NF-κB Restricts Inflammasome Activation via Elimination of Damaged Mitochondria.

Nuclear factor κB (NF-κB), a key activator of inflammation, primes the NLRP3-inflammasome for activation by inducing pro-IL-1ß and NLRP3 expression. NF-κB, however, also prevents excessive inflammation and restrains NLRP3-inflammasome activation through a poorly defined mechanism. We now show that NF-κB exerts its anti-inflammatory activity by inducing delayed accumulation of the autophagy receptor p62/SQSTM1. External NLRP3-activating stimuli trigger a form of mitochondrial (mt) damage that is caspase-1- and NLRP3-independent and causes release of direct NLRP3-inflammasome activators, including mtDNA and mtROS. Damaged mitochondria undergo Parkin-dependent ubiquitin conjugation and are specifically recognized by p62, which induces their mitophagic clearance. Macrophage-specific p62 ablation causes pronounced accumulation of damaged mitochondria and excessive IL-1ß-dependent inflammation, enhancing macrophage death. Therefore, the "NF-κB-p62-mitophagy" pathway is a macrophage-intrinsic regulatory loop through which NF-κB restrains its own inflammation-promoting activity and orchestrates a self-limiting host response that maintains homeostasis and favors tissue repair.

Evolving and Expanding the Roles of Mitophagy as a Homeostatic and Pathogenic Process.

The central functions fulfilled by mitochondria as both energy generators essential for tissue homeostasis and gateways to programmed apoptotic and necrotic cell death mandate tight control over the quality and quantity of these ubiquitous endosymbiotic organelles. Mitophagy, the targeted engulfment and destruction of mitochondria by the cellular autophagy apparatus, has conventionally been considered as the mechanism primarily responsible for mitochondrial quality control. However, our understanding of how, why, and under what specific conditions mitophagy is activated has grown tremendously over the past decade. Evidence is accumulating that nonmitophagic mitochondrial quality control mechanisms are more important to maintaining normal tissue homeostasis whereas mitophagy is an acute tissue stress response. Moreover, previously unrecognized mitophagic regulation of mitochondrial quantity control, metabolic reprogramming, and cell differentiation suggests that the mechanisms linking genetic or acquired defects in mitophagy to neurodegenerative and cardiovascular diseases or cancer are more complex than simple failure of normal mitochondrial quality control. Here, we provide a comprehensive overview of mitophagy in cellular homeostasis and disease and examine the most revolutionary concepts in these areas. In this context, we discuss evidence that atypical mitophagy and nonmitophagic pathways play central roles in mitochondrial quality control, functioning that was previously considered to be the primary domain of mitophagy.

Cancer-cell-secreted extracellular vesicles target p53 to impair mitochondrial function in muscle.

Skeletal muscle loss and weakness are associated with bad prognosis and poorer quality of life in cancer patients. Tumor-derived factors have been implicated in muscle dysregulation by inducing cachexia and apoptosis. Here, we show that extracellular vesicles secreted by breast cancer cells impair mitochondrial homeostasis and function in skeletal muscle, leading to decreased mitochondrial content and energy production and increased oxidative stress. Mechanistically, miR-122-5p in cancer-cell-secreted EVs is transferred to myocytes, where it targets the tumor suppressor TP53 to decrease the expression of TP53 target genes involved in mitochondrial regulation, including Tfam, Pgc-1α, Sco2, and 16S rRNA. Restoration of Tp53 in muscle abolishes mitochondrial myopathology in mice carrying breast tumors and partially rescues their impaired running capacity without significantly affecting muscle mass. We conclude that extracellular vesicles from breast cancer cells mediate skeletal muscle mitochondrial dysfunction in cancer and may contribute to muscle weakness in some cancer patients.

Mitochondrial quality surveillance: mitophagy in cardiovascular health and disease.

