/reperfusion, and thereby represents a novel therapeutic approach for cardioprotection.

Communication between cells is a foundational concept for understanding the physiology and pathology of biological systems. Paracrine/autocrine signaling, direct cell-to-cell interplay, and extracellular matrix interactions are three types of cell communication that regulate responses to different stimuli. In the heart, cardiomyocytes, fibroblasts, and endothelial cells interact to form the cardiac tissue. Under pathological conditions, such as myocardial infarction, humoral factors released by these cells may induce tissue damage or protection, depending on the type and concentration of molecules secreted. Cardiac remodeling is also mediated by the factors secreted by cardiomyocytes and fibroblasts that are involved in the extensive reciprocal interactions between these cells. Identifying the molecules and cellular signal pathways implicated in these processes will be crucial for creating effective tissue-preserving treatments during or after reperfusion. Numerous therapies to protect cardiac tissue from reperfusion-induced injury have been explored, and ample pre-clinical research has attempted to identify drugs or techniques to mitigate cardiac damage. However, despite great success in animal models, it has not been possible to completely translate these cardioprotective effects to human applications. This review provides a current summary of the principal molecules, pathways, and mechanisms underlying cardiomyocyte and cardiac fibroblast crosstalk during ischemia/reperfusion injury. We also discuss pre-clinical molecules proposed as treatments for myocardial infarction and provide a clinical perspective on these potential therapeutic agents.

Heart Neonatal rat ventricular

Cardiac hypertrophy is an adaptive response triggered by pathological stimuli. Regulation of the synthesis and the degradation of the Ca2+ channel inositol 1,4,5-trisphosphate receptor (IP3R) affects progression to cardiac hypertrophy. Herpud1, a component of the endoplasmic reticulum-associated degradation (ERAD) complex, participates in IP3R1 degradation and Ca2+ signaling, but the cardiac function of Herpud1 remains unknown. We hypothesize that Herpud1 acts as a negative regulator of cardiac hypertrophy by regulating IP3R protein levels. Our results show that Herpud1-knockout mice exhibit cardiac hypertrophy and dysfunction and that decreased Herpud1 protein levels lead to elevated levels of hypertrophic markers in cultured rat cardiomyocytes. In addition, IP3R levels were elevated both in Herpud1-knockout mice and Herpud1 siRNA-treated rat cardiomyocytes. The latter treatment also led to elevated cytosolic and nuclear Ca2+ levels. In summary, the absence of Herpud1 generates a pathological hypertrophic phenotype by regulating IP3R protein levels. Herpud1 is a novel negative regulator of pathological cardiac hypertrophy.

Polycystin-1 (PC1) is a transmembrane protein found in different cell types, including cardiomyocytes. Alterations in PC1 expression have been linked to mitochondrial damage in renal tubule cells and in patients with autosomal dominant polycystic kidney disease. However, to date, the regulatory role of PC1 in cardiomyocyte mitochondria is not well understood. The analysis of mitochondrial morphology from cardiomyocytes of heterozygous PC1 mice (PDK1+/- ) using transmission electron microscopy showed that cardiomyocyte mitochondria were smaller with increased mitochondria density and circularity. These parameters were consistent with mitochondrial fission. We knocked-down PC1 in cultured rat cardiomyocytes and human-induced pluripotent stem cells (iPSC)-derived cardiomyocytes to evaluate mitochondrial function and morphology. The results showed that downregulation of PC1 expression results in reduced protein levels of sub-units of the OXPHOS complexes and less functional mitochondria (reduction of mitochondrial membrane potential, mitochondrial respiration, and ATP production). This mitochondrial dysfunction activates the elimination of defective mitochondria by mitophagy, assessed by an increase of autophagosome adapter protein LC3B and the recruitment of the Parkin protein to the mitochondria. siRNA-mediated PC1 knockdown leads to a loss of the connectivity of the mitochondrial network and a greater number of mitochondria per cell, but of smaller sizes, which characterizes mitochondrial fission. PC1 silencing also deregulates the AKT-FoxO1 signaling pathway, which is involved in the regulation of mitochondrial metabolism, mitochondrial morphology, and processes that are part of cell quality control, such as mitophagy. Together, these data provide new insights about the controls that PC1 exerts on mitochondrial morphology and function in cultured cardiomyocytes dependent on the AKT-FoxO1 signaling pathway.

