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Background: Cellular redox control is maintained by generation of reactive oxygen/nitrogen species balanced by activation of antioxidative pathways. Disruption of redox balance leads to oxidative stress, a central causative event in numerous diseases including heart failure. Redox control in the heart exposed to hemodynamic stress, however, remains to be fully elucidated. Methods: Pressure overload was triggered by transverse aortic constriction in mice. Transcriptomic and metabolomic regulations were evaluated by RNA-sequencing and metabolomics, respectively. Stable isotope tracer labeling experiments were conducted to determine metabolic flux in vitro. Neonatal rat ventricular myocytes and H9c2 cells were used to examine molecular mechanisms. Results: We show that production of cardiomyocyte NADPH, a key factor in redox regulation, is decreased in pressure overload-induced heart failure. As a consequence, the level of reduced glutathione is downregulated, a change associated with fibrosis and cardiomyopathy. We report that the pentose phosphate pathway and mitochondrial serine/glycine/folate metabolic signaling, 2 NADPH-generating pathways in the cytosol and mitochondria, respectively, are induced by transverse aortic constriction. We identify ATF4 (activating transcription factor 4) as an upstream transcription factor controlling the expression of multiple enzymes in these 2 pathways. Consistently, joint pathway analysis of transcriptomic and metabolomic data reveal that ATF4 preferably controls oxidative stress and redox-related pathways. Overexpression of ATF4 in neonatal rat ventricular myocytes increases NADPH-producing enzymes‚ whereas silencing of ATF4 decreases their expression. Further, stable isotope tracer experiments reveal that ATF4 overexpression augments metabolic flux within these 2 pathways. In vivo, cardiomyocyte-specific deletion of ATF4 exacerbates cardiomyopathy in the setting of transverse aortic constriction and accelerates heart failure development, attributable, at least in part, to an inability to increase the expression of NADPH-generating enzymes. Conclusions: Our findings reveal that ATF4 plays a critical role in the heart under conditions of hemodynamic stress by governing both cytosolic and mitochondrial production of NADPH.

Supplemental Digital Content is available in the text., Background: Cardiac hypertrophy is an independent risk factor for heart failure, a leading cause of morbidity and mortality globally. The calcineurin/NFAT (nuclear factor of activated T cells) pathway and the MAPK (mitogen-activated protein kinase)/Erk (extracellular signal-regulated kinase) pathway contribute to the pathogenesis of cardiac hypertrophy as an interdependent network of signaling cascades. How these pathways interact remains unclear and few direct targets responsible for the prohypertrophic role of NFAT have been described. Methods: By engineering cardiomyocyte-specific ETS2 (a member of the E26 transformation-specific sequence [ETS] domain family) knockout mice, we investigated the role of ETS2 in cardiac hypertrophy. Primary cardiomyocytes were used to evaluate ETS2 function in cell growth. Results: ETS2 is phosphorylated and activated by Erk1/2 on hypertrophic stimulation in both mouse (n=3) and human heart samples (n=8 to 19). Conditional deletion of ETS2 in mouse cardiomyocytes protects against pressure overload–induced cardiac hypertrophy (n=6 to 11). Silencing of ETS2 in the hearts of calcineurin transgenic mice significantly attenuates hypertrophic growth and contractile dysfunction (n=8). As a transcription factor, ETS2 is capable of binding to the promoters of hypertrophic marker genes, such as ANP, BNP, and Rcan1.4 (n=4). We report that ETS2 forms a complex with NFAT to stimulate transcriptional activity through increased NFAT binding to the promoters of at least 2 hypertrophy-stimulated genes: Rcan1.4 and microRNA-223 (=n4 to 6). Suppression of microRNA-223 in cardiomyocytes inhibits calcineurin-mediated cardiac hypertrophy (n=6), revealing microRNA-223 as a novel prohypertrophic target of the calcineurin/NFAT and Erk1/2-ETS2 pathways. Conclusions: Our findings point to a critical role for ETS2 in calcineurin/NFAT pathway-driven cardiac hypertrophy and unveil a previously unknown molecular connection between the Erk1/2 activation of ETS2 and expression of NFAT/ETS2 target genes.

Mammalian germline cells, embryonic stem cells, and adult stem cells hold the potential for defining, constructing, and maintaining a unique individual through the respective processes of gametogenesis, embryogenesis, and tissue repair over the span of a lifetime. Autophagy carries out critical functions in these cells using a variety of mechanisms. Of particular importance to the integrity of germline and stem cells are (1) the removal of damaged and/or excess mitochondria, thereby reducing the potential for generating reactive oxygen species that can damage genomic content; (2) the removal and recycling of cellular structures to facilitate the extensive remodeling that occurs during the initial establishment and subsequent differentiation of these cell populations; and (3) the supply of substrates and energy sources when needed under stress and in the environment of the “hypoxic niche” that these cells often occupy.

Down syndrome (DS) is the most frequently occurring human chromosomal disorder and is responsible for a range of both congenital defects and progressive, degenerative conditions. For instance, an estimated 50% DS neonates are born with congenital heart defects (CHD) and more than 50% of DS adults develop early onset Alzheimer’s. Using induced pluripotent stem cells (iPSCs) derived from DS patients and isogenic controls we previously demonstrated the presence of a hyper-metabolic, hyper-fused mitochondrial network in trisomic iPSCs (3S-iPSCs) compared to disomic (2S-iPSCs) controls. Furthermore, mitochondrial function was normalized by siRNA depletion of RCAN1, an inhibitor of the protein phosphatase calcineurin (CN). Both CN signaling and mitochondrial metabolism have been implicated in a variety of steps during the progression from embryonic stem cells to cardiac progenitors, including self-renewal, exit from pluripotency, and commitment to cardiac verses hematopoietic lineages. Based on this, we hypothesized that the dynamics of many of these processes will be altered over the course of differentiation of 3S-iPSCs to cardiomyocytes when compared to 2S-iPSCs. Here, we investigate the temporal expression of pluripotency associated genes and lineage associated genes as well as cardiac mesoderm and mature cardiomyocyte specific genes. We also define and compare changes in CN activity, expression of specific CN isoforms, mitochondrial expansion, ROS generation, and activation of stress responses. Our study identifies early developmental and metabolic sequelae capable of contributing to CHD in DS that may result from a disruption in the normal balance in crosstalk between CN and RCAN1.

