Summary Mitochondrial respiratory dysfunction is linked to the pathogenesis of multiple diseases including heart failure but the specific mechanisms for this link remain largely elusive. We modeled the impairment of mitochondrial respiration by inactivation of the Ndufs4 gene, a protein critical for Complex I (C-I) assembly, in the mouse heart (cKO). While C-I supported respiration decreased by >40%, the cKO mice maintained normal cardiac function in vivo and high-energy phosphate content in isolated perfused hearts. However, the cKO mice developed accelerated heart failure after pressure overload or repeated pregnancy. Decreased NAD+/NADH ratio by C-I deficiency inhibited Sirt3 activity, leading to increase in protein acetylation, and sensitization of the permeability transition in mitochondria (mPTP). NAD+ precursor supplementation to cKO mice partially normalized the NAD+/NADH ratio, protein acetylation and mPTP sensitivity. These findings describe a mechanism connecting mitochondrial dysfunction to the susceptibility to diseases and propose a potential therapeutic target.

Changes in the cardiac metabolic milieu during the perinatal period redirect mitochondrial substrate preference from carbohydrates to fatty acids. Mechanisms responsible for this mitochondrial plasticity are unknown. Here we found that PINK1-Mfn2-Parkin mediated mitophagy directs this metabolic transformation in mice. A mitofusin (Mfn) 2 mutant lacking PINK1 phosphorylation sites necessary for Parkin binding (Mfn2 AA) inhibited mitochondrial Parkin translocation, suppressing mitophagy without impairing mitochondrial fusion. Cardiac Parkin deletion or expression of Mfn2 AA from birth, but not after weaning, prevented postnatal mitochondrial maturation essential to survival. Five week old Mfn2 AA hearts retained a fetal mitochondrial transcriptional signature without normal increases in fatty acid metabolism and mitochondrial biogenesis genes. Myocardial fatty acylcarnitine levels and cardiomyocyte respiration induced by palmitoylcarnitine were concordantly depressed. Thus, instead of transcriptional reprogramming, fetal cardiomyocyte mitochondria undergo perinatal Parkin-mediated mitophagy and replacement by mature adult mitochondria. Mitophagic mitochondrial removal underlies developmental cardiomyocyte mitochondrial plasticity and metabolic transitioning of perinatal hearts.

Summary Mitochondria are dynamic organelles, remodeling and exchanging contents during cyclic fusion and fission. Genetic mutations of mitofusin (Mfn) 2 interrupt mitochondrial fusion and cause the untreatable neurodegenerative condition, Charcot Marie Tooth disease type 2A (CMT2A). It has not been possible to directly modulate mitochondrial fusion, in part because the structural basis of mitofusin function is incompletely understood. Here we show that mitofusins adopt either a fusion-constrained or fusion-permissive molecular conformation directed by specific intramolecular binding interactions, and demonstrate that mitofusin-dependent mitochondrial fusion can be regulated by targeting these conformational transitions. Based on this model we engineered a cell-permeant minipeptide to destabilize fusion-constrained mitofusin and promote the fusion-permissive conformation, reversing mitochondrial abnormalities in cultured fibroblasts and neurons harboring CMT2A gene defects. The relationship between mitofusin conformational plasticity and mitochondrial dynamism uncovers a central mechanism regulating mitochondrial fusion whose manipulation can correct mitochondrial pathology triggered by defective or imbalanced mitochondrial dynamics.

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 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 very 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/SQSTM1 and processed LC3. 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 markedly upregulates Parkin, thus provoking mitophagic mitochondrial depletion that contributes to the lethal cardiomyopathy.

Rationale Mitochondrial reactive oxygen species (ROS) are implicated in aging, chronic degenerative neurological syndromes, and myopathies. Based on the free radical hypothesis, dietary, pharmacological, and genetic ROS suppression has been tested to minimize tissue damage, with remarkable therapeutic efficacy. The effects of mitochondrial-specific ROS suppression in primary mitophagic dysfunction are unknown. Objective An in vivo dose-ranging analysis of ROS suppression in an experimental cardiomyopathy provoked by defective mitochondrial clearance. Methods and Results Mice lacking mitofusin (Mfn) 2 in hearts have impaired Parkin-mediated mitophagy leading to accumulation of damaged ROS-producing organelles and progressive heart failure. As expected, cardiomyocyte-directed expression of mitochondrial targeted catalase (CAT) at modest levels normalized mitochondrial ROS production and prevented mitochondrial depolarization, respiratory impairment, and structural degeneration in Mfn2 null hearts. In contrast, CAT expression at higher levels that super-suppressed mitochondrial ROS failed to improve either mitochondrial fitness or cardiomyopathy, revealing that ROS toxicity is not the primary mechanism for cardiac degeneration. Lack of benefit from super-suppressing ROS was associated with failure to invoke secondary autophagic pathways of mitochondrial quality control, revealing a role for ROS signaling in mitochondrial clearance. Mitochondrial permeability transition pore (MPTP) function was normal, and genetic inhibition of MPTP function did not alter mitochondrial or cardiac degeneration, in Mfn2 null hearts. Conclusions Local mitochondrial ROS: 1. Contribute to mitochondrial degeneration and 2. Activate mitochondrial quality control mechanisms. A therapeutic window for mitochondrial ROS suppression should minimize the former while retaining the latter, which we achieved by expressing lower levels of CAT.

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Mitochondrial permeability transition pore (mPTP) is involved in cardiac dysfunction during chronic β-adrenergic receptor (β-AR) stimulation. The mechanism by which chronic β-AR stimulation leads to mPTP openings is elusive. Here, we show that chronic administration of isoproterenol (ISO) persistently increases the frequency of mPTP openings followed by mitochondrial damage and cardiac dysfunction. Mechanistically, this effect is mediated by phosphorylation of mitochondrial fission protein, dynamin-related protein 1 (Drp1), by Ca2+/calmodulin-dependent kinase II (CaMKII) at a serine 616 (S616) site. Mutating this phosphorylation site or inhibiting Drp1 activity blocks CaMKII- or ISO-induced mPTP opening and myocyte death in vitro and rescues heart hypertrophy in vivo. In human failing hearts, Drp1 phosphorylation at S616 is increased. These results uncover a pathway downstream of chronic β-AR stimulation that links CaMKII, Drp1 and mPTP to bridge cytosolic stress signal with mitochondrial dysfunction in the heart.

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