Antisense Oligonucleotide Therapy for Calmodulinopathy.

BACKGROUND: Calmodulinopathies are rare inherited arrhythmia syndromes caused by dominant heterozygous variants in CALM1, CALM2, or CALM3, which each encode the identical CaM (calmodulin) protein. We hypothesized that antisense oligonucleotide (ASO)-mediated depletion of an affected calmodulin gene would ameliorate disease manifestations, whereas the other 2 calmodulin genes would preserve CaM level and function. METHODS: We tested this hypothesis using human induced pluripotent stem cell-derived cardiomyocyte and mouse models of CALM1 pathogenic variants. RESULTS: Human CALM1F142L/+ induced pluripotent stem cell-derived cardiomyocytes exhibited prolonged action potentials, modeling congenital long QT syndrome. CALM1 knockout or CALM1-depleting ASOs did not alter CaM protein level and normalized repolarization duration of CALM1F142L/+ induced pluripotent stem cell-derived cardiomyocytes. Similarly, an ASO targeting murine Calm1 depleted Calm1 transcript without affecting CaM protein level. This ASO alleviated drug-induced bidirectional ventricular tachycardia in CalmN98S/+ mice without a deleterious effect on cardiac electrical or contractile function. CONCLUSIONS: These results provide proof of concept that ASOs targeting individual calmodulin genes are potentially effective and safe therapies for calmodulinopathies.

Loss of MCL-1 leads to impaired autophagy and rapid development of heart failure.

Myeloid cell leukemia-1 (MCL-1) is an anti-apoptotic BCL-2 protein that is up-regulated in several human cancers. MCL-1 is also highly expressed in myocardium, but its function in myocytes has not been investigated. We generated inducible, cardiomyocyte-specific Mcl-1 knockout mice and found that ablation of Mcl-1 in the adult heart led to rapid cardiomyopathy and death. Although MCL-1 is known to inhibit apoptosis, this process was not activated in MCL-1-deficient hearts. Ultrastructural analysis revealed disorganized sarcomeres and swollen mitochondria in myocytes. Mitochondria isolated from MCL-1-deficient hearts exhibited reduced respiration and limited Ca(2+)-mediated swelling, consistent with opening of the mitochondrial permeability transition pore (mPTP). Double-knockout mice lacking MCL-1 and cyclophilin D, an essential regulator of the mPTP, exhibited delayed progression to heart failure and extended survival. Autophagy is normally induced by myocardial stress, but induction of autophagy was impaired in MCL-1-deficient hearts. These data demonstrate that MCL-1 is essential for mitochondrial homeostasis and induction of autophagy in the heart. This study also raises concerns about potential cardiotoxicity for chemotherapeutics that target MCL-1.

Hypoxia Prevents Mitochondrial Dysfunction and Senescence in Human c-Kit+ Cardiac Progenitor Cells.

Senescence-associated dysfunction deleteriously affects biological activities of human c-Kit+ cardiac progenitor cells (hCPCs), particularly under conditions of in vitro culture. In comparison, preservation of self-renewal and decreases in mitochondrial reactive oxygen species (ROS) are characteristics of murine CPCs in vivo that reside within hypoxic niches. Recapitulating hypoxic niche oxygen tension conditions of ∼1% O2 in vitro for expansion of hCPCs rather than typical normoxic cell culture conditions (21% O2 ) could provide significant improvement of functional and biological activities of hCPCs. hCPCs were isolated and expanded under permanent hypoxic (hCPC-1%) or normoxic (hCPC-21%) conditions from left ventricular tissue explants collected during left ventricular assist device implantation. hCPC-1% exhibit increased self-renewal and suppression of senescence characteristics relative to hCPC-21%. Oxidative stress contributed to higher susceptibility to apoptosis, as well as decreased mitochondrial function in hCPC-21%. Hypoxia prevented accumulation of dysfunctional mitochondria, supporting higher oxygen consumption rates and mitochondrial membrane potential. Mitochondrial ROS was an upstream mediator of senescence since treatment of hCPC-1% with mitochondrial inhibitor antimycin A recapitulated mitochondrial dysfunction and senescence observed in hCPC-21%. NAD+ /NADH ratio and autophagic flux, which are key factors for mitochondrial function, were higher in hCPC-1%, but hCPC-21% were highly dependent on BNIP3/NIX-mediated mitophagy to maintain mitochondrial function. Overall, results demonstrate that supraphysiological oxygen tension during in vitro expansion initiates a downward spiral of oxidative stress, mitochondrial dysfunction, and cellular energy imbalance culminating in early proliferation arrest of hCPCs. Senescence is inhibited by preventing ROS through hypoxic culture of hCPCs. Stem Cells 2019;37:555-567.