Small extracellular vesicles are nanosized vesicles (30–200 nm) that can ferry proteins, nucleic acids, and lipids between cells and therefore, have significant potential as biomarkers, drug delivery tools or therapeutic agents. SEVs of endothelial origin have been shown to -among other functions-reduce in vitro ischemia/reperfusion (I/R) injury in cardiomyocytes, but whether a pro-inflammatory state of the endothelium impairs the functionality of these SEVs remains to be elucidated. To test this, human umbilical vein endothelial cells cells were treated with TNF-α 10 ng/mL and the expression of the pro-inflammatory parameters VCAM-1, ICAM-1 and eNOS were determined by Western blot. SEVs were isolated from endothelial cells treated with or without TNF-α 10 ng/mL using size exclusion chromatography. The size and concentration of SEVs was measured by Nanoparticle Tracking Analysis. The expression of the surface marker CD81 was determined by immunoassay, whereas their morphology was assessed by electron microscopy. The function of endothelial SEVs was assessed by evaluating their cardioprotective effect in an ex vivo model of global I/R using isolated hearts from adult C57BL/6 mice. Treatment of HUVECs with TNF-α induced the expression of VCAM-1 and ICAM-1, whereas eNOS levels were decreased. TNF-α did not affect the production, size, morphology, or expression of CD81. SEVs significantly reduced the infarct size as compared with untreated mice hearts, but SEVs isolated from TNF-α treated cells were unable to achieve this effect. Therefore, a pro-inflammatory state induced by TNF-α does not alter the production of endothelial SEVs but impairs their function in the setting of I/R injury.

Cardiac fibroblasts (CF) are mesenchymal-type cells responsible for maintaining the homeostasis of the heart's extracellular matrix (ECM). Their dysfunction leads to excessive secretion of ECM proteins, tissue stiffening, impaired nutrient and oxygen exchange, and electrical abnormalities in the heart. Additionally, CF act as sentinel cells in the cardiac tissue microenvironment, responding to various stimuli that may affect heart function. Deleterious stimuli induce an inflammatory response in CF, increasing the secretion of cytokines such as IL-1β and TNF-α and the expression of cell adhesion molecules like ICAM1 and VCAM1, initially promoting damage resolution by recruiting immune cells. However, constant harmful stimuli lead to a chronic inflammatory process and heart dysfunction. Therefore, it is necessary to study the mechanisms that govern CF inflammation. NFκB is a key regulator of the cardiac inflammatory process, making the search for mechanisms of NFκB regulation and CF inflammatory response crucial for developing new treatment options for cardiovascular diseases. SGK1, a serine-threonine protein kinase, is one of the regulators of NFκB and is involved in the fibrotic effects of angiotensin II and aldosterone, as well as in CF differentiation. However, its role in the CF inflammatory response is unknown. On the other hand, many bioactive natural products have demonstrated anti-inflammatory effects, but their role in CF inflammation is unknown. One such molecule is boldine, an alkaloid obtained from Boldo (Peumus boldus), a Chilean endemic tree with proven cytoprotective effects. However, its involvement in the regulation of SGK1 and CF inflammation is unknown. In this study, we evaluated the role of SGK1 and boldine in the inflammatory response in CF isolated from neonatal Sprague-Dawley rats. The involvement of SGK1 was analyzed using GSK650394, a specific SGK1 inhibitor. Our results demonstrate that SGK1 is crucial for LPS- and IFN-γ-induced inflammatory responses in CF (cytokine expression, cell adhesion molecule expression, and leukocyte adhesion). Furthermore, a conditioned medium (intracellular content of CF subject to freeze/thaw cycles) was used to simulate a sterile inflammation condition. The conditioned medium induced a potent inflammatory response in CF, which was completely prevented by the SGK1 inhibitor. Finally, our results indicate that boldine inhibits both SGK1 activation and the CF inflammatory response induced by LPS, IFN-γ, and CF-conditioned medium. Taken together, our results position SGK1 as an important regulator of the CF inflammatory response and boldine as a promising anti-inflammatory drug in the context of cardiovascular diseases.