Ischemic disease is among the deadliest and most disabling illnesses. Prominent examples include myocardial infarction and stroke. Most, if not all, underlying pathological changes, including oxidative stress, inflammation, and nutrient deprivation, are potent inducers of the integrated stress response (ISR). Four upstream kinases are involved in ISR signaling that sense a myriad of input stress signals and converge on the phosphorylation of serine 51 of eukaryotic translation initiation factor 2α (eIF2α). As a result, translation initiation is halted, creating a window of opportunity for the cell to repair itself and restore homeostasis. A growing number of studies show strong induction of the ISR in ischemic disease. Genetic and pharmacological evidence suggests that the ISR plays critical roles in disease initiation and progression. Here, we review the basic regulation of the ISR, particularly in response to ischemia, and summarize recent findings relevant to the actions of the ISR in ischemic disease. We then discuss therapeutic opportunities by modulating the ISR to treat ischemic heart disease, brain ischemia, ischemic liver disease, and ischemic kidney disease. Finally, we propose that the ISR represents a promising therapeutic target for alleviating symptoms of ischemic disease and improving clinical outcomes.

A major factor in the progression to heart failure in humans is the inability of the adult heart to repair itself after injury. Our group recently demonstrated that the early postnatal mammalian heart is capable of regeneration following injury through proliferation of preexisting cardiomyocytes1,2. We recently also showed that Meis1, a TALE family homeodomain transcription factor, translocates to cardiomyocyte nuclei shortly after birth and mediates postnatal cell cycle arrest3. Here we report that Hoxb13 acts as a Meis1 cofactor in postnatal cardiomyocytes. Cardiomyocyte-specific deletion of Hoxb13 can both extend the postnatal window of cardiomyocyte proliferation and reactivate cardiomyocyte cell cycle in the adult heart. Moreover, adult Meis1/Hoxb13 double knockout hearts display widespread cardiomyocyte mitosis, sarcomere disassembly and an improvement in left ventricular systolic function following myocardial infarction both by echocardiography and MRI. ChIP-seq analysis demonstrates that Meis1 and Hoxb13 act cooperatively to regulate cardiomyocyte maturation and cell cycle. Finally, we show that the calcium-activated protein phosphatase calcineurin dephosphorylates Hoxb13 at serine-204 (S204), resulting in its nuclear localization and cell cycle arrest. Collectively, these results demonstrate that Meis1 and Hoxb13 act cooperatively to regulate cardiomyocyte maturation and proliferation and provide mechanistic insights into the link between hyperplastic and hypertrophic growth of cardiomyocytes.

A major factor in the progression to heart failure in humans is the inability of the adult heart to repair itself after injury. We recently demonstrated that the early postnatal mammalian heart is capable of regeneration following injury through proliferation of preexisting cardiomyocytes1,2 and that Meis1, a three amino acid loop extension (TALE) family homeodomain transcription factor, translocates to cardiomyocyte nuclei shortly after birth and mediates postnatal cell cycle arrest3. Here we report that Hoxb13 acts as a cofactor of Meis1 in postnatal cardiomyocytes. Cardiomyocyte-specific deletion of Hoxb13 can extend the postnatal window of cardiomyocyte proliferation and reactivate the cardiomyocyte cell cycle in the adult heart. Moreover, adult Meis1-Hoxb13 double-knockout hearts display widespread cardiomyocyte mitosis, sarcomere disassembly and improved left ventricular systolic function following myocardial infarction, as demonstrated by echocardiography and magnetic resonance imaging. Chromatin immunoprecipitation with sequencing demonstrates that Meis1 and Hoxb13 act cooperatively to regulate cardiomyocyte maturation and cell cycle. Finally, we show that the calcium-activated protein phosphatase calcineurin dephosphorylates Hoxb13 at serine-204, resulting in its nuclear localization and cell cycle arrest. These results demonstrate that Meis1 and Hoxb13 act cooperatively to regulate cardiomyocyte maturation and proliferation and provide mechanistic insights into the link between hyperplastic and hypertrophic growth of cardiomyocytes.

Mitochondrial damage is implicated as a major contributing factor for a number of noncommunicable chronic diseases such as cardiovascular diseases, cancer, obesity, and insulin resistance/type 2 diabetes. Here, we discuss the role of mitochondria in maintaining cellular and whole-organism homeostasis, the mechanisms that promote mitochondrial dysfunction, and the role of this phenomenon in noncommunicable chronic diseases. We also review the state of the art regarding the preclinical evidence associated with the regulation of mitochondrial function and the development of current mitochondria-targeted therapeutics to treat noncommunicable chronic diseases. Finally, we give an integrated vision of how mitochondrial damage is implicated in these metabolic diseases.

Angiotensin-(1-9) is a peptide from the noncanonical renin-angiotensin system with anti-hypertrophic effects in cardiomyocytes via an unknown mechanism. In the present study we aimed to elucidate it, basing us initially on previous work from our group and colleagues who proved a relationship between disturbances in mitochondrial morphology and calcium handling, associated with the setting of cardiac hypertrophy. Our first finding was that angiotensin-(1-9) can induce mitochondrial fusion through DRP1 phosphorylation. Secondly, angiotensin-(1-9) blocked mitochondrial fission and intracellular calcium dysregulation in a model of norepinephrine-induced cardiomyocyte hypertrophy, preventing the activation of the calcineurin/NFAT signaling pathway. To further investigate angiotensin-(1-9) anti-hypertrophic mechanism, we performed RNA-seq studies, identifying the upregulation of miR-129 under angiotensin-(1-9) treatment. miR-129 decreased the transcript levels of the protein kinase A inhibitor (PKIA), resulting in the activation of the protein kinase A (PKA) signaling pathway. Finally, we showed that PKA activity is necessary for the effects of angiotensin-(1-9) over mitochondrial dynamics, calcium handling and its anti-hypertrophic effects.

Background: Cardiac dysfunction is a major component of sepsis-induced multiorgan failure in critical care units. Changes in cardiac autophagy and its role during sepsis pathogenesis have not been clearly defined. Targeted autophagy-based therapeutic approaches for sepsis are not yet developed. Methods: Beclin-1-dependent autophagy in the heart during sepsis and the potential therapeutic benefit of targeting this pathway were investigated in a mouse model of lipopolysaccharide (LPS)-induced sepsis. Results: LPS induced a dose-dependent increase in autophagy at low doses, followed by a decline that was in conjunction with mammalian target of rapamycin activation at high doses. Cardiac-specific overexpression of Beclin-1 promoted autophagy, suppressed mammalian target of rapamycin signaling, improved cardiac function, and alleviated inflammation and fibrosis after LPS challenge. Haplosufficiency for beclin 1 resulted in opposite effects. Beclin-1 also protected mitochondria, reduced the release of mitochondrial danger-associated molecular patterns, and promoted mitophagy via PTEN-induced putative kinase 1-Parkin but not adaptor proteins in response to LPS. Injection of a cell-permeable Tat-Beclin-1 peptide to activate autophagy improved cardiac function, attenuated inflammation, and rescued the phenotypes caused by beclin 1 deficiency in LPS-challenged mice. Conclusions: These results suggest that Beclin-1 protects the heart during sepsis and that the targeted induction of Beclin-1 signaling may have important therapeutic potential.

