Mcl-1-mediated mitochondrial fission protects against stress but impairs cardiac adaptation to exercise.

Myeloid cell leukemia-1 (Mcl-1) is a structurally and functionally unique anti-apoptotic Bcl-2 protein. While elevated levels of Mcl-1 contribute to tumor cell survival and drug resistance, loss of Mcl-1 in cardiac myocytes leads to rapid mitochondrial dysfunction and heart failure development. Although Mcl-1 is an anti-apoptotic protein, previous studies indicate that its functions extend beyond regulating apoptosis. Mcl-1 is localized to both the mitochondrial outer membrane and matrix. Here, we have identified that Mcl-1 in the outer mitochondrial membrane mediates mitochondrial fission, which is independent of its anti-apoptotic function. We demonstrate that Mcl-1 interacts with Drp1 to promote mitochondrial fission in response to various challenges known to perturb mitochondria morphology. Induction of fission by Mcl-1 reduces nutrient deprivation-induced cell death and the protection is independent of its BH3 domain. Finally, cardiac-specific overexpression of Mcl-1OM, but not Mcl-1Matrix, contributes to a shift in the balance towards fission and leads to reduced exercise capacity, suggesting that a pre-existing fragmented mitochondrial network leads to decreased ability to adapt to an acute increase in workload and energy demand. Overall, these findings highlight the importance of Mcl-1 in maintaining mitochondrial health in cells.

Parkin does not prevent accelerated cardiac aging in mitochondrial DNA mutator mice.

The E3 ubiquitin ligase Parkin plays an important role in regulating clearance of dysfunctional or unwanted mitochondria in tissues, including the heart. However, whether Parkin also functions to prevent cardiac aging by maintaining a healthy population of mitochondria is still unclear. Here, we have examined the role of Parkin in the context of mtDNA damage and myocardial aging using a mouse model carrying a proofreading defective mitochondrial DNA polymerase gamma (POLG). We observed both decreased Parkin protein levels and development of cardiac hypertrophy in POLG hearts with age; however, cardiac hypertrophy in POLG mice was neither rescued, nor worsened by cardiac specific overexpression or global deletion of Parkin, respectively. Unexpectedly, mitochondrial fitness did not substantially decline with age in POLG mice when compared to WT. We found that baseline mitophagy receptor-mediated mitochondrial turnover and biogenesis were enhanced in aged POLG hearts. We also observed the presence of megamitochondria in aged POLG hearts. Thus, these processes may limit the accumulation of dysfunctional mitochondria as well as the degree of cardiac functional impairment in the aging POLG heart. Overall, our results demonstrate that Parkin is dispensable for constitutive mitochondrial quality control in a mtDNA mutation model of cardiac aging.

BNIP3L/NIX and FUNDC1-mediated mitophagy is required for mitochondrial network remodeling during cardiac progenitor cell differentiation.