During ischemia/reperfusion (I/R), cardiomyocytes activate pathways that regulate cell survival and death and release factors that modulate fibroblast-to-myofibroblast differentiation. The mechanisms underlying these effects are not fully understood. Polycystin­1 (PC1) is a mechanosensor crucial for cardiac function. This work aims to assess the role of PC1 in cardiomyocyte survival, its role in profibrotic factor expression in cardiomyocytes, and its paracrine effects on I/R-induced cardiac fibroblast function. In vivo and ex vivo I/R and simulated in vitro I/R (sI/R) were induced in wild-type and PC1-knockout (PC1 KO) mice and PC1-knockdown (siPC1) neonatal rat ventricular myocytes (NRVM), respectively. Neonatal rat cardiac fibroblasts (NRCF) were stimulated with conditioned medium (CM) derived from NRVM or siPC1-NRVM supernatant after reperfusion and fibroblast-to-myofibroblast differentiation evaluated. Infarcts were larger in PC1-KO mice subjected to in vivo and ex vivo I/R, and necrosis rates were higher in siPC1-NRVM than control after sI/R. PC1 activated the pro-survival AKT protein during sI/R and induced PC1-AKT-pathway-dependent CTGF expression. Furthermore, conditioned media from sI/R-NRVM induced PC1-dependent fibroblast-to-myofibroblast differentiation in NRCF. This novel evidence shows that PC1 mitigates cardiac damage during I/R, likely through AKT activation, and regulates CTGF expression in cardiomyocytes via AKT. Moreover, PC1-NRVM regulates fibroblast-to-myofibroblast differentiation during sI/R. PC1, therefore, may emerge as a new key regulator of I/R injury-induced cardiac remodeling.

The right and left ventricles have traditionally been studied as individual entities. Furthermore, modifications found in diseased left ventricles are assumed to influence on right ventricle alterations, but the connection is poorly understood. In this review, we describe the differences between ventricles under physiological and pathological conditions. Understanding the mechanisms that differentiate both ventricles would facilitate a more effective use of therapeutics and broaden our knowledge of right ventricle (RV) dysfunction. RV failure is the strongest predictor of mortality in pulmonary arterial hypertension, but at present, there are no definitive therapies directly targeting RV failure. We further explore the current state of drugs and molecules that improve RV failure in experimental therapeutics and clinical trials to treat pulmonary arterial hypertension and provide evidence of their potential benefits in heart failure.

Inhibitors of the ubiquitin-proteasome system improve hemodynamic parameters and decrease the infarct size after ischemia reperfusion. The molecular basis of this protection is not fully understood since most available data report inhibition of the 26 proteasome after ischemia reperfusion. The decrease in cellular ATP levels during ischemia leads to the dissociation of the 26S proteasome into the 19S regulatory complex and the 20S catalytic core, which results in protein degradation independently of ubiquitination. There is scarce information on the activity of the 20S proteasome during cardiac ischemia. Accordingly, the aim of this work was to determine the effects of 30 minutes of ischemia, or 30 min of ischemia followed by 60 minutes of reperfusion on the three main peptidase activities of the 20S proteasome in Langendorff perfused rat hearts. We found that 30 min of ischemia produced a significant increase in the chymotrypsin-like activity of the proteasome, without changes in its caspase-like or trypsin-like activities. In contrast, all three activities were decreased upon reperfusion. Ixazomib, perfused before ischemia at a concentration that reduced the chymotrypsin-like activity to 50% of the control values, without affecting the other proteasomal activities, improved the hemodynamic parameters upon reperfusion and decreased the infarct size. Ixazomib also prevented the 50% reduction in RyR2 content observed after ischemia. The protection was lost, however, when simultaneous inhibition of chymotrypsin-like and caspase-like activities of the proteasome was achieved at higher concentration of ixazomib. Our results suggest that selective inhibition of chymotrypsin-like activity of the proteasome during ischemia preserves key proteins for cardiomyocyte function and exerts a positive impact on cardiac performance after reperfusion.

Dihydropyridines are widely used for the treatment of several cardiac diseases due to their blocking activity on L-type Ca2 + channels and their renowned antioxidant properties. We synthesized six novel dihydropyridine molecules and performed docking studies on the binding site of the L-type Ca2 + channel. We used biochemical techniques on isolated adult rat cardiomyocytes to assess the efficacy of these molecules on their Ca2 + channel-blocking activity and antioxidant properties. The Ca2 + channel-blocking activity was evaluated by confocal microscopy on fluo-3AM loaded cardiomyocytes, as well as using patch clamp experiments. Antioxidant properties were evaluated by flow cytometry using the ROS sensitive dye 1,2,3 DHR. Our docking studies show that a novel compound with 3-OH substitution inserts into the active binding site of the L-type Ca2 + channel previously described for nitrendipine. In biochemical assays, the novel meta-OH group in the aryl in C4 showed a high blocking effect on L-type Ca2 + channel as opposed to para-substituted compounds. In the tests we performed, none of the molecules showed antioxidant properties. Only substitutions in C2, C3 and C5 of the aryl ring render dihydropyridine compounds with the capacity of blocking LTCC. Based on our docking studies, we postulate that the antioxidant activity requires a larger group than the meta-OH substitution in C2, C3 or C5 of the dihydropyridine ring.