Accumulation of Mitochondrial DNA Mutations Disrupts Cardiac Progenitor Cell Function and Reduces Survival.

Transfer of cardiac progenitor cells (CPCs) improves cardiac function in heart failure patients. However, CPC function is reduced with age, limiting their regenerative potential. Aging is associated with numerous changes in cells including accumulation of mitochondrial DNA (mtDNA) mutations, but it is unknown how this impacts CPC function. Here, we demonstrate that acquisition of mtDNA mutations disrupts mitochondrial function, enhances mitophagy, and reduces the replicative and regenerative capacities of the CPCs. We show that activation of differentiation in CPCs is associated with expansion of the mitochondrial network and increased mitochondrial oxidative phosphorylation. Interestingly, mutant CPCs are deficient in mitochondrial respiration and rely on glycolysis for energy. In response to differentiation, these cells fail to activate mitochondrial respiration. This inability to meet the increased energy demand leads to activation of cell death. These findings demonstrate the consequences of accumulating mtDNA mutations and the importance of mtDNA integrity in CPC homeostasis and regenerative potential.

Metabolic dysfunction consistent with premature aging results from deletion of Pim kinases.

RATIONALE: The senescent cardiac phenotype is accompanied by changes in mitochondrial function and biogenesis causing impairment in energy provision. The relationship between myocardial senescence and Pim kinases deserves attention because Pim-1 kinase is cardioprotective, in part, by preservation of mitochondrial integrity. Study of the pathological effects resulting from genetic deletion of all Pim kinase family members could provide important insight about cardiac mitochondrial biology and the aging phenotype. OBJECTIVE: To demonstrate that myocardial senescence is promoted by loss of Pim leading to premature aging and aberrant mitochondrial function. METHODS AND RESULTS: Cardiac myocyte senescence was evident at 3 months in Pim triple knockout mice, where all 3 isoforms of Pim kinase family members are genetically deleted. Cellular hypertrophic remodeling and fetal gene program activation were followed by heart failure at 6 months in Pim triple knockout mice. Metabolic dysfunction is an underlying cause of cardiac senescence and instigates a decline in cardiac function. Altered mitochondrial morphology is evident consequential to Pim deletion together with decreased ATP levels and increased phosphorylated AMP-activated protein kinase, exposing an energy deficiency in Pim triple knockout mice. Expression of the genes encoding master regulators of mitochondrial biogenesis, PPARγ (peroxisome proliferator-activated receptor gamma) coactivator-1 α and ß, was diminished in Pim triple knockout hearts, as were downstream targets included in mitochondrial energy transduction, including fatty acid oxidation. Reversal of the dysregulated metabolic phenotype was observed by overexpressing c-Myc (Myc proto-oncogene protein), a downstream target of Pim kinases. CONCLUSIONS: Pim kinases prevent premature cardiac aging and maintain a healthy pool of functional mitochondria leading to efficient cellular energetics.

Cellular redox status determines sensitivity to BNIP3-mediated cell death in cardiac myocytes.