Cell-based therapies represent a very promising strategy to repair and regenerate the injured heart to prevent progression to heart failure. To date, these therapies have had limited success due to a lack of survival and retention of the infused cells. Therefore, it is important to increase our understanding of the biology of these cells and utilize this information to enhance their survival and function in the injured heart. Mitochondria are critical for progenitor cell function and survival. Here, we demonstrate the importance of mitochondrial autophagy, or mitophagy, in the differentiation process in adult cardiac progenitor cells (CPCs). We found that mitophagy was rapidly induced upon initiation of differentiation in CPCs. We also found that mitophagy was mediated by mitophagy receptors, rather than the PINK1-PRKN/PARKIN pathway. Mitophagy mediated by BNIP3L/NIX and FUNDC1 was not involved in regulating progenitor cell fate determination, mitochondrial biogenesis, or reprogramming. Instead, mitophagy facilitated the CPCs to undergo proper mitochondrial network reorganization during differentiation. Abrogating BNIP3L- and FUNDC1-mediated mitophagy during differentiation led to sustained mitochondrial fission and formation of donut-shaped impaired mitochondria. It also resulted in increased susceptibility to cell death and failure to survive the infarcted heart. Finally, aging is associated with accumulation of mitochondrial DNA (mtDNA) damage in cells and we found that acquiring mtDNA mutations selectively disrupted the differentiation-activated mitophagy program in CPCs. These findings demonstrate the importance of BNIP3L- and FUNDC1-mediated mitophagy as a critical regulator of mitochondrial network formation during differentiation, as well as the consequences of accumulating mtDNA mutations. Abbreviations: Baf: bafilomycin A1; BCL2L13: BCL2 like 13; BNIP3: BCL2 interacting protein 3; BNIP3L: BCL2 interacting protein 3 like; CPCs: cardiac progenitor cells; DM: differentiation media; DNM1L: dynamin 1 like; EPCs: endothelial progenitor cells; FCCP: carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone; FUNDC1: FUN14 domain containing 1; HSCs: hematopoietic stem cells; MAP1LC3B/LC3: microtubule-associated protein 1 light chain 3 beta; MFN1/2: mitofusin 1/2; MSCs: mesenchymal stem cells; mtDNA: mitochondrial DNA; OXPHOS: oxidative phosphorylation; PPARGC1A: PPARG coactivator 1 alpha; PHB2: prohibitin 2; POLG: DNA polymerase gamma, catalytic subunit; SQSTM1: sequestosome 1; TEM: transmission electron microscopy; TMRM: tetramethylrhodamine methyl ester.

Decline in cellular function of aged mouse c-kit+ cardiac progenitor cells.

KEY POINTS: While autologous stem cell-based therapies are currently being tested on elderly patients, there are limited data on the function of aged stem cells and in particular c-kit+ cardiac progenitor cells (CPCs). We isolated c-kit+ cells from young (3 months) and aged (24 months) C57BL/6 mice to compare their biological properties. Aged CPCs have increased senescence, decreased stemness and reduced capacity to proliferate or to differentiate following dexamethasone (Dex) treatment in vitro, as evidenced by lack of cardiac lineage gene upregulation. Aged CPCs fail to activate mitochondrial biogenesis and increase proteins involved in mitochondrial oxidative phosphorylation in response to Dex. Aged CPCs fail to upregulate paracrine factors that are potentially important for proliferation, survival and angiogenesis in response to Dex. The results highlight marked differences between young and aged CPCs, which may impact future design of autologous stem cell-based therapies. ABSTRACT: Therapeutic use of c-kit+ cardiac progenitor cells (CPCs) is being evaluated for regenerative therapy in older patients with ischaemic heart failure. Our understanding of the biology of these CPCs has, however, largely come from studies of young cells and animal models. In the present study we examined characteristics of CPCs isolated from young (3 months) and aged (24 months) mice that could underlie the diverse outcomes reported for CPC-based therapeutics. We observed morphological differences and altered senescence indicated by increased senescence-associated markers ß-galactosidase and p16 mRNA in aged CPCs. The aged CPCs also proliferated more slowly than their young counterparts and expressed lower levels of the stemness marker LIN28. We subsequently treated the cells with dexamethasone (Dex), routinely used to induce commitment in CPCs, for 7 days and analysed expression of cardiac lineage marker genes. While MEF2C, GATA4, GATA6 and PECAM mRNAs were significantly upregulated in response to Dex treatment in young CPCs, their expression was not increased in aged CPCs. Interestingly, Dex treatment of aged CPCs also failed to increase mitochondrial biogenesis and expression of the mitochondrial proteins Complex III and IV, consistent with a defect in mitochondria complex assembly in the aged CPCs. Dex-treated aged CPCs also had impaired ability to upregulate expression of paracrine factor genes and the conditioned media from these cells had reduced ability to induce angiogenesis in vitro. These findings could impact the design of future CPC-based therapeutic approaches for the treatment of older patients suffering from cardiac injury.

