Young Investigators Competition251Mitofusin 2 controls calcium transmission between the SR and mitochondria and regulates the bioenergetic feedback response in cardiac myocytes

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Abstract

Background

Mitochondrial Ca2+ uptake stimulates key enzymes of the Krebs cycle, which produces NADH for ATP production and NADPH that is required for elimination of H2O2. In heart failure, dysregulation of cytosolic Ca2+ and Na+ homeostasis accounts for reduced mitochondrial Ca2+ uptake which results in energetic mismatch and oxidative stress. It is unclear, however, whether these defects play a causal role for the development of heart failure. Close association between the sarcoplasmic reticulum (SR) and mitochondria are thought to constitute a "microdomain" that could explain efficient mitochondrial Ca2+ uptake despite a low Ca2+ affinity of the mitochondrial Ca2+ uniporter. The molecular determinants of such a microdomain, however, are presently unknown.

Methods and Results

We generated mice with postnatal and cardiomyocyte-specific deletion (KO) of mitofusin (Mfn) 1 or 2, proteins originally known to mediate mitochondrial outer membrane fusion. Compared to their wild-type littermates (WT), cardiac size and contractile function were normal in 6 week old Mfn1 and Mfn2 KO mice. Mitochondrial-SR architecture (determined by electron microscopy) was normal, but mean contact length between SR and adjacent mitochondria was decreased by 50%, and SR-mitochondrial mean gap distance was increased by 38% in Mfn2-KO, but not Mfn1-KO hearts, demonstrating Mfn2 involvement in SR-mitochondrial tethering. Isolated cardiac myocytes were paced at 0.5 Hz, and then a physiological increase in workload was simulated by applying the β-adrenergic agonist isoproterenol (30 nM) and increasing stimulation frequency to 5 Hz for 3 min. During this transition, mitochondrial Ca2+ accumulation (measured by rhod-2 using a patch-clamp based approach) was blunted at 0.5 Hz in Mfn2-KO myocytes compared to WT, despite unchanged mitochondrial membrane potential (as determined by TMRM). This was associated with an oxidation of NAD(P)H and FADH2, the main products of the Krebs cycle, and a slight increase in mitochondrial superoxide formation in Mfn2-KO vs. WT myocytes (p < 0.05). In contrast, no such changes were detected in Mfn1-KO mice. By the age of 16 weeks, Mfn2-KO mice developed dilated cardiomyopathy, while Mfn1-KO mice developed normal.

Conclusions

Mfn2 (but not Mfn1) tethers mitochondria to the SR and thus, is an important molecular determinant of the mitochondrial Ca2+ microdomain. This has important implications for energy supply and demand matching and the control of oxidative stress. Defects in mitochondria-SR Ca2+ transmission play a causal role for the development of heart failure.

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