ACTIN Anchors the Highly Oligomeric DRP1 at Mitochondria-Sarcoplasmic Reticulum Contact Sites in Adult Murine Heart: Its Functional Implication

Rationale Mitochondrial fission and fusion are relatively infrequent in adult cardiomyocytes compared to another cell types[1][1]–[3][2]. This is surprising considering that proteins involved in mitochondrial dynamics are highly expressed in the heart. It has been previously reported that dynamin-related protein 1 (DRP1) has a critical role in mitochondrial fitness and cardiac protection[1][1], [4][3]. Cardiac DRP1 ablation in the adult heart evokes a progressive dilated cardiac myopathy and lethal heart failure[1][1]. Nevertheless, the conditional cardiac-specific DRP1 knock-out animals present a significantly longer survival rate compared with global DRP1 KO models[1][1], [4][3], [5][4]. We have described before the great importance for cardiac physiology of the strategic positioning of mitochondrial proteins in the cardiac tissue[6][5], [7][6]. Therefore, we hypothesize that DRP1 plays a regulatory role in cardiac physiology and mitochondrial fitness by preferentially accumulating at mitochondria and junctional sarcoplasmic reticulum (jSR) contact sites, where the high Ca2+ microdomain is formed during excitation-contraction (EC) coupling. Objective This study aims to determine whether mitochondria-associated DRP1 is preferentially accumulated in the mitochondria and jSR contact sites, the mechanism responsible for such a biased distribution, and its functional implication. Methods and Results Using high-resolution imaging approaches, we found that mitochondria-associated DRP1 in cardiomyocytes was localized in the discrete regions where T-tubule, jSR, and mitochondria are adjacent to each other. Western blot results showed that mitochondria-bound DRP1 was restricted to the mitochondria-associated membranes (MAM), with undetectable levels in purified mitochondria. Furthermore, in comparison to the cytosolic DRP1, the membrane-bound DRP1 in SR and MAM fractions formed high molecular weight oligomers demosntratd by 2D blue native technique. In both electrically paced adult cardiomyocytes and Langendorff-perfused beating hearts, the oscillatory Ca2+ pulses preserved MAM-associated DRP1 accumulation. Interestingly, similar to DRP1, all mitochondria-bound β-ACTIN only exists in MAM and not in the purified mitochondria. Additionally, co-immunoprecipitation pulls down both DRP1 and β-ACTIN together. Inhibition of β-ACTIN polymerization with Cytochalasin D disrupts the tight association between DRP1 and β-ACTIN. In cardiac-specific DRP1 knock-out mouse after 6 weeks of tamoxifen induction (DRP1icKo), the cardiomyocytes show disarray of sarcomere, a decrease of cardiac contraction, loss of mitochondrial membrane potential, significantly decreased spare respiratory capacity, and frequent occurrence of early after contraction (EAC), suggesting the heart is susceptible to arrhythmias and heart failure. Despite of this phenotype, DRP1icKo animals have longer life span than other DRP1 KO models. Strikingly, DRP1 levels are only modestly decreased in the MAM when compared with the rest of the cellular fractions. These preserved levels were accompanied by the preservation of the mitochondrial pool in the MAM fraction obtained from the DRP1icKO hearts. Conclusions The results show that in adult cardiomyocytes, mitochondria bound DRP1 clusters in high molecular weight protein complexes at MAM. This clustering is fortified by EC coupling mediated Ca2+ transients and requires its interaction with β-ACTIN. Together with the better preserved DRP1 levels in the DRP1icKO model in the MAM, we conclude that DRP1 is anchored at the mitochondria-SR interface through β-ACTIN and positions itself to play a fundamental role in regulating mitochondrial quality control in the working heart. ### Competing Interest Statement The authors have declared no competing interest. [1]: #ref-1 [2]: #ref-3 [3]: #ref-4 [4]: #ref-5 [5]: #ref-6 [6]: #ref-7