Selective autophagy of mitochondria, known as mitophagy, is a major quality control pathway in the heart that is involved in removing unwanted or dysfunctional mitochondria from the cell. Baseline mitophagy is critical for maintaining fitness of the mitochondrial network by continuous turnover of aged and less-functional mitochondria. Mitophagy is also critical in adapting to stress associated with mitochondrial damage or dysfunction. The removal of damaged mitochondria prevents reactive oxygen species-mediated damage to proteins and DNA and suppresses activation of inflammation and cell death. Impairments in mitophagy are associated with the pathogenesis of many diseases, including cancers, inflammatory diseases, neurodegeneration, and cardiovascular disease. Mitophagy is a highly regulated and complex process that requires the coordination of labeling dysfunctional mitochondria for degradation while simultaneously promoting de novo autophagosome biogenesis adjacent to the cargo. In this review, we provide an update on our current understanding of these steps in mitophagy induction and discuss the physiological and pathophysiological consequences of altered mitophagy in the heart.

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.

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.

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.

Mitochondria and autophagy in adult stem cells: proliferate or differentiate.

Adult stem cells are undifferentiated cells that are found in many different tissues after development. They are responsible for regenerating and repairing tissues after injury, as well as replacing cells when needed. Adult stem cells maintain a delicate balance between self-renewal to prevent depletion of the stem cell pool and differentiation to continually replenish downstream lineages. The important role of mitochondria in generating energy, calcium storage and regulating cell death is well established. However, new research has linked mitochondria to stem cell maintenance and fate. In addition, efficient mitochondrial quality control is critical for stem cell homeostasis to ensure their long-term survival in tissues. In this review, we discuss the latest evidence linking mitochondrial function, remodeling and turnover via autophagy to regulation of adult stem cell self-renewal and differentiation.

Loss of Parkin Results in Altered Muscle Stem Cell Differentiation during Regeneration.

The high capacity of the skeletal muscle to regenerate is due to the presence of muscle stem cells (MuSCs, or satellite cells). The E3 ubiquitin ligase Parkin is a key regulator of mitophagy and is recruited to mitochondria during differentiation of mouse myoblast cell line. However, the function of mitophagy during regeneration has not been investigated in vivo. Here, we have utilized Parkin deficient (Parkin-/-) mice to investigate the role of Parkin in skeletal muscle regeneration. We found a persistent deficiency in skeletal muscle regeneration in Parkin-/- mice after cardiotoxin (CTX) injury with increased area of fibrosis and decreased cross-sectional area (CSA) of myofibres post-injury. There was also a significant modulation of MuSCs differentiation and mitophagic markers, with altered mitochondrial proteins during skeletal muscle regeneration in Parkin-/- mice. Our data suggest that Parkin-mediated mitophagy plays a key role in skeletal muscle regeneration and is necessary for MuSCs differentiation.

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.

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.

Interdependence of Parkin-Mediated Mitophagy and Mitochondrial Fission in Adult Mouse Hearts.

RATIONALE: The role of Parkin in hearts is unclear. Germ-line Parkin knockout mice have normal hearts, but Parkin is protective in cardiac ischemia. Parkin-mediated mitophagy is reportedly either irrelevant, or a major factor, in the lethal cardiomyopathy evoked by cardiac myocyte-specific interruption of dynamin-related protein 1 (Drp1)-mediated mitochondrial fission. OBJECTIVE: To understand the role of Parkin-mediated mitophagy in normal and mitochondrial fission-defective adult mouse hearts. METHODS AND RESULTS: Parkin mRNA and protein were present at low levels in normal mouse hearts, but were upregulated after cardiac myocyte-directed Drp1 gene deletion in adult mice. Alone, forced cardiac myocyte Parkin overexpression activated mitophagy without adverse effects. Likewise, cardiac myocyte-specific Parkin deletion evoked no adult cardiac phenotype, revealing no essential function for, and tolerance of, Parkin-mediated mitophagy in normal hearts. Concomitant conditional Parkin deletion with Drp1 ablation in adult mouse hearts prevented Parkin upregulation in mitochondria of fission-defective hearts, also increasing 6-week survival, improving ventricular ejection performance, mitigating adverse cardiac remodeling, and decreasing cardiac myocyte necrosis and replacement fibrosis. Underlying the Parkin knockout rescue was suppression of Drp1-induced hyper-mitophagy, assessed as ubiquitination of mitochondrial proteins and mitochondrial association of autophagosomal p62/sequestosome 1 (SQSTM1) and processed microtubule-associated protein 1 light chain 3 (LC3-II). Consequently, mitochondrial content of Drp1-deficient hearts was preserved. Parkin deletion did not alter characteristic mitochondrial enlargement of Drp1-deficient cardiac myocytes. CONCLUSIONS: Parkin is rare in normal hearts and dispensable for constitutive mitophagic quality control. Ablating Drp1 in adult mouse cardiac myocytes not only interrupts mitochondrial fission, but also markedly upregulates Parkin, thus provoking mitophagic mitochondrial depletion that contributes to the lethal cardiomyopathy.