The atypical BH3-only protein Bcl-2/adenovirus E1B 19-kDa interacting protein 3 (BNIP3) is an important regulator of hypoxia-mediated cell death. Interestingly, the susceptibility to BNIP3-mediated cell death differs between cells. In this study we examined whether there are mechanistic differences in BNIP3-mediated cell death between neonatal and adult cardiac myocytes. We discovered that BNIP3 is a potent inducer of cell death in neonatal myocytes, whereas adult myocytes are remarkably resistant to BNIP3. When exploring the potential underlying basis for the resistance, we discovered that adult myocytes express significantly higher levels of the mitochondrial antioxidant manganese superoxide dismutase (MnSOD) than neonatal myocytes. Overexpression of MnSOD confers resistance to BNIP3-mediated cell death in neonatal myocytes. In contrast, the presence of a pharmacological MnSOD inhibitor, 2-methoxyestradiol, results in increased sensitivity to BNIP3-mediated cell death in adult myocytes. Cotreatment with the mitochondria-targeted antioxidant MitoTEMPO or the MnSOD mimetic manganese (III) tetrakis (4-benzoic acid) porphyrin chloride abrogates the increased cell death by 2-methoxyestradiol. Moreover, increased oxidative stress also restores the ability of BNIP3 to induce cell death in adult myocytes. Taken together, these data indicate that redox status determines cell susceptibility to BNIP3-mediated cell death. These findings are clinically relevant, given that pediatric hearts are known to be more vulnerable than the adult heart to ischemic injury. Our studies provide important insight into why pediatric hearts are more sensitive to ischemic injury and may help in the clinical management of childhood heart disease.

Parkin protein deficiency exacerbates cardiac injury and reduces survival following myocardial infarction.

It is known that loss-of-function mutations in the gene encoding Parkin lead to development of Parkinson disease. Recently, Parkin was found to play an important role in the removal of dysfunctional mitochondria via autophagy in neurons. Although Parkin is expressed in the heart, its functional role in this tissue is largely unexplored. In this study, we have investigated the role of Parkin in the myocardium under normal physiological conditions and in response to myocardial infarction. We found that Parkin-deficient (Parkin(-/-)) mice had normal cardiac function for up to 12 months of age as determined by echocardiographic analysis. Although ultrastructural analysis revealed that Parkin-deficient hearts had disorganized mitochondrial networks and significantly smaller mitochondria, mitochondrial function was unaffected. However, Parkin(-/-) mice were much more sensitive to myocardial infarction when compared with wild type mice. Parkin(-/-) mice had reduced survival and developed larger infarcts when compared with wild type mice after the infarction. Interestingly, Parkin protein levels and mitochondrial autophagy (mitophagy) were rapidly increased in the border zone of the infarct in wild type mice. In contrast, Parkin(-/-) myocytes had reduced mitophagy and accumulated swollen, dysfunctional mitochondria after the infarction. Overexpression of Parkin in isolated cardiac myocytes also protected against hypoxia-mediated cell death, whereas nonfunctional Parkinson disease-associated mutants ParkinR42P and ParkinG430D had no effect. Our results suggest that Parkin plays a critical role in adapting to stress in the myocardium by promoting removal of damaged mitochondria.

Mitochondria and mitophagy: the yin and yang of cell death control.

Mitochondria are primarily responsible for providing the contracting cardiac myocyte with a continuous supply of ATP. However, mitochondria can rapidly change into death-promoting organelles. In response to changes in the intracellular environment, mitochondria become producers of excessive reactive oxygen species and release prodeath proteins, resulting in disrupted ATP synthesis and activation of cell death pathways. Interestingly, cells have developed a defense mechanism against aberrant mitochondria that can cause harm to the cell. This mechanism involves selective sequestration and subsequent degradation of the dysfunctional mitochondrion before it causes activation of cell death. Induction of mitochondrial autophagy, or mitophagy, results in selective clearance of damaged mitochondria in cells. In response to stress such as ischemia/reperfusion, prosurvival and prodeath pathways are concomitantly activated in cardiac myocytes. Thus, there is a delicate balance between life and death in the myocytes during stress, and the final outcome depends on the complex cross-talk between these pathways. Mitophagy functions as an early cardioprotective response, favoring adaptation to stress by removing damaged mitochondria. In contrast, increased oxidative stress and apoptotic proteases can inactivate mitophagy, allowing for the execution of cell death. Herein, we discuss the importance of mitochondria and mitophagy in cardiovascular health and disease and provide a review of our current understanding of how these processes are regulated.

Therapeutic inhibition of RBM20 improves diastolic function in a murine heart failure model and human engineered heart tissue.