Angiotensin-(19), a peptide of the non-classical renin angiotensin system, has been shown to prevent and revert hypertension and cardiac hypertrophy. We hypothetized that systemic delivery of angiotensin-(1-9) following myocardial infarction will also be protective and extend to provide protection during reperfusion of the ischemic heart. Adult Sprague Dawley rats were subjected to left anterior descending artery ligation and treated with angiotensin-(1-9) via osmotic mini-pump for 2 weeks in the presence or absence of Mas receptor or AT2R antagonists (A779 and PD123319, respectively). Myocardial death and left ventricular function were evaluated after infarction. Infarct size and functional parameters were determined in isolated rat hearts after global ischemia/reperfusion in the presence of angiotensin-(1-9) plus receptor antagonists or Akt inhibitor at reperfusion. in vitro, neonatal rat ventricular cardiomyocytes underwent simulated ischemia/reperfusion and angiotensin-(1-9) was co-incubated with A779, PD123319 or Akt inhibitor. Systemic delivery of angiotensin-(1-9) significantly decreased cell death and improved left ventricular recovery after in vivo myocardial infarction. Perfusion with the peptide reduced the infarct size and improved functional recovery after ex vivo ischemia/reperfusion. In vitro, angiotensin-(1-9) decreased cell death in isolated neonatal rat ventricular cardiomyocytes subjected to simulated ischemia/reperfusion. The cardioprotective effects of angiotensin-(1-9) were blocked by PD123319 and Akti VIII but not by A779. Angiotensin-(1-9) limits reperfusion-induced cell death by an AT2R- and Aktdependent mechanism. Angiotensin-(1-9) is a novel strategy to protect against cardiac ischemia/reperfusion injury.

Calcineurin, also known as PP2B or PPP3, is a member of the PPP family of protein phosphatases that also includes PP1 and PP2A. Together these three phosphatases carryout the majority of dephosphorylation events in the heart. Calcineurin is distinct in that it is activated by the binding of calcium/calmodulin (Ca2+/CaM) and therefore acts as a node for integrating Ca2+ signals with changes in phosphorylation, two fundamental intracellular signaling cascades. In the heart, calcineurin is primarily thought of in the context of pathological cardiac remodeling, acting through the Nuclear Factor of Activated T-cell (NFAT) family of transcription factors. However, calcineurin activity is also essential for normal heart development and homeostasis in the adult heart. Furthermore, it is clear that NFAT-driven changes in transcription are not the only relevant processes initiated by calcineurin in the setting of pathological remodeling. There is a growing appreciation for the diversity of calcineurin substrates that can impact cardiac function as well as the diversity of mechanisms for targeting calcineurin to specific sub-cellular domains in cardiomyocytes and other cardiac cell types. Here, we will review the basics of calcineurin structure, regulation, and function in the context of cardiac biology. Particular attention will be given to: the development of improved tools to identify and validate new calcineurin substrates; recent studies identifying new calcineurin isoforms with unique properties and targeting mechanisms; and the role of calcineurin in cardiac development and regeneration.

Forkhead box O (FoxO) proteins and thyroid hormone (TH) have well established roles in cardiovascular morphogenesis and remodeling. However, specific role(s) of individual FoxO family members in stress-induced growth and remodeling of cardiomyocytes remains unknown. Here, we report that FoxO1, but not FoxO3, activity is essential for reciprocal regulation of types II and III iodothyronine deiodinases (Dio2 and Dio3, respectively), key enzymes involved in intracellular TH metabolism. We further show that Dio2 is a direct transcriptional target of FoxO1, and the FoxO1–Dio2 axis governs TH-induced hypertrophic growth of neonatal cardiomyocytes in vitro and in vivo. Utilizing transverse aortic constriction as a model of hemodynamic stress in wild-type and cardiomyocyte-restricted FoxO1 knockout mice, we unveil an essential role for the FoxO1–Dio2 axis in afterload-induced pathological cardiac remodeling and activation of TRα1. These findings demonstrate a previously unrecognized FoxO1–Dio2 signaling axis in stress-induced cardiomyocyte growth and remodeling and intracellular TH homeostasis., Disease stress-induced cardiac hypertrophy is a major mechanism of pathological cardiac remodeling. Here, the authors unveil a previously unrecognized role of a FoxO1–Dio2 signaling axis in maladaptive, afterload-induced cardiac hypertrophy and intracellular thyroid hormone homeostasis.

The calcium-activated protein phosphatase, calcineurin, lies at the intersection of protein phosphorylation and calcium signaling cascades, where it provides an essential nodal point for coordination between these two fundamental modes of intracellular communication. In excitatory cells, such as neurons and cardiomyocytes, that experience rapid and frequent changes in cytoplasmic calcium, calcineurin protein levels are exceptionally high, suggesting that these cells require high levels of calcineurin activity. Yet, it is widely recognized that excessive activation of calcineurin in the heart contributes to pathological hypertrophic remodeling and the progression to failure. How does a calcium activated enzyme function in the calcium-rich environment of the continuously contracting heart without pathological consequences? This review will discuss the wide range of calcineurin substrates relevant to cardiovascular health and the mechanisms calcineurin uses to find and act on appropriate substrates in the appropriate location while potentially avoiding others. Fundamental differences in calcineurin signaling in neonatal verses adult cardiomyocytes will be addressed as well as the importance of maintaining heterogeneity in calcineurin activity across the myocardium. Finally, we will discuss how circadian oscillations in calcineurin activity may facilitate integration with other essential but conflicting processes, allowing a healthy heart to reap the benefits of calcineurin signaling while avoiding the detrimental consequences of sustained calcineurin activity that can culminate in heart failure.

Increasing non‐shivering thermogenesis (NST), which expends calories as heat rather than storing them as fat, is championed as an effective way to combat obesity and metabolic disease. Innate mechanisms constraining the capacity for NST present a fundamental limitation to this approach, yet are not well understood. Here, we provide evidence that Regulator of Calcineurin 1 (RCAN1), a feedback inhibitor of the calcium‐activated protein phosphatase calcineurin (CN), acts to suppress two distinctly different mechanisms of non‐shivering thermogenesis (NST): one involving the activation of UCP1 expression in white adipose tissue, the other mediated by sarcolipin (SLN) in skeletal muscle. UCP1 generates heat at the expense of reducing ATP production, whereas SLN increases ATP consumption to generate heat. Gene expression profiles demonstrate a high correlation between Rcan1 expression and metabolic syndrome. On an evolutionary timescale, in the context of limited food resources, systemic suppression of prolonged NST by RCAN1 might have been beneficial; however, in the face of caloric abundance, RCAN1‐mediated suppression of these adaptive avenues of energy expenditure may now contribute to the growing epidemic of obesity.

