Mitofusin 2 Is Essential for IP₃-Mediated SR/Mitochondria Metabolic Feedback in Ventricular Myocytes

Aim: Endothelin-1 (ET-1) and angiotensin II (Ang II) are multifunctional peptide hormones that regulate the function of the cardiovascular and renal systems. Both hormones increase the intracellular production of inositol-1,4,5-trisphosphate (IP₃) by activating their membrane-bound receptors. We have previously demonstrated that IP₃-mediated sarcoplasmic reticulum (SR) Ca²⁺ release results in mitochondrial Ca²⁺ uptake and activation of ATP production. In this study, we tested the hypothesis that intact SR/mitochondria microdomains are required for metabolic IP₃-mediated SR/mitochondrial feedback in ventricular myocytes. Methods: As a model for disrupted mitochondrial/SR microdomains, cardio-specific tamoxifen-inducible mitofusin 2 (Mfn2) knock out (KO) mice were used. Mitochondrial Ca²⁺ uptake, membrane potential, redox state, and ATP generation were monitored in freshly isolated ventricular myocytes from Mfn2 KO mice and their control wild-type (WT) littermates. Results: Stimulation of ET-1 receptors in healthy control myocytes increases mitochondrial Ca²⁺ uptake, maintains mitochondrial membrane potential and redox balance leading to the enhanced ATP generation. Mitochondrial Ca²⁺ uptake upon ET-1 stimulation was significantly higher in interfibrillar (IFM) and perinuclear (PNM) mitochondria compared to subsarcolemmal mitochondria (SSM) in WT myocytes. Mfn2 KO completely abolished mitochondrial Ca²⁺ uptake in IFM and PNM mitochondria but not in SSM. However, mitochondrial Ca²⁺ uptake induced by beta-adrenergic receptors activation with isoproterenol (ISO) was highest in SSM, intermediate in IFM, and smallest in PNM regions. Furthermore, Mfn2 KO did not affect ISO-induced mitochondrial Ca²⁺ uptake in SSM and IFM mitochondria; however, enhanced mitochondrial Ca²⁺ uptake in PNM. In contrast to ET-1, ISO induced a decrease in ATP levels in WT myocytes. Mfn2 KO abolished ATP generation upon ET-1 stimulation but increased ATP levels upon ISO application with highest levels observed in PNM regions. Conclusion: When the physical link between SR and mitochondria by Mfn2 was disrupted, the SR/mitochondrial metabolic feedback mechanism was impaired resulting in the inability of the IP₃-mediated SR Ca²⁺ release to induce ATP production in ventricular myocytes from Mfn2 KO mice. Furthermore, we revealed the difference in Mfn2-mediated SR-mitochondrial communication depending on mitochondrial location and type of communication (IP₃R-mRyR1 vs. ryanodine receptor type 2-mitochondrial calcium uniporter).