Heart failure with preserved ejection fraction (HFpEF) is prevalent and deadly, but so far, there is no targeted therapy. A main contributor to the disease is impaired ventricular filling, which we improved with antisense oligonucleotides (ASOs) targeting the cardiac splice factor RBM20. In adult mice with increased wall stiffness, weekly application of ASOs over 2 months increased expression of compliant titin isoforms and improved cardiac function as determined by echocardiography and conductance catheter. RNA sequencing confirmed RBM20-dependent isoform changes and served as a sensitive indicator of potential side effects, largely limited to genes related to the immune response. We validated our approach in human engineered heart tissue, showing down-regulation of RBM20 to less than 50% within 3 weeks of treatment with ASOs, resulting in adapted relaxation kinetics in the absence of cardiac pathology. Our data suggest anti-RBM20 ASOs as powerful cardiac splicing regulators for the causal treatment of human HFpEF.

Cardiac interstitial tetraploid cells can escape replicative senescence in rodents but not large mammals.

Cardiomyocyte ploidy has been described but remains obscure in cardiac interstitial cells. Ploidy of c-kit+ cardiac interstitial cells was assessed using confocal, karyotypic, and flow cytometric technique. Notable differences were found between rodent (rat, mouse) c-kit+ cardiac interstitial cells possessing mononuclear tetraploid (4n) content, compared to large mammals (human, swine) with mononuclear diploid (2n) content. In-situ analysis, confirmed with fresh isolates, revealed diploid content in human c-kit+ cardiac interstitial cells and a mixture of diploid and tetraploid content in mouse. Downregulation of the p53 signaling pathway provides evidence why rodent, but not human, c-kit+ cardiac interstitial cells escape replicative senescence. Single cell transcriptional profiling reveals distinctions between diploid versus tetraploid populations in mouse c-kit+ cardiac interstitial cells, alluding to functional divergences. Collectively, these data reveal notable species-specific biological differences in c-kit+ cardiac interstitial cells, which could account for challenges in extrapolation of myocardial from preclinical studies to clinical trials.

Bnip3 mediates mitochondrial dysfunction and cell death through Bax and Bak.

Bnip3 is a pro-apoptotic member of the Bcl-2 family that is down-regulated in pancreatic cancers, which correlates with resistance to chemotherapy and a worsened prognosis. In contrast, Bnip3 is up-regulated in heart failure and contributes to loss of myocardial cells during I/R (ischaemia/reperfusion). Bnip3 exerts its action at the mitochondria, but the mechanism by which Bnip3 mediates mitochondrial dysfunction is not clear. In the present study, we have identified Bax and Bak as downstream effectors of Bnip3-mediated mitochondrial dysfunction. Bnip3 plays a role in hypoxia-mediated cell death, but MEFs (mouse embryonic fibroblasts) derived from mice deficient in Bax and Bak were completely resistant to hypoxia even with substantial up-regulation of Bnip3. These cells were also resistant to Bnip3 overexpression, but re-expression of Bax or Bak restored susceptibility to Bnip3, suggesting that Bnip3 can act via either Bax or Bak. In contrast, Bnip3 overexpression in wild-type MEFs induced mitochondrial dysfunction with loss of membrane potential and release of cytochrome c. Cell death by Bnip3 was reduced in the presence of mPTP (mitochondrial permeability transition pore) inhibitors, but did not prevent Bnip3-mediated activation of Bax or Bak. Moreover, overexpression of Bnip3DeltaTM, a dominant-negative form of Bnip3, reduced translocation of GFP (green fluorescent protein)-Bax to mitochondria during sI/R (simulated I/R) in HL-1 myocytes. Similarly, down-regulation of Bnip3 using RNA interference decreased activation of Bax in response to sI/R in HL-1 myocytes. These results suggest that Bnip3 mediates mitochondrial dysfunction through activation of Bax or Bak which is independent of mPTP opening.

Eat, breathe, ROS: controlling stem cell fate through metabolism.