Rationale and Objective: The Regulator of Calcineurin 1 (RCAN1) inhibits calcineurin (CN), a Ca 2+ -activated protein phosphatase important in cardiac remodeling. The hearts and brains of Rcan1 KO mice are more susceptible to damage from ischemia/reperfusion (I/R). As mitochondria are known to be key mediators of I/R damage, the goal of our studies was to determine the impact of the CN/RCAN1 signaling axis on the mitochondrial network and function. Methods and Results: Using both neonatal and isolated adult cardiomyocytes, we found that, the mitochondrial network was more fragmented in the absence of RCAN1, due to increased CN-dependent activation of the fission protein DRP1. RCAN1-deficient cardiomyocytes had reduced mitochondrial membrane potential, consumed less O 2 , and generated lower levels of ROS. Importantly, they also displayed a reduced capacity for mitochondrial Ca 2+ uptake. RCAN1-depleted cardiomyocytes were more sensitive to I/R, however, pharmacological inhibition of CN, DRP1, or calpains (Ca 2+ -activated proteases) restored protection, suggesting that, in the absence of RCAN1, calpain-mediated damage following I/R is greater because of a decrease in the ability of mitochondria to buffer cytoplasmic Ca 2+ . Over-expression of RCAN1 was sufficient to enhance fusion. In addition to its impact on mitochondrial dynamics, CN activation also promoted mitochondrial biogenesis. We demonstrate that expression of the 1b isoform of PGC1α (a master regulator of mitochondrial biogenesis) is directly under CN control . In vivo, a cardiomyocyte-specific CN transgene caused a more rapid increase in: transcript levels for P gc 1α-1b and PolG (the mitochondrial DNA polymerase), mtDNA content, and the abundance of electron transport complex proteins. Despite the increase in mitochondrial content, changes in metabolic enzymes were consistent with a shift toward glycolysis rather than fatty acid oxidation. Conclusions: The CN/RCAN1 signaling axis helps determine both the number of mitochondria (DRP1-dependent fission) and the total mitochondrial mass ( Pgc1α -dependent biogenesis). Maintaining the proper level of these two processes is essential to cardiovascular health and metabolic flexibility.

Angiotensin-(19), a peptide of the non-classical renin angiotensin system, has been shown to prevent and revert hypertension and cardiac hypertrophy. We hypothetized that systemic delivery of angiotensin-(1-9) following myocardial infarction will also be protective and extend to provide protection during reperfusion of the ischemic heart. Adult Sprague Dawley rats were subjected to left anterior descending artery ligation and treated with angiotensin-(1-9) via osmotic mini-pump for 2 weeks in the presence or absence of Mas receptor or AT

Rationale: The regulator of calcineurin 1 (RCAN1) inhibits CN (calcineurin), a Ca 2+ -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. Objective: 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. Methods and Results: 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, O 2 consumption, and generation of reactive oxygen species, as well as a reduced capacity for mitochondrial Ca 2+ uptake. RCAN1-depleted cardiomyocytes were more sensitive to I/R; however, pharmacological inhibition of CN, DRP1, or CAPN (calpains; Ca 2+ -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 Ca 2+ . 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 O 2 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. Conclusions: RCAN1 helps maintain a more interconnected mitochondrial network, and maintaining appropriate RCAN1 levels is important to human health and disease.

Key points Two-pore channels (TPCs) were identified as a novel family of endolysosome-targeted calcium release channels gated by nicotinic acid adenine dinucleotide phosphate, as also as intracellular Na+ channels able to control endolysosomal fusion, a key process in autophagic flux. Autophagy, an evolutionarily ancient response to cellular stress, has been implicated in the pathogenesis of a wide range of cardiovascular pathologies, including heart failure. We report direct evidence indicating that TPCs are involved in regulating autophagy in cardiomyocytes, and that TPC knockout mice show alterations in the cardiac lysosomal system. TPC downregulation implies a decrease in the viability of cardiomyocytes under starvation conditions. In cardiac tissues from both humans and rats, TPC transcripts and protein levels were higher in females than in males, and correlated negatively with markers of autophagy. We conclude that the endolysosomal channels TPC1 and TPC2 are essential for appropriate basal and induced autophagic flux in cardiomyocytes, and also that they are differentially expressed in male and female hearts. Abstract Autophagy participates in physiological and pathological remodelling of the heart. The endolysosomal two-pore channels (TPCs), TPC1 and TPC2, have been implicated in the regulation of autophagy. The present study aimed to investigate the role of TPC1 and TPC2 in basal and induced cardiac autophagic activity. In cultured cardiomyocytes, starvation induced a significant increase in TPC1 and TPC2 transcripts and protein levels that paralleled the increase in autophagy identified by increased LC3-II and decreased p62 levels. Small interfering RNA depletion of TPC2 alone or together with TPC1 increased both LC3II and p62 levels under basal conditions and in response to serum starvation, suggesting that, under conditions of severe energy depletion (serum plus glucose starvation), changes in the autophagic flux (as assessed by use of bafilomycin A1) occurred either when TPC1 or TPC2 were downregulated. The knockdown of TPCs diminished cardiomyocyte viability under starvation and simulated ischaemia. Electron micrographs of hearts from TPC1/2 double knockout mice showed that cardiomyocytes contained large numbers of immature lysosomes with diameters significantly smaller than those of wild-type mice. In cardiac tissues from humans and rats, TPC1 and TPC2 transcripts and protein levels were higher in females than in males. Furthermore, transcript levels of TPCs correlated negatively with p62 levels in heart tissues. TPC1 and TPC2 are essential for appropriate basal and induced autophagic flux in cardiomyocytes (i.e. there is a negative effect on cell viability under stress conditions in their absence) and they are differentially expressed in male and female human and murine hearts, where they correlate with markers of autophagy.