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.

Functional Effect of Pim1 Depends upon Intracellular Localization in Human Cardiac Progenitor Cells.

Human cardiac progenitor cells (hCPC) improve heart function after autologous transfer in heart failure patients. Regenerative potential of hCPCs is severely limited with age, requiring genetic modification to enhance therapeutic potential. A legacy of work from our laboratory with Pim1 kinase reveals effects on proliferation, survival, metabolism, and rejuvenation of hCPCs in vitro and in vivo. We demonstrate that subcellular targeting of Pim1 bolsters the distinct cardioprotective effects of this kinase in hCPCs to increase proliferation and survival, and antagonize cellular senescence. Adult hCPCs isolated from patients undergoing left ventricular assist device implantation were engineered to overexpress Pim1 throughout the cell (PimWT) or targeted to either mitochondrial (Mito-Pim1) or nuclear (Nuc-Pim1) compartments. Nuc-Pim1 enhances stem cell youthfulness associated with decreased senescence-associated ß-galactosidase activity, preserved telomere length, reduced expression of p16 and p53, and up-regulation of nucleostemin relative to PimWT hCPCs. Alternately, Mito-Pim1 enhances survival by increasing expression of Bcl-2 and Bcl-XL and decreasing cell death after H2O2 treatment, thereby preserving mitochondrial integrity superior to PimWT. Mito-Pim1 increases the proliferation rate by up-regulation of cell cycle modulators Cyclin D, CDK4, and phospho-Rb. Optimal stem cell traits such as proliferation, survival, and increased youthful properties of aged hCPCs are enhanced after targeted Pim1 localization to mitochondrial or nuclear compartments. Targeted Pim1 overexpression in hCPCs allows for selection of the desired phenotypic properties to overcome patient variability and improve specific stem cell characteristics.

Therapeutic targeting of autophagy: potential and concerns in treating cardiovascular disease.

Autophagy is an evolutionarily conserved process by which long-lived proteins and organelles are sequestered by autophagosomes and subsequently degraded by lysosomes for recycling. Autophagy is important for maintaining cardiac homeostasis and is a survival mechanism that is upregulated during stress or starvation. Accumulating evidence suggests that dysregulated or reduced autophagy is associated with heart failure and aging. Thus, modulating autophagy represents an attractive future therapeutic target for treating cardiovascular disease. Activation of autophagy is generally considered to be cardioprotective, whereas excessive autophagy can lead to cell death and cardiac atrophy. It is important to understand how autophagy is regulated to identify ideal therapeutic targets for treating disease. Here, we discuss the key proteins in the core autophagy machinery and describe upstream regulators that respond to extracellular and intracellular signals to tightly coordinate autophagic activity. We review various genetic and pharmacological studies that demonstrate the important role of autophagy in the heart and consider the advantages and limitations of approaches that modulate autophagy.

Cell death in the myocardium: my heart won't go on.

Loss of cardiomyocytes plays a critical role in the pathogenesis of heart failure. With fewer myocytes, the heart is unable to sustain efficient contraction. Much attention has been focused on understanding mechanisms of cell death in myocytes with the ultimate goal being to reduce the extent of injury and improve function in the failing myocardium. Both necrosis and apoptosis contribute to loss of myocytes, and this loss of cells is a hallmark of cardiac pathologies, including ischemia/reperfusion, myocardial infarction, and heart failure. Apoptosis is a highly regulated process that is activated via death receptors in the plasma membrane or via permeabilization of the mitochondria. Necrosis is generally viewed as an uncontrolled process that leads to mitochondrial swelling, cell rupture, and subsequent inflammation. However, recent studies have uncovered a signaling pathway that mediates regulated necrosis or necroptosis. Mitochondria play an important role in both apoptosis and necrosis, and changes in their morphology can affect the cells' susceptibility to stress. This review focuses on the various modes of cell death in the myocardium and highlights how they contribute to loss of myocytes in response to stress.