The heart is critically dependent on mitochondrial respiration for energy supply. Ischemia decreases oxygen availability, with catastrophic consequences for cellular energy systems. After a few minutes of ischemia, the mitochondrial respiratory chain halts, ATP levels drop and ion gradients across cell membranes collapse. Activation of cellular proteases and generation of reactive oxygen species by mitochondria during ischemia alter mitochondrial membrane permeability, causing mitochondrial swelling and fragmentation and eventually cell death. The mitochondria, therefore, are important targets of cardioprotection against ischemic injury. We have previously shown that ixazomib (IXA), a proteasome inhibitor used for treating multiple myeloma, effectively reduced the size of the infarct produced by global ischemia in isolated rat hearts and prevented degradation of the sarcoplasmic reticulum calcium release channel RyR2. The aim of this work was to further characterize the protective effect of IXA by determining its effect on mitochondrial morphology and function after ischemia. We also quantified the effect of IXA on levels of mitofusin-2, a protein involved in maintaining mitochondrial morphology and mitochondria-SR communication. We found that mitochondria were significantly preserved and functional parameters such as oxygen consumption, the ability to generate a membrane potential, and glutathione content were improved in mitochondria isolated from hearts perfused with IXA prior to ischemia. IXA also blocked the release of cytochrome c observed in ischemia and significantly preserved mitofusin-2 integrity. These beneficial effects resulted in a significant decrease in the left ventricular end diastolic pressure upon reperfusion and a smaller infarct in isolated hearts.

Cardiomyopathy is commonly observed in patients with autosomal dominant polycystic kidney disease (ADPKD), even when they have normal renal function and arterial pressure. The role of cardiomyocyte polycystin-1 (PC1) in cardiovascular pathophysiology remains unknown. PC1 is a potential regulator of BIN1 that maintains T-tubule structure, and alterations in BIN1 expression induce cardiac pathologies. We used a cardiomyocyte-specific PC1-silenced (PC1-KO) mouse model to explore the relevance of cardiomyocyte PC1 in the development of heart failure (HF), considering reduced BIN1 expression induced T-tubule remodeling as a potential mechanism. PC1-KO mice exhibited an impairment of cardiac function, as measured by echocardiography, but no signs of HF until 7-9 months of age. Of the PC1-KO mice, 43% died suddenly at 7 months of age, and 100% died after 9 months with dilated cardiomyopathy. Total BIN1 mRNA, protein levels, and its localization in plasma membrane-enriched fractions decreased in PC1-KO mice. Moreover, the BIN1 + 13 isoform decreased while the BIN1 + 13 + 17 isoform was overexpressed in mice without signs of HF. However, BIN1 + 13 + 17 overexpression was not observed in mice with HF. T-tubule remodeling and BIN1 score measured in plasma samples were associated with decreased PC1-BIN1 expression and HF development. Our results show that decreased PC1 expression in cardiomyocytes induces dilated cardiomyopathy associated with diminished BIN1 expression and T-tubule remodeling. In conclusion, positive modulation of BIN1 expression by PC1 suggests a novel pathway that may be relevant to understanding the pathophysiological mechanisms leading to cardiomyopathy in ADPKD patients.

Cardiac hypertrophy is an adaptive response to manage an excessive cardiac workload and maintain normal cardiac function. However, sustained hypertrophy leads to cardiomyopathy, cardiac failure, and death. Adrenergic receptors play a key role in regulating cardiac function under normal and pathological conditions. Mitochondria are responsible for 90% of ATP production in cardiomyocytes. Mitochondrial function is dynamically regulated by fusion and fission processes. Changes in mitochondrial dynamics and metabolism are central issues in cardiac hypertrophy. Stimulating cardiomyocytes with adrenergic agonists generates hypertrophy and increases mitochondrial fission, which in turn is associated with decreased ATP synthesis. Miro1 is a mitochondrial outer membrane protein involved in mitochondrial dynamics and transport in neurons. The objective of this work was to evaluate whether Miro1 regulates cardiomyocyte hypertrophy through changes in mitochondrial dynamics. In neonatal rat ventricular myocytes, we showed that phenylephrine induced cardiomyocyte hypertrophy and increased Miro1 mRNA and protein levels. Moreover, alpha-adrenergic stimulation provoked a mitochondrial fission pattern in the cardiomyocytes. Miro1 knockdown prevented both the cardiomyocyte hypertrophy and mitochondrial fission pattern. Our results suggest that Miro1 participates in phenylephrine-induced cardiomyocyte hypertrophy through mitochondrial fission.