Cardiac hypertrophy is often initiated as an adaptive response to haemodynamic stress or myocardial injury, and allows the heart to meet an increased demand for oxygen. Although initially beneficial, hypertrophy can ultimately contribute to the progression of cardiac disease, leading to an increase in interstitial fibrosis and a decrease in ventricular function. Metabolic changes have emerged as key mechanisms involved in the development and progression of pathological remodelling. As the myocardium is a highly oxidative tissue, mitochondria play a central role in maintaining optimal performance of the heart. 'Mitochondrial dynamics', the processes of mitochondrial fusion, fission, biogenesis and mitophagy that determine mitochondrial morphology, quality and abundance have recently been implicated in cardiovascular disease. Studies link mitochondrial dynamics to the balance between energy demand and nutrient supply, suggesting that changes in mitochondrial morphology may act as a mechanism for bioenergetic adaptation during cardiac pathological remodelling. Another critical function of mitochondrial dynamics is the removal of damaged and dysfunctional mitochondria through mitophagy, which is dependent on the fission/fusion cycle. In this article, we discuss the latest findings regarding the impact of mitochondrial dynamics and mitophagy on the development and progression of cardiovascular pathologies, including diabetic cardiomyopathy, atherosclerosis, damage from ischaemia-reperfusion, cardiac hypertrophy and decompensated heart failure. We will address the ability of mitochondrial fusion and fission to impact all cell types within the myocardium, including cardiac myocytes, cardiac fibroblasts and vascular smooth muscle cells. Finally, we will discuss how these findings can be applied to improve the treatment and prevention of cardiovascular diseases.

Adipose-derived stem cells (ADSCs) have myocardial regeneration potential, and transplantation of these cells following myocardial infarction (MI) in animal models leads to modest improvements in cardiac function. We hypothesized that pharmacological priming of pre-transplanted ADSCs would further improve left ventricular functional recovery after MI. We previously identified a compound from a family of 3,5-disubstituted isoxazoles, ISX1, capable of activating an Nkx2-5-driven promoter construct. Here, using ADSCs, we found that ISX1 (20 mM, 4 days) triggered a robust, dose-dependent, fourfold increase in Nkx2-5 expression, an early marker of cardiac myocyte differentiation and increased ADSC viability in vitro. Co-culturing neonatal cardiomyocytes with ISX1-treated ADSCs increased early and late cardiac gene expression. Whereas ISX1 promoted ADSC differentiation toward a cardiogenic lineage, it did not elicit their complete differentiation or their differentiation into mature adipocytes, osteoblasts, or chondrocytes, suggesting that re-programming is cardiomyocyte specific. Cardiac transplantation of ADSCs improved left ventricular functional recovery following MI, a response which was significantly augmented by transplantation of ISX1- pretreated cells. Moreover, ISX1-treated and transplanted ADSCs engrafted and were detectable in the myocardium 3 weeks following MI, albeit at relatively small numbers. ISX1 treatment increased histone acetyltransferase (HAT) activity in ADSCs, which was associated with histone 3 and histone 4 acetylation. Finally, hearts transplanted with ISX1-treated ADSCs manifested significant increases in neovascularization, which may account for the improved cardiac function. These findings suggest that a strategy of drug-facilitated initiation of myocyte differentiation enhances exogenously transplanted ADSC persistence in vivo, and consequent tissue neovascularization, to improve cardiac function.

Eukaryotic cells contain a variety of subcellular organelles, each of which performs unique tasks. Thus follows that in order to coordinate these different intracellular functions, a highly dynamic system of communication must exist between the various compartments. Direct endoplasmic reticulum (ER)–mitochondria communication is facilitated by the physical interaction of their membranes in dedicated structural domains known as mitochondria-associated membranes (MAMs), which facilitate calcium (Ca2+) and lipid transfer between organelles and also act as platforms for signaling. Numerous studies have demonstrated the importance of MAM in ensuring correct function of both organelles, and recently MAMs have been implicated in the genesis of various human diseases. Here, we review the salient structural features of interorganellar communication via MAM and discuss the most common experimental techniques employed to assess functionality of these domains. Finally, we will highlight the contribution of MAM to a variety of cellular functions and consider the potential role of MAM in the genesis of metabolic diseases. In doing so, the importance for cell functions of maintaining appropriate communication between ER and mitochondria will be emphasized.

Diabetic cardiomyopathy (DCM) is a common consequence of longstanding type 2 diabetes mellitus (T2DM) and encompasses structural, morphological, functional, and metabolic abnormalities in the heart. Myocardial energy metabolism depends on mitochondria, which must generate sufficient ATP to meet the high energy demands of the myocardium. Dysfunctional mitochondria are involved in the pathophysiology of diabetic heart disease. A large body of evidence implicates myocardial insulin resistance in the pathogenesis of DCM. Recent studies show that insulin signaling influences myocardial energy metabolism by impacting cardiomyocyte mitochondrial dynamics and function under physiological conditions. However, comprehensive understanding of molecular mechanisms linking insulin signaling and changes in the architecture of the mitochondrial network in diabetic cardiomyopathy is lacking. This review summarizes our current understanding of how defective insulin signaling impacts cardiac function in diabetic cardiomyopathy and discusses the potential role of mitochondrial dynamics.

Close contacts between endoplasmic reticulum and mitochondria enable reciprocal Ca2+ exchange, a key mechanism in the regulation of mitochondrial bioenergetics. During the early phase of endoplasmic reticulum stress, this inter-organellar communication increases as an adaptive mechanism to ensure cell survival. The signalling pathways governing this response, however, have not been characterized. Here we show that caveolin-1 localizes to the endoplasmic reticulum–mitochondria interface, where it impairs the remodelling of endoplasmic reticulum–mitochondria contacts, quenching Ca2+ transfer and rendering mitochondrial bioenergetics unresponsive to endoplasmic reticulum stress. Protein kinase A, in contrast, promotes endoplasmic reticulum and mitochondria remodelling and communication during endoplasmic reticulum stress to promote organelle dynamics and Ca2+ transfer as well as enhance mitochondrial bioenergetics during the adaptive response. Importantly, caveolin-1 expression reduces protein kinase A signalling, as evidenced by impaired phosphorylation and alterations in organelle distribution of the GTPase dynamin-related protein 1, thereby enhancing cell death in response to endoplasmic reticulum stress. In conclusion, caveolin-1 precludes stress-induced protein kinase A-dependent remodelling of endoplasmic reticulum–mitochondria communication.

Cardiovascular disease is the leading cause of death worldwide. As such, there is great interest in identifying novel mechanisms that govern the cardiovascular response to disease-related stress. First described in failing hearts, autophagy within the cardiovascular system has been widely characterized in cardiomyocytes, cardiac fibroblasts, endothelial cells, vascular smooth muscle cells, and macrophages. In all cases, a window of optimal autophagic activity appears to be critical to the maintenance of cardiovascular homeostasis and function; excessive or insufficient levels of autophagic flux can each contribute to heart disease pathogenesis. In this Review, we discuss the potential for targeting autophagy therapeutically and our vision for where this exciting biology may lead in the future.