Microtubule-associated protein 1 light chain 3 (LC3) interacts with Bnip3 protein to selectively remove endoplasmic reticulum and mitochondria via autophagy.

Autophagy plays an important role in cellular quality control and is responsible for removing protein aggregates and dysfunctional organelles. Bnip3 is an atypical BH3-only protein that is known to cause mitochondrial dysfunction and cell death. Interestingly, Bnip3 can also protect against cell death by inducing mitochondrial autophagy. The mechanism for this process, however, remains poorly understood. Bnip3 contains a C-terminal transmembrane domain that is essential for homodimerization and proapoptotic function. In this study, we show that homodimerization of Bnip3 is also a requirement for induction of autophagy. Several Bnip3 mutants that do not interfere with its mitochondrial localization but disrupt homodimerization failed to induce autophagy in cells. In addition, we discovered that endogenous Bnip3 is localized to both mitochondria and the endoplasmic reticulum (ER). To investigate the effects of Bnip3 at mitochondria or the ER on autophagy, Bnip3 was targeted specifically to each organelle by substituting the Bnip3 transmembrane domain with that of Acta or cytochrome b(5). We found that Bnip3 enhanced autophagy in cells from both sites. We also discovered that Bnip3 induced removal of both ER (ERphagy) and mitochondria (mitophagy) via autophagy. The clearance of these organelles was mediated in part via binding of Bnip3 to LC3 on the autophagosome. Although ablation of the Bnip3-LC3 interaction by mutating the LC3 binding site did not impair the prodeath activity of Bnip3, it significantly reduced both mitophagy and ERphagy. Our data indicate that Bnip3 regulates the apoptotic balance as an autophagy receptor that induces removal of both mitochondria and ER.

Construction and Consensus Performance of (Q)SAR Models for Predicting Phospholipidosis Using a Dataset of 743 Compounds.

Drug-induced phospholipidosis (PLD) continues to be a safety concern for pharmaceutical companies and regulatory agencies, prompting the FDA/CDER Phospholipidosis Working Group to develop a database of PLD findings that was recently expanded to contain a total of 743 compounds (385 positive and 358 negative). Three commercial (quantitative) structure-activity relationship [(Q)SAR)] software platforms [MC4PC, Leadscope Predictive Data Miner (LPDM), and Derek for Windows (DfW)] were used to build and/or test models with the database and evaluated individually and together for their ability to predict PLD induction. Models constructed with MC4PC showed improved sensitivity over previous models constructed with an earlier version of the database and software (61.2 % vs. 50.0 %), but lower specificity in cross-validation experiments (58.2 % vs. 91.9 %) due in part to the more balanced ratio of positives to negatives in the training set. A new model created with LPDM gave good cross-validation statistics (79.0 % sensitivity, 78.0 % specificity) and the single DfW structural alert for PLD was found to have high positive predictivity (83.3 %) but low sensitivity (10.4 %) when tested with the entire PLD database. Combining the predictions of MC4PC, LPDM and/or DfW resulted in increased sensitivity and coverage over using one software platform alone, although it did not enhance the overall prediction accuracy beyond that of the best performing individual software platform. The comparison across software platforms, however, facilitated the identification and analysis of chemicals that were consistently predicted incorrectly by all platforms. The prevalence of cationic amphiphilic drug (CAD) structural motifs in the database contributed heavily to many of the structural alerts and discriminating features in the models, but the subset of incorrectly predicted structures across all models underscores the need to account for mitigating features and/or additional filtering criteria to assess PLD, in particular for PLD-inducing non-CADs and non-PLD-inducing CADs. (Q)SAR tools may be used as part of an early screening battery or regulatory risk assessment approach to identify those compounds with the greatest chance of inducing PLD and potentially toxicity.