Ang-(1-9) peptide is a bioactive vasodilator peptide that prevents cardiomyocyte hypertrophy in vitro and in vivo as well as lowers blood pressure and pathological cardiovascular remodeling; however, it has a reduced half-life in circulation, requiring a suitable carrier for its delivery. In this work, hybrid nanoparticles composed of polymeric nanoparticles (pNPs) based on Eudragit® E/Alginate (EE/Alg), and gold nanospheres (AuNS), were developed to evaluate their encapsulation capacity and release of Ang-(1-9) under different experimental conditions. Hybrid pNPs were characterized by dynamic light scattering, zeta potential, transmission and scanning electron microscopy, size distribution, and concentration by nanoparticle tracking analysis. Nanometric pNPs, with good polydispersity index and colloidally stable, produced high association efficiency of Ang-(1-9) and controlled release. Finally, the treatment of neonatal cardiomyocytes in culture with EE/Alg/AuNS 2% + Ang-(1-9) 20% pNPs decreased the area and perimeter, demonstrating efficacy in preventing norepinephrine-induced cardiomyocyte hypertrophy. On the other hand, the incorporation of AuNS did not cause negative effects either on the cytotoxicity or on the association capacity of Ang-(1-9), suggesting that the hybrid carrier EE/Alg/AuNS pNPs could be used for the delivery of Ang-(1-9) in the treatment of cardiovascular hypertrophy.

Cardiac hypertrophy is the result of responses to various physiological or pathological stimuli. Recently, we showed that polycystin-1 participates in cardiomyocyte hypertrophy elicited by pressure overload and mechanical stress. Interestingly, polycystin-1 knockdown does not affect phenylephrine-induced cardiomyocyte hypertrophy, suggesting that the effects of polycystin-1 are stimulus-dependent. In this study, we aimed to identify the role of polycystin-1 in insulin-like growth factor-1 (IGF-1) signaling in cardiomyocytes. Polycystin-1 knockdown completely blunted IGF-1-induced cardiomyocyte hypertrophy. We then investigated the molecular mechanism underlying this result. We found that polycystin-1 silencing impaired the activation of the IGF-1 receptor, Akt, and ERK1/2 elicited by IGF-1. Remarkably, IGF-1-induced IGF-1 receptor, Akt, and ERK1/2 phosphorylations were restored when protein tyrosine phosphatase 1B was inhibited, suggesting that polycystin-1 knockdown deregulates this phosphatase in cardiomyocytes. Moreover, protein tyrosine phosphatase 1B inhibition also restored IGF-1-dependent cardiomyocyte hypertrophy in polycystin-1-deficient cells. Our findings provide the first evidence that polycystin-1 regulates IGF-1-induced cardiomyocyte hypertrophy through a mechanism involving protein tyrosine phosphatase 1B.

Resumen La patologia cardiovascular es la primera causa de muerte en Chile y en el mundo. Desde el punto de vistaquirurgico, anestesiologos y cirujanos enfrentan mas frecuentemente pacientes mayores con patologia cardiovascular. La incidencia de isquemia miocardica en pacientes de alto riesgo, sometidos a cirugia no-cardiaca, es cercana al 40% durante el perioperatorio. La incidencia de infarto miocardico y muerte en cirugia no-cardiaca, oscila entre 1 y 5%. Existe una estrecha relacion entre los eventos isquemicos perioperatorios y el aumento de la morbimortalidad cardiovascular. Por este motivo, se han desarrollado medidas terapeuticas orientadas a disminuir la incidencia de isquemia perioperatoria y aminorar el dano asociado a ella. La adecuada identificacion de pacientes de riesgo, la optimizacion del tratamiento medico depatologias asociadas y el uso de farmacos cardioprotectores durante el perioperatorio, han mostrado disminuir la incidencia de complicaciones cardiacas.