The heart must function continuously as it is responsible for both supplying oxygen and nutrients throughout the entire body, as well as for the transport of waste products to excretory organs. When facing either a physiological or pathological increase in cardiac demand, the heart undergoes structural and functional remodeling as a means of adapting to increased workload. These adaptive responses can include changes in gene expression, protein composition, and structure of sub-cellular organelles involved in energy production and metabolism. Mitochondria are essential for cardiac function, as they supply the ATP necessary to support continuous cycles of contraction and relaxation. In addition, mitochondria carry out other important processes, including synthesis of essential cellular components, calcium buffering, and initiation of cell death signals. Not surprisingly, mitochondrial dysfunction has been linked to several cardiovascular disorders, including hypertension, cardiac hypertrophy, ischemia/reperfusion and heart failure. The present chapter will discuss how changes in mitochondrial cristae structure, fusion/fission dynamics, fatty acid oxidation, ATP production, and the generation of reactive oxygen species might impact cardiac structure and function, particularly in the context of pathological hypertrophy and fibrotic response. In addition, the mechanistic role of mitochondria in autophagy and programmed cell death of cardiomyocytes will be addressed. Here we will also review strategies to improve mitochondrial function and discuss their cardioprotective potential.

The mammalian heart has a remarkable regenerative capacity for a short period of time after birth, after which the majority of cardiomyocytes permanently exit cell cycle. We sought to determine the primary post-natal event that results in cardiomyocyte cell-cycle arrest. We hypothesized that transition to the oxygen rich postnatal environment is the upstream signal that results in cell cycle arrest of cardiomyocytes. Here we show that reactive oxygen species (ROS), oxidative DNA damage, and DNA damage response (DDR) markers significantly increase in the heart during the first postnatal week. Intriguingly, postnatal hypoxemia, ROS scavenging, or inhibition of DDR all prolong the postnatal proliferative window of cardiomyocytes, while hyperoxemia and ROS generators shorten it. These findings uncover a previously unrecognized protective mechanism that mediates cardiomyocyte cell cycle arrest in exchange for utilization of oxygen dependent aerobic metabolism. Reduction of mitochondrial-dependent oxidative stress should be important component of cardiomyocyte proliferation-based therapeutic approaches.

SummaryThe 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.

Insulin regulates heart metabolism through the regulation of insulin-stimulated glucose uptake. Studies have indicated that insulin can also regulate mitochondrial function. Relevant to this idea, mitochondrial function is impaired in diabetic individuals. Furthermore, the expression of Opa-1 and mitofusins, proteins of the mitochondrial fusion machinery, is dramatically altered in obese and insulin-resistant patients. Given the role of insulin in the control of cardiac energetics, the goal of this study was to investigate whether insulin affects mitochondrial dynamics in cardiomyocytes. Confocal microscopy and the mitochondrial dye MitoTracker Green were used to obtain three-dimensional images of the mitochondrial network in cardiomyocytes and L6 skeletal muscle cells in culture. Three hours of insulin treatment increased Opa-1 protein levels, promoted mitochondrial fusion, increased mitochondrial membrane potential, and elevated both intracellular ATP levels and oxygen consumption in cardiomyocytes in vitro and in vivo. Consequently, the silencing of Opa-1 or Mfn2 prevented all the metabolic effects triggered by insulin. We also provide evidence indicating that insulin increases mitochondrial function in cardiomyocytes through the Akt-mTOR-NFκB signaling pathway. These data demonstrate for the first time in our knowledge that insulin acutely regulates mitochondrial metabolism in cardiomyocytes through a mechanism that depends on increased mitochondrial fusion, Opa-1, and the Akt-mTOR-NFκB pathway.

The heart must function continuously as it is responsible for both supplying oxygen and nutrients throughout the entire body, as well as for the transport of waste products to excretory organs. When facing either a physiological or pathological increase in cardiac demand, the heart undergoes structural and functional remodeling as a means of adapting to increased workload. These adaptive responses can include changes in gene expression, protein composition, and structure of sub-cellular organelles involved in energy production and metabolism. Mitochondria are essential for cardiac function, as they supply the ATP necessary to support continuous cycles of contraction and relaxation. In addition, mitochondria carry out other important processes, including synthesis of essential cellular components, calcium buffering, and initiation of cell death signals. Not surprisingly, mitochondrial dysfunction has been linked to several cardiovascular disorders, including hypertension, cardiac hypertrophy, ischemia/reperfusion and heart failure. The present chapter will discuss how changes in mitochondrial cristae structure, fusion/fission dynamics, fatty acid oxidation, ATP production, and the generation of reactive oxygen species might impact cardiac structure and function, particularly in the context of pathological hypertrophy and fibrotic response. In addition, the mechanistic role of mitochondria in autophagy and programmed cell death of cardiomyocytes will be addressed. Here we will also review strategies to improve mitochondrial function and discuss their cardioprotective potential.

We recently identified a brief time period during postnatal development when the mammalian heart retains significant regenerative potential after amputation of the ventricular apex. However, one major unresolved question is whether the neonatal mouse heart can also regenerate in response to myocardial ischemia, the most common antecedent of heart failure in humans. Here, we induced ischemic myocardial infarction (MI) in 1-d-old mice and found that this results in extensive myocardial necrosis and systolic dysfunction. Remarkably, the neonatal heart mounted a robust regenerative response, through proliferation of preexisting cardiomyocytes, resulting in full functional recovery within 21 d. Moreover, we show that the miR-15 family of microRNAs modulates neonatal heart regeneration through inhibition of postnatal cardiomyocyte proliferation. Finally, we demonstrate that inhibition of the miR-15 family from an early postnatal age until adulthood increases myocyte proliferation in the adult heart and improves left ventricular systolic function after adult MI. We conclude that the neonatal mammalian heart can regenerate after myocardial infarction through proliferation of preexisting cardiomyocytes and that the miR-15 family contributes to postnatal loss of cardiac regenerative capacity.

Stress-induced hypertrophic growth of the heart predisposes the heart to arrhythmia, contractile dysfunction, and clinical heart failure. FHL2 (four-and-a-half LIM domain protein 2) is expressed predominantly in the heart, and inactivation of the gene coding for FHL2 leads to exaggerated responsiveness to adrenergic stress. Activation of calcineurin occurs downstream of β-adrenergic signaling and is required for isoproterenol-induced myocardial hypertrophy. Based on these facts, we hypothesized that FHL2 suppresses stress-induced activation of calcineurin. FHL2 is upregulated in mouse hearts exposed to isoproterenol, a β-adrenergic agonist, and isoproterenol-induced increases in the NFAT target genes RCAN1.4 and BNP were amplified significantly in FHL2 knockout (FHL2(-/-)) mice compared with levels in wild-type (WT) mice. To determine whether the effect of FHL2 on NFAT target gene transcript levels occurred at the level of transcription, HEK 293 cells and neonatal rat ventricular myocytes (NRVMs) were transfected with a luciferase reporter construct harboring the NFAT-dependent promoters of either RCAN1 or interleukin 2 (IL-2). Consistent with the in vivo data, small interfering RNA (siRNA) knockdown of FHL2 led to increased activation of these promoters by constitutively active calcineurin or the calcium ionophore ionomycin. Importantly, activation of the RCAN1 promoter by ionomycin, in control and FHL2 knockdown cells, was abolished by the calcineurin inhibitor cyclosporine, confirming the calcineurin dependence of the response. Overexpression of FHL2 inhibited activation of both NFAT reporter constructs. Furthermore, NRVMs overexpressing FHL2 exhibited reduced hypertrophic growth in response to constitutively active calcineurin, as measured by cell cross-sectional area and fetal gene expression. Finally, immunostaining in isolated adult cardiomyocytes revealed colocalization of FHL2 and calcineurin predominantly at the sarcomere and activation of calcineurin by endothelin-1-facilitated interaction between FHL2 and calcineurin. FHL2 is an endogenous, agonist-dependent suppressor of calcineurin.