Harnessing the Power of Integrated Mitochondrial Biology and Physiology: A Special Report on the NHLBI Mitochondria in Heart Diseases Initiative.

Mitochondrial biology is the sum of diverse phenomena from molecular profiles to physiological functions. A mechanistic understanding of mitochondria in disease development, and hence the future prospect of clinical translations, relies on a systems-level integration of expertise from multiple fields of investigation. Upon the successful conclusion of a recent National Institutes of Health, National Heart, Lung, and Blood Institute initiative on integrative mitochondrial biology in cardiovascular diseases, we reflect on the accomplishments made possible by this unique interdisciplinary collaboration effort and exciting new fronts on the study of these remarkable organelles.

Staying young at heart: autophagy and adaptation to cardiac aging.

Aging is a predominant risk factor for developing cardiovascular disease. Therefore, the cellular processes that contribute to aging are attractive targets for therapeutic interventions that can delay or prevent the development of age-related diseases. Our understanding of the underlying mechanisms that contribute to the decline in cell and tissue functions with age has greatly advanced over the past decade. Classical hallmarks of aging cells include increased levels of reactive oxygen species, DNA damage, accumulation of dysfunctional organelles, oxidized proteins and lipids. These all contribute to a progressive decline in the normal physiological function of the cell and to the onset of age-related conditions. A major cause of the aging process is progressive loss of cellular quality control. Autophagy is an important quality control pathway and is necessary to maintain cardiac homeostasis and to adapt to stress. A reduction in autophagy has been observed in a number of aging models and there is compelling evidence that enhanced autophagy delays aging and extends life span. Enhancing autophagy counteracts age-associated accumulation of protein aggregates and damaged organelles in cells. In this review, we discuss the functional role of autophagy in maintaining homeostasis in the heart, and how a decline is associated with accelerated cardiac aging. We also evaluate therapeutic approaches being researched in an effort to maintain a healthy young heart.

Evaluating mitochondrial autophagy in the mouse heart.

Mitochondrial autophagy plays an important role in mediating mitochondrial quality control. Evaluating the extent of mitochondrial autophagy is challenging in the adult heart in vivo. Keima is a fluorescent protein that emits different colored signals at acidic and neutral pHs. Keima targeted to mitochondria (Mito-Keima) is useful in evaluating the extent of mitochondrial autophagy in cardiomyocytes in vitro. In order to evaluate the level of mitochondrial autophagy in the heart in vivo, we generated adeno-associated virus (AAV) serotype 9 harboring either Mito-Keima or Lamp1-YFP. AAV9-Mito-Keima and AAV9-Lamp1-YFP were administered intravenously and mice were subjected to either forty-eight hours of fasting or normal chow. Thin slices of the heart prepared within cold PBS were subjected to confocal microscopic analyses. The acidic dots Mito-Keima elicited by 561nm excitation were co-localized with Lamp1-YFP dots (Pearson's correlation, 0.760, p<0.001), confirming that the acidic dots of Mito-Keima were localized in lysosomes. The area co-occupied by Mito-Keima puncta with 561nm excitation and Lamp1-YFP was significantly greater 48h after fasting. Electron microscopic analyses indicated that autophagosomes containing only mitochondria were observed in the heart after fasting. The mitochondrial DNA content and the level of COX1/GAPDH, indicators of mitochondrial mass, were significantly smaller in the fasting group than in the control group, consistent with the notion that lysosomal degradation of mitochondria is stimulated after fasting. In summary, the level of mitochondrial autophagy in the adult heart can be evaluated with intravenous injection of AAV-Mito-Keima and AAV-Lamp1-YFP and confocal microscopic analyses.

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.