Whereas reperfusion injury accounts for up to 50% of the injury of ischemia/reperfusion (I/R), no therapies are available targeting this. Histone deacetylases (HDAC) inhibitor reduces infarct size by 50% in mouse I/R models. Translating these results to human, however, requires large animal testing

Abstract Aims Two‐dimensional speckle‐tracking echocardiography can assess left atrial (LA) function by measuring atrial volumes and deformation parameters (strain, strain rate). This cross‐sectional analysis explores the association between ideal CV health (CVH), LA function, and systemic biomarkers in healthy individuals from the Chilean MAUCO Cohort. Methods We enrolled 95 MAUCO participants with different levels of CVH (mean age: 51 ± 8 years). We categorized participants into low or high CVH groups: A: 0‐2, or B: 3‐6 CVH risk factors. 2D echocardiography, glucose, insulin, total cholesterol, triglycerides, proBNP, hsCRP, insulin resistance index (HOMA), and right and left atrial strain (RASs and LASs, respectively) were determined. Results LASs was lower in Group A, while systolic and diastolic blood pressure (BP), body mass index (BMI), insulin, HOMA, total cholesterol, triglycerides, and LV and RV end‐diastolic volume were significantly higher in Group A than Group B ( P < .01). Change in LASs was inversely correlated with insulin ( P = .040), HOMA ( P = .013), total cholesterol ( P = .039), glycemia ( P = .018), and BMI ( P = .0.037). Conclusion LASs during the reservoir phase was diminished in subjects with a lower level of CVH. Higher insulin, HOMA, total cholesterol, glycemia, and BMI values were associated with decreased LA deformation during the reservoir phase. Morphofunctional alterations of the LA were also identified in the group with suboptimal CVH, as well as BP values in the range of hypertension. LA dysfunction in an asymptomatic population, along with metabolic syndrome, could be an early event in the continuum of CV damage.

El infarto del miocardio es una de las principales causas de muerte a nivel mundial y se produce a consecuencia de procesos de isquemia-reperfusion (IR). El dano miocardico generado por IR puede ser atenuado a traves del pre-condicionamiento isque-mico (PI) temprano, mediado por la via RISK o PI tardio, que se asocia a una respuesta genomica en la que se activan proteinas como oxido nitrico sintasa inducible (iNOS). Las vias de senalizacion que median el PI tambien pue-den ser activadas farmacologicamente. Dexmedetomi-dina (Dex) es un agonista alfa2-adrenergico, que se ha descrito como un potente agente cardioprotector frente a IR. Recientemente, nuestro grupo describio que Dex requiere el endotelio y la activacion de la via oxido ni-trico sintasa endotelial (eNOS)-oxido nitrico (NO) para pre-condicionar el miocardio. Sin embargo, no existen estudios que muestren la posible participacion de iNOS en la proteccion conferida por Dex.La presente adenda tiene por objetivo evaluar si Dex activa iNOS en el corazon y en cardiomiocitos. Para esto, corazones de rata adulta fueron estimulados con Dex 10 nM y se observo que el farmaco aumento la produccion de NO medida por cuantificacion de nitri-tos, mas no estimulo la activacion de iNOS medida por Western blot. Ademas, Dex tampoco indujo el aumento de mRNA de iNOS en cardiomiocitos adultos. Por lo tanto, Dex genera NO independiente a iNOS durante su efecto pre-condicionante agudo. Sin embargo, se re-quieren mas estudios que clarifiquen su papel en una posible proteccion a largo plazo frente a IR generada por Dex.

Altered calcium handling and mitochondrial function are hallmarks of heart disease and both influence heart susceptibility to ischemia reperfusion (I/R). Regulator of Calcineurin 1 (RCAN1) is a feedback inhibitor of calcineurin, a calcium-activated phosphatase involved in cardiac remodeling. We used advanced imaging techniques and an in vitro model of I/R (sim-I/R) to dissect RCAN1's role in mitochondrial dynamics and cell survival. SiRNA was used to deplete RCAN1 from neonatal rat ventricular myocytes (NRVMs). Confocal imaging showed an increase in the number of mitochondria per cell and a decreased individual mitochondrial size, indicative of increased fission in the absence of RCAN1 (See Figure). There was a corresponding decrease in the phosphorylation of the fission protein DRP1, a calcineurin substrate. Consistent with increased fission, the siRCAN1-depleted NRVMs showed decreases in ATP production, mitochondrial membrane potential, oxygen consumption, and mitochondrial calcium uptake. Death following sim-I/R was significantly greater in siRCAN1-depleted NRVMs compared to controls. Importantly, we found an increase in both cytosolic calcium levels and calpain activity in the SiRCAN1-depleted NRVMs subjected to sim-I/R. Resistance to I/R was restored in siRCAN1-depleted NRVMs by pharmacological inhibition of calcineurin (FK506), mitochondrial fission (Mdivi), or calpain. We hypothesize that RCAN1 preserves mitochondrial fusion by inhibiting calcineurin-dependent translocation of DRP1 to mitochondria, preserving mitochondrial network connectivity and cacium buffering capacity. These findings have important implications regarding cell metabolism and survival in all tissues. Funding: AHA and NIH

