MICU3 Regulates Mitochondrial Calcium and Cardiac Hypertrophy.

Abstract

Calcium (Ca²⁺) uptake by mitochondria occurs via the mitochondrial Ca²⁺ uniporter. Mitochondrial Ca²⁺ uniporter exists as a complex, regulated by 3 MICU (mitochondrial Ca²⁺ uptake) proteins localized in the intermembrane space: MICU1, MICU2, and MICU3. Although MICU3 is present in the heart, its role is largely unknown. We used CRISPR-Cas9 to generate a mouse with global deletion of MICU3 and an adeno-associated virus (AAV9) to overexpress MICU3 in wild-type mice. We examined the role of MICU3 in regulating mitochondrial calcium ([Ca²⁺]_m) in ex vivo hearts using an optical method following adrenergic stimulation in perfused hearts loaded with a Ca²⁺-sensitive fluorophore. Additionally, we studied how deletion and overexpression of MICU3, respectively, impact cardiac function in vivo by echocardiography and the molecular composition of the mitochondrial Ca²⁺ uniporter complex via Western blot, immunoprecipitation, and Blue native-PAGE analysis. Finally, we measured MICU3 expression in failing human hearts. MICU3 knock out hearts and cardiomyocytes exhibited a significantly smaller increase in [Ca²⁺]_m than wild-type hearts following acute isoproterenol infusion. In contrast, heart with overexpression of MICU3 exhibited an enhanced increase in [Ca²⁺]_m compared with control hearts. Echocardiography analysis showed no significant difference in cardiac function in knock out MICU3 mice relative to wild-type mice at baseline. However, mice with overexpression of MICU3 exhibited significantly reduced ejection fraction and fractional shortening compared with control mice. We observed a significant increase in the ratio of heart weight to tibia length in hearts with overexpression of MICU3 compared with controls, consistent with hypertrophy. We also found a significant decrease in MICU3 protein and expression in failing human hearts. Our results indicate that increased and decreased expression of MICU3 enhances and reduces, respectively, the uptake of [Ca²⁺]_m in the heart. We conclude that MICU3 plays an important role in regulating [Ca²⁺]_m physiologically, and overexpression of MICU3 is sufficient to induce cardiac hypertrophy, making MICU3 a possible therapeutic target.