Toshiro Saito

Drp1-Dependent Mitochondrial Autophagy Plays a Protective Role Against Pressure Overload–Induced Mitochondrial Dysfunction and Heart Failure

Background— Mitochondrial autophagy is an important mediator of mitochondrial quality control in cardiomyocytes. The occurrence of mitochondrial autophagy and its significance during cardiac hypertrophy are not well understood. Methods and Results— Mice were subjected to transverse aortic constriction (TAC) and observed at multiple time points up to 30 days. Cardiac hypertrophy developed after 5 days, the ejection fraction was reduced after 14 days, and heart failure was observed 30 days after TAC. General autophagy was upregulated between 1 and 12 hours after TAC but was downregulated below physiological levels 5 days after TAC. Mitochondrial autophagy, evaluated by electron microscopy, mitochondrial content, and Keima with mitochondrial localization signal, was transiently activated at ≈3 to 7 days post-TAC, coinciding with mitochondrial translocation of Drp1. However, it was downregulated thereafter, followed by mitochondrial dysfunction. Haploinsufficiency of Drp1 abolished mitochondrial autophagy and exacerbated the development of both mitochondrial dysfunction and heart failure after TAC. Injection of Tat-Beclin 1, a potent inducer of autophagy, but not control peptide, on day 7 after TAC, partially rescued mitochondrial autophagy and attenuated mitochondrial dysfunction and heart failure induced by overload. Haploinsufficiency of either drp1 or beclin 1 prevented the rescue by Tat-Beclin 1, suggesting that its effect is mediated in part through autophagy, including mitochondrial autophagy. Conclusions— Mitochondrial autophagy is transiently activated and then downregulated in the mouse heart in response to pressure overload. Downregulation of mitochondrial autophagy plays an important role in mediating the development of mitochondrial dysfunction and heart failure, whereas restoration of mitochondrial autophagy attenuates dysfunction in the heart during pressure overload.

Molecular Mechanisms of Mitochondrial Autophagy/Mitophagy in the Heart

Mitochondrial quality is a crucial determinant of cell viability, and mitochondrial autophagy plays a central role in this control mechanism. Based on studies in yeast, numerous investigations of this process have been conducted, and the framework of mammalian mitochondrial autophagy is progressively appearing. However, many enigmas about the molecular mechanisms involved remain unsolved. Furthermore, the pathological significance of mitochondrial autophagy in the heart remains largely unclear. In this review, we discuss the current understanding of mitochondrial autophagy in mammals with reference to that in yeast. Regarding the process in yeast, some points of uncertainty have arisen. We also summarize recent advances in the research of autophagy and mitochondrial autophagy in the heart. This article is a part of a review series on Autophagy in Health and Disease.

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 (Pearsons 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.

Unexpected Functional Consequences of the Loss of the Autophagy-Related Conjugation System.

We discuss the recent finding by Mizishimas group regarding the role of the autophagy-related conjugation system in mediating the closure of autophagosomes and its implication in the study of autophagy in mammalian cells. The study not only shows a novel function of the autophagy-related conjugation system but also indicates that mammalian cells are capable of generating autophagosomes even without it.

Response by Shirakabe et al to Letter Regarding Article, “Drp1-Dependent Mitochondrial Autophagy Plays a Protective Role Against Pressure Overload–Induced Mitochondrial Dysfunction and Heart Failure”

We thank Drs Papalia and Okonko for their interest in our work.1 Autophagy is an essential mechanism by which cells maintain the quality of proteins and organelles. Although pressure overload (PO) sequentially activates nonselective autophagy (hereafter autophagy) and mitochondria-selective autophagy (hereafter mitophagy), these autophagic activities do not last long, and mitochondrial dysfunction develops thereafter. Understanding the molecular mechanism by which autophagy and mitophagy are activated transiently but then inactivated during PO should provide a key to sustaining the level of autophagy and delaying the development of heart failure. Drs Papalia and Okonko propose that iron-dependent mechanisms may explain the transient nature of autophagy and the consequent development of mitochondrial dysfunction. The hypothesis that suppression …