Background: The hexosamine biosynthetic pathway (HBP) generates UDP-GlcNAc (uridine diphosphate N-acetylglucosamine) for glycan synthesis and O-linked GlcNAc (O-GlcNAc) protein modifications. Despite the established role of the HBP in glucose metabolism and multiple diseases, regulation of the HBP remains largely undefined. Methods & Results: Here, we show that spliced Xbp1 (Xbp1s), the most conserved signal transducer of the unfolded protein response (UPR), is a direct transcriptional activator of the HBP. We demonstrate that the UPR triggers activation of the HBP by means of Xbp1s-dependent transcription of genes coding for key, rate-limiting enzymes. We establish that this previously unrecognized UPR-HBP axis is triggered in a variety of stress conditions known to promote O-GlcNAc modification. We go on to demonstrate that Xbp1s, acutely stimulated by ischemia/reperfusion (I/R) in heart, confers robust cardioprotection against I/R injury. We also show that HBP induction is required for this cardioprotective response. Mechanistically, HBP may mediate the adaptive branch of the UPR by activating autophagy and ER-associated degradation. Conclusion: These studies reveal that Xbp1s couples the UPR to the HBP, promoting robust cardioprotection during I/R.

Atrial fibrillation is the most common arrhythmia, and it has a significant effect on morbidity and mortality. It is a significant risk factor for stroke and heart failure. Despite extensive interest in and research effort for this disease, the understanding of its mechanisms and triggers is still incomplete. Autophagy is a catabolic process involved in protein degradation, organelle turnover, and nonselective breakdown of cytoplasmic components during nutrient starvation or stress. In the heart, autophagy is observed in a variety of human pathologies, where it can be either adaptive or maladaptive, depending on the context. Specifically in patients with atrial fibrillation, autophagy seems to be impaired. In this chapter, we review autophagy regulation in the onset of atrial fibrillation; also we will discuss the potential usefulness of different pharmacological modulators of autophagy in the treatment of atrial fibrillation.

Introduction: Histone deacetylases (HDACs) participate in pathological cardiac growth and failure, and small molecule inhibitors of HDACs slow, and even regress, pathological hypertrophy. mTOR (mam...

Introduction: Diabetic cardiomyopathy is typified by alterations in cardiac metabolism, morphology, and function independent of hypertension and coronary artery disease. We have shown previously th...

Zhao Wang, 2013 Finalist and Presenting Author

Dysregulation of calcium handling and mitochondrial function are hallmarks of heart disease. Furthermore, mitochondrial dynamics and metabolism influence the susceptibility of the heart to ischemia reperfusion (I/R). Regulator of Calcineurin 1 (RCAN1) is an endogenous feedback inhibitor of calcineurin, a calcium-activated phosphatase involved in pathological cardiac remodeling. The hearts of mice with a targeted disruption of Rcan1 are more susceptible to damage from I/R in vivo. Here we have used an in vitro model of simulated I/R (sim-I/R) and advanced mitochondrial imaging techniques to dissect RCAN1’s cardiomyocyte-autonomous role in mitochondrial dynamics and cell survival. SiRNA was used to deplete the RCAN1.4 and RCAN1.1 isoforms from neonatal rat ventricular myocytes (NRVMs). Cultures were subjected to 6 hours of sim-I/R followed by 12 hours of reperfusion. Death, measured as LDH release, was significantly greater in the siRCAN1-depeleted cultures than in si-controls. Si-depleted NRVMs were dyed with Mitotracker-green and three-dimensional images obtained by confocal microscopy. Z-stacks of thresholded images were volume-reconstituted. Our results show that siRCAN1-depleted NRVMs have a decrease in the mean volume of mitochondrial particles and an increase in the total number of these organelles. Consistent with increased mitochondrial fission, the rate of ATP production and the mitochondrial membrane potential were lower in siRCAN1-depleted NRVMs compared to control siRNA treated cells. There was also a profound redistribution of the mitochondrial network, decreasing the functional coupling between mitochondria and endoplasmic reticulum, assayed as a decrease in mitochondrial calcium uptake capacity. In vivo and in vitro ultraestructural EM studies showed increased mitochondrial fragmentation and a loss of the cristae structure. Finally, there was evidence of sarcomeric hypertrophy in NRVMs depleted of RCAN1, correlating with a switch to a more glycolytic metabolism. These studies suggest that RCAN1 helps preserve a functional mitochondrial network, providing new insights into the role of mitochondrial dynamics and calcineurin/RCAN1 regulation in adaptive and pathological cardiac remodeling.