Alterations in intracellular Ca 2+ homeostasis are an important trigger of pathological cardiac remodeling; however, mechanisms governing context-dependent changes in Ca 2+ influx are poorly understood. Store-operated Ca 2+ entry (SOCE) is a major mechanism regulating Ca 2+ trafficking in numerous cell types, yet its prevalence in adult heart and possible role in physiology and disease are each unknown. The Ca 2+ -binding protein, stromal interaction molecule 1 (STIM1), is a Ca 2+ sensor in the sarcoplasmic reticulum (SR), capable of triggering SOCE by interacting with plasma membrane Ca 2+ channels. We report that SOCE is abundant and robust in neonatal cardiomyocytes; however, SOCE is absent from adult cardiomyocytes. Levels of STIM1 transcript and protein correlate with the amplitude of SOCE, and manipulation of STIM1 protein levels (via shRNA) or activity (via expression of constitutively active or dominant-negative mutants) reveals a critical role for STIM1 in activating SOCE in cardiac myocytes. In neonatal hearts a recently identified STIM1 splice variant (STIM1L) is predominant but diminishes with maturation, only to reemerge with agonist- or afterload-induced cardiac stress. To test for pathophysiological relevance, we evaluated both in vitro and in vivo models of cardiac hypertrophy, finding that STIM1 expression is re-activated by pathological stress to trigger significant SOCE-dependent Ca 2+ influx. STIM1 amplifies agonist-induced hypertrophy via activation of the calcineurin–NFAT pathway. Importantly, inhibition of STIM1 suppresses agonist-triggered hypertrophy, pointing to a requirement for SOCE in this remodeling response. Stress-triggered STIM1 re-expression, and consequent SOCE activation, are critical elements in the upstream, Ca 2+ -dependent control of pathological cardiac hypertrophy.

Following publication of the article, the authors wrote to point out the following mistake

Mitochondria are highly dynamic organelles, capable of undergoing constant fission and fusion events, forming networks. These dynamic events allow the transmission of chemical and physical messengers and the exchange of metabolites within the cell. In this article we review the signaling mechanisms controlling mitochondrial fission and fusion, and its relationship with cell bioenergetics, especially in the heart. Furthermore we also discuss how defects in mitochondrial dynamics might be involved in the pathogenesis of metabolic cardiac diseases.

In response to changes in workload, the heart grows or shrinks. Indeed, the myocardium is capable of robust and rapid structural remodeling. In the setting of normal, physiological demand, the heart responds with hypertrophic growth of individual cardiac myocytes, a process that serves to maintain cardiac output and minimize wall stress. However, disease-related stresses, such as hypertension or myocardial infarction, provoke a series of changes that culminate in heart failure and/or sudden death. At the other end of the spectrum, cardiac unloading, such as occurs with prolonged bed rest or weightlessness, causes the heart to shrink. In recent years, considerable strides have been made in deciphering the molecular and cellular events governing pro- and anti-growth events in the heart. Prominent among these mechanisms are those mediated by FoxO (Forkhead box-containing protein, O subfamily) transcription factors. In many cell types, these proteins are critical regulators of cell size, viability, and metabolism, and their importance in the heart is just emerging. Also in recent years, evidence has emerged for a pivotal role for autophagy, an evolutionarily conserved pathway of lysosomal degradation of damaged proteins and organelles, in cardiac growth and remodeling. Indeed, evidence for activated autophagy has been detected in virtually every form of myocardial disease. Now, it is clear that FoxO is an upstream regulator of both autophagy and the ubiquitin-proteasome system. Here, we discuss recent advances in our understanding of cardiomyocyte autophagy, its governance by FoxO, and the roles each of these plays in cardiac remodeling.

In response to hypertension, the heart manifests robust hypertrophic growth, which offsets load-induced elevations in wall stress. If sustained, this hypertrophic response is a major risk factor for systolic dysfunction and heart failure. Extensive research efforts have focused on the progression from hypertrophy to failure; however, precise understanding of underlying mechanisms remains elusive. Recently, autophagy, a process of cellular cannibalization, has been implicated. Autophagy is activated during ventricular hypertrophy, serving to maintain cellular homeostasis. Excessive autophagy eliminates, however, essential cellular elements and possibly provokes cell death, which together contribute to hypertension-related heart disease.

Cardiac hypertrophy develops in response to a variety of cardiovascular stresses and results in activation of numerous signaling cascades and proteins. In the present study, we demonstrate that cytoglobin is a stress-responsive hemoprotein in the hypoxia-induced hypertrophic myocardium and it is transcriptionally regulated by calcineurin-dependent transcription factors. The cytoglobin transcript level is abundantly expressed in the adult heart and in response to hypoxia cytoglobin expression is markedly up-regulated within the hypoxia-induced hypertrophic heart. To define the molecular mechanism resulting in the induction of cytoglobin, we undertook a transcriptional analysis of the 5′ upstream regulatory region of the cytoglobin gene. Evolutionarily conserved binding elements for transcription factors HIF-1, AP-1, and NFAT are located within the upstream region of the cytoglobin gene. Transcriptional assays demonstrated that calcineurin activity modulates cytoglobin transcription. Increased calcineurin activity enhances the ability of NFAT and AP-1 to bind to the putative cytoglobin promoter, especially under hypoxic conditions. In addition, inhibition of calcineurin, NFAT, and/or AP-1 activities decreases endogenous cytoglobin transcript and protein levels. Thus, the regulation of cytoglobin transcription by calcineurin-dependent transcription factors suggests that cytoglobin may have a functional role in calcium-dependent events accompanying cardiac remodeling.