INTRODUCTION: Research reveals cardiac regeneration exists at levels previously deemed unattainable. Clinical trials using stem cells demonstrate promising cardiomyogenic and regenerative potential but insufficient contractile recovery. Incomplete understanding of the biology of administered cells likely contributes to inconsistent patient outcomes. Metabolism is a core component of many well-characterized stem cell types, and metabolic changes fundamentally alter stem cell fate from self-renewal to lineage commitment, and vice versa. However, the metabolism of stem cells currently studied for cardiac regeneration remains incompletely understood. Areas covered: Key metabolic features of stem cells are reviewed and unique stem cell metabolic characteristics are discussed. Metabolic changes altering stem cell fate are considered from quiescence and self-renewal to lineage commitment. Key metabolic concepts are applied toward examining cardiac regeneration through stem cell-based approaches, and clinical implications of current cell therapies are evaluated to identify potential areas of improvement. Expert commentary: The metabolism and biology of stem cells used for cardiac therapy remain poorly characterized. A growing appreciation for the fundamental relationship between stem cell functionality and metabolic phenotype is developing. Future studies unraveling links between cardiac stem cell metabolism and regenerative potential may considerably improve treatment strategies and therapeutic outcomes.

Peptidyl-Prolyl Isomerase 1 Regulates Ca2+ Handling by Modulating Sarco(Endo)Plasmic Reticulum Calcium ATPase and Na2+/Ca2+ Exchanger 1 Protein Levels and Function.

BACKGROUND: Aberrant Ca2+ handling is a prominent feature of heart failure. Elucidation of the molecular mechanisms responsible for aberrant Ca2+ handling is essential for the development of strategies to blunt pathological changes in calcium dynamics. The peptidyl-prolyl cis-trans isomerase peptidyl-prolyl isomerase 1 (Pin1) is a critical mediator of myocardial hypertrophy development and cardiac progenitor cell cycle. However, the influence of Pin1 on calcium cycling regulation has not been explored. On the basis of these findings, the aim of this study is to define Pin1 as a novel modulator of Ca2+ handling, with implications for improving myocardial contractility and potential for ameliorating development of heart failure. METHODS AND RESULTS: Pin1 gene deletion or pharmacological inhibition delays cytosolic Ca2+ decay in isolated cardiomyocytes. Paradoxically, reduced Pin1 activity correlates with increased sarco(endo)plasmic reticulum calcium ATPase (SERCA2a) and Na2+/Ca2+ exchanger 1 protein levels. However, SERCA2a ATPase activity and calcium reuptake were reduced in sarcoplasmic reticulum membranes isolated from Pin1-deficient hearts, suggesting that Pin1 influences SERCA2a function. SERCA2a and Na2+/Ca2+ exchanger 1 associated with Pin1, as revealed by proximity ligation assay in myocardial tissue sections, indicating that regulation of Ca2+ handling within cardiomyocytes is likely influenced through Pin1 interaction with SERCA2a and Na2+/Ca2+ exchanger 1 proteins. CONCLUSIONS: Pin1 serves as a modulator of SERCA2a and Na2+/Ca2+ exchanger 1 Ca2+ handling proteins, with loss of function resulting in impaired cardiomyocyte relaxation, setting the stage for subsequent investigations to assess Pin1 dysregulation and modulation in the progression of heart failure.

Unbreak my heart: targeting mitochondrial autophagy in diabetic cardiomyopathy.

SIGNIFICANCE: Diabetes is strongly associated with increased incidence of heart disease and mortality due to development of diabetic cardiomyopathy. Even in the absence of cardiovascular disease, cardiomyopathy frequently arises in diabetic patients. Current treatment options for cardiomyopathy in diabetic patients are the same as for nondiabetic patients and do not address the causes underlying the loss of contractility. RECENT ADVANCES: Although there are numerous distinctions between Type 1 and Type 2 diabetes, recent evidence suggests that the two disease states converge on mitochondria as an epicenter for cardiomyocyte damage. CRITICAL ISSUES: Accumulation of dysfunctional mitochondria contributes to cardiac tissue injury in both acute and chronic conditions. Removal of damaged mitochondria by macroautophagy, termed "mitophagy," is critical for maintaining cardiomyocyte health and contractility both under normal conditions and during stress. However, very little is known about the involvement of mitophagy in the pathogenesis of diabetic cardiomyopathy. A growing interest in this topic has given rise to a wave of publications that aim at deciphering the status of autophagy and mitophagy in Type 1 and Type 2 diabetes. FUTURE DIRECTIONS: This review summarizes these recent studies with the goal of drawing conclusions about the activation or suppression of autophagy and mitophagy in the diabetic heart. A better understanding of how autophagy and mitophagy are affected in the diabetic myocardium is still needed, as well as whether they can be targeted therapeutically.