Background: Autophagy is a ubiquitous process of intracellular protein and organelle recycling, critical to numerous forms of stress responsiveness. Polycystins are integral membrane proteins local...

Antecedentes: La muerte de los cardiomiocitos es determinante en el desarrollo de patologías cardiacas posteriores al infarto del miocardio y la insuficiencia cardiaca. Las variaciones en la expresión de la familia de proteínas BCL-2 regulan vías, tanto de muerte, como de sobrevida celular. Así, BCL-2 es una proteína anti- apoptótica y NIX una proteína que induce la necrosis y/o la apoptosis celular. La Policistina-1 (PC1) es un mecanosensor vital para la función contráctil cardiaca; sin embargo, se desconoce su papel en la sobrevida de los cardiomiocitos durante el estrés mecánico.

Introducción: Las células de la musculatura lisa vascular (CMLV) se caracterizan por mantener cierto grado de desdiferenciación, variando su fenotipo entre el contráctil y el secretor, de acuerdo con las necesidades del tejido, y el contráctil predominante en condiciones fisiológicas. Cualquier alteración del estímulo mecánico, ya sea en el flujo sanguíneo o la tensión mecánica ejercida sobre las CMLV, conducen a cambios de su fenotipo y remodelamiento de la vasculatura, lo que puede constituir el punto de inflexión de varias patologías relevantes en la salud pública como, por ejemplo, la hipertensión arterial.

Background: Transverse tubules (T-tubules) play a key role in cardiac contractility. The expression of Bridging integrator 1 (BIN1), specifically, the cardiac isoforms BIN1+13 and BIN1+ 13+17 promote the formation and ultrastructure of T-tubules and it has been described that the transcriptional factor c-Myc may be a negative regulator of BIN1 expression. Polycystin-1 (PC1) is a mechanosensor in cardiomyocytes, with a crucial role to maintain cardiac function. Our aim was to determine whether PC1 regulates BIN1-induced T-tubule formation by a c-Myc-regulated mechanism. Methods: We used adult C57BL/6 cardiomyocyte-specific knockout mice (PC1 KO). Cardiac function was tested by echocardiography, whereas protein and mRNA content of BIN1, PC1 and c-Myc were measured using western blot and qRT-PCR, respectively. T-tubules were analyzed by transmission electron microscopy (TEM). For statistical analyses, t-test or one-way ANOVA followed by Tukey's test were used. Differences were considered significant at p < 0.05. Results: Survival of PC1 KO mice decreased dramatically after 7 months of age, with clear symptoms of dilated cardiomyopathy and heart failure (decreased fractional shortening and ejection fraction). Ventricular cardiac tissue of PC1 KO mice without (< 7-month-old mice) and with symptoms (7-9-month-old mice) of heart failure (HF) was associated with reduced levels of BIN1 protein content. Total BIN1 mRNA in PC1 KO mice without symptoms did not show differences as compared to controls, but it was significantly decreased in mice with symptoms of HF. Moreover, PC1 KO mice with and without symptoms showed decreased BIN1+13 mRNA levels, whereas BIN1+13+17 mRNA was only increased in mice without symptoms. These changes in BIN1 isoforms were related with T-tubules, which showed increased lumen and decreased intra-luminal density in PC1 KO mice with and without symptoms. Furthermore, c-Myc protein content was increased in cardiac tissue of PC1 KO mice. Conclusion: PC1 may be a regulator of the differential expression of cardiac isoforms of BIN1 in cardiomyocytes by a mechanism that involves negative regulation of c-Myc, related to loss of T-tubule ultrastructure and heart failure development.