The heart is a highly plastic organ capable of remodeling in response to changes in physiological or pathological demand. For example, when workload increases, compensatory hypertrophic growth of individual cardiomyocytes occurs to increase cardiac output. Sustained stress, however, such as that occurring with hypertension or following myocardial infarction, triggers changes in energy metabolism and sarcomeric protein composition, loss of cardiomyocytes, ventricular dilation, reduced pump function, and ultimately heart failure. It has been known for some time that autophagy is active in cardiomyocytes, occurring at increased levels in disease. Now, with recent advances in our understanding of molecular mechanisms governing autophagy, the potential contributions of cardiomyocyte autophagy to ventricular remodeling and disease pathogenesis are being explored. As part of this work, several recent studies have focused on autophagy in heart disease elicited by changes in hemodynamic load. Pressure overload stress elicits a robust autophagic response in cardiomyocytes that is maladaptive, contributing to disease progression. In this context, load-induced aggregation of intracellular proteins is a proximal event triggering autophagic clearance mechanisms. These findings in the setting of pressure overload contrast with protein aggregation occurring in a model of protein chaperone malfunction, where activation of autophagy is beneficial, antagonizing disease progression. Here, we review recent studies of cardiomyocyte autophagy in load-induced disease and address molecular mechanisms and unanswered questions.

Background— Recent reports demonstrate that multiple forms of cardiovascular stress, including pressure overload, chronic ischemia, and infarction-reperfusion injury, provoke an increase in autophagic activity in cardiomyocytes. However, nothing is known regarding molecular events that stimulate autophagic activity in stressed myocardium. Because autophagy is a highly conserved process through which damaged proteins and organelles can be degraded, we hypothesized that stress-induced protein aggregation is a proximal trigger of cardiomyocyte autophagy. Methods and Results— Here, we report that pressure overload promotes accumulation of ubiquitinated protein aggregates in the left ventricle, development of aggresome-like structures, and a corresponding induction of autophagy. To test for causal links, we induced protein accumulation in cultured cardiomyocytes by inhibiting proteasome activity, finding that aggregation of polyubiquitinated proteins was sufficient to induce cardiomyocyte autophagy. Furthermore, attenuation of autophagic activity dramatically enhanced both aggresome size and abundance, consistent with a role for autophagic activity in protein aggregate clearance. Conclusions— We conclude that protein aggregation is a proximal trigger of cardiomyocyte autophagy and that autophagic activity functions to attenuate aggregate/aggresome formation in heart. Findings reported here are the first to demonstrate that protein aggregation occurs in response to hemodynamic stress, situating pressure-overload heart disease in the category of proteinopathies.

Recent work has demonstrated the importance of chromatin remodeling, especially histone acetylation, in the control of gene expression in the heart. Studies in preclinical models suggest that inhibition of histone deacetylase (HDAC) activity - using compounds that show promise in ongoing oncology trials - blunts pathologic growth of cardiac myocytes. Indeed, small-molecule inhibitors of HDACs are members of an evolving class of pharmacologic agents in development for the treatment of several diseases. If proved effective in the treatment of heart disease, HDAC inhibitors could have a significant impact on public health, as cardiovascular disease remains the leading cause of death in the US. This paper reviews understanding of the mechanisms of action of HDAC inhibitors in the heart and summarizes emerging data regarding their effects on disease-related cardiac remodeling and function.

Altering chromatin structure through histone posttranslational modifications has emerged as a key driver of transcriptional responses in cells. Modulation of these transcriptional responses by pharmacological inhibition of class I histone deacetylases (HDACs), a group of chromatin remodeling enzymes, has been successful in blocking the growth of some cancer cell types. These inhibitors also attenuate the pathogenesis of pathological cardiac remodeling by blunting and even reversing pathological hypertrophy. The mechanistic target of rapamycin (mTOR) is a critical sensor and regulator of cell growth that, as part of mTOR complex 1 (mTORC1), drives changes in protein synthesis and metabolism in both pathological and physiological hypertrophy. We demonstrated through pharmacological and genetic methods that inhibition of class I HDACs suppressed pathological cardiac hypertrophy through inhibition of mTOR activity. Mice genetically silenced for HDAC1 and HDAC2 had a reduced hypertrophic response to thoracic aortic constriction (TAC) and showed reduced mTOR activity. We determined that the abundance of tuberous sclerosis complex 2 (TSC2), an mTOR inhibitor, was increased through a transcriptional mechanism in cardiomyocytes when class I HDACs were inhibited. In neonatal rat cardiomyocytes, loss of TSC2 abolished HDAC-dependent inhibition of mTOR activity, and increased expression of TSC2 was sufficient to reduce hypertrophy in response to phenylephrine. These findings point to mTOR and TSC2-dependent control of mTOR as critical components of the mechanism by which HDAC inhibitors blunt pathological cardiac growth. These results also suggest a strategy to modulate mTOR activity and facilitate the translational exploitation of HDAC inhibitors in heart disease.

Efficient mitochondrial Ca2+ uptake takes place at contact points between the ER and mitochondria, and represents a key regulator of many cell functions. In a previous study with HeLa cells, we showed that ER-to-mitochondria Ca2+ transfer increases during the early phase of ER stress induced by tunicamycin as an adaptive response to stimulate mitochondrial bioenergetics. It remains unknown whether other types of stress signals trigger similar responses. Here we observed that rapamycin, which inhibits the nutrient-sensing complex mTORC1, increased ER-mitochondria coupling in HeLa cells to a similar extent as did tunicamycin. Interestingly, although global responses to both stressors were comparable, there were notable differences in the spatial distribution of such changes. While tunicamycin increased organelle proximity primarily in the perinuclear region, rapamycin increased organelle contacts throughout the entire cell. These differences were paralleled by dissimilar alterations in the distribution of regulatory proteins of the ER-mitochondria interface, heterogeneities in mitochondrial Ca2+ uptake, and the formation of domains within the mitochondrial network with varying mitochondrial transmembrane potential. Collectively, these data suggest that while increasing ER-mitochondria coupling appears to represent a general response to cell stress, the intracellular distribution of the associated responses needs to be tailored to meet specific cellular requirements.

The prevalence of heart failure is increasing rapidly worldwide, and yet effective treatments remain elusive. Pathological remodeling of the ventricle – and associated hypertrophic growth, fibrotic change, cavity dilatation and electrophysiological remodeling – is a significant contributor to the pathogenesis of this prevalent disorder. As a consequence, there is great interest in developing new therapies to target pathological remodeling of the heart with the intent to prevent, arrest, or possibly reverse the otherwise inexorable progression of disease. To tackle this problem, numerous models of disease have been developed, both in vivo and in vitro. Here, we review models of cardiac hypertrophy and failure, compare and contrast their strengths and limitations, and on occasion cite recent works where the use of these models has contributed to significant scientific advances.