PINK1 Is Dispensable for Mitochondrial Recruitment of Parkin and Activation of Mitophagy in Cardiac Myocytes.

Myocyte function and survival relies on the maintenance of a healthy population of mitochondria. The PINK1/Parkin pathway plays an important role in clearing defective mitochondria via autophagy in cells. However, how the PINK1/Parkin pathway regulates mitochondrial quality control and whether it coordinates with other mitophagy pathways are still unclear. Therefore, the objective of this study was to investigate the effect of PINK1-deficiency on mitochondrial quality control in myocytes. Using PINK1-deficient (PINK1-/-) mice, we found that Parkin is recruited to damaged cardiac mitochondria in hearts after treatment with the mitochondrial uncoupler FCCP or after a myocardial infarction even in the absence of PINK1. Parkin recruitment to depolarized mitochondria correlates with increased ubiquitination of mitochondrial proteins and activation of mitophagy in PINK1-/- myocytes. In addition, induction of mitophagy by the atypical BH3-only protein BNIP3 is unaffected by lack of PINK1. Overall, these data suggest that Parkin recruitment to depolarized cardiac mitochondria and subsequent activation of mitophagy is independent of PINK1. Moreover, alternative mechanisms of Parkin activation and pathways of mitophagy remain functional in PINK1-/- myocytes and could compensate for the PINK1 deficiency.

Cardiomyocyte health: adapting to metabolic changes through autophagy.

Autophagy is important in the heart for maintaining homeostasis when changes in nutrient levels occur. Autophagy is involved in the turnover of cellular components, and is rapidly upregulated during stress. Studies have found that autophagy is reduced in metabolic disorders including obesity and diabetes. This leads to accumulation of protein aggregates and dysfunctional organelles, which contributes to the pathogenesis of cardiovascular disease. Autophagy is primarily regulated by two components: the mechanistic target of rapamycin (mTOR) and AMP-activated protein kinase (AMPK). Although mTOR integrates information about growth factors and nutrients and is a negative regulator of autophagy, AMPK is an energy sensor and activates autophagy when energy levels are low. These pathways therefore present targets for the development of autophagy-modulating therapies.

Autophagy and mitophagy in the myocardium: therapeutic potential and concerns.

The autophagic-lysosomal degradation pathway is critical for cardiac homeostasis, and defects in this pathway are associated with development of cardiomyopathy. Autophagy is responsible for the normal turnover of organelles and long-lived proteins. Autophagy is also rapidly up-regulated in response to stress, where it rapidly clears dysfunctional organelles and cytotoxic protein aggregates in the cell. Autophagy is also important in clearing dysfunctional mitochondria before they can cause harm to the cell. This quality control mechanism is particularly important in cardiac myocytes, which contain a very high volume of mitochondria. The degradation of proteins and organelles also generates free fatty acids and amino acids, which help maintain energy levels in myocytes during stress conditions. Increases in autophagy have been observed in various cardiovascular diseases, but a major question that remains to be answered is whether enhanced autophagy is an adaptive or maladaptive response to stress. This review discusses the regulation and role of autophagy in the myocardium under baseline conditions and in various aetiologies of heart disease. It also discusses whether this pathway represents a new therapeutic target to treat or prevent cardiovascular disease and the concerns associated with modulating autophagy.

Parkin deficiency results in accumulation of abnormal mitochondria in aging myocytes.

Autophagy plays a critical role in cellular quality control and is involved in removing damaged or excess organelles. Dysfunctional mitochondria are quickly cleared from the cell by autophagosomes before they can cause damage to the cell. Parkin is an E3 ubiquitin ligase that selectively ubiquitinates proteins on dysfunctional mitochondria, thereby marking those mitochondria for degradation by autophagosomes. In our recent study, we investigated the functional role of Parkin in the myocardium and discovered that Parkin is dispensable in the adult heart under normal conditions. Instead, our findings suggest that Parkin plays an important role in clearing damaged mitochondria in myocytes during stress. Here, we report that Parkin deficiency results in the accumulation of abnormal mitochondria in myocytes with age.