Data for Roman et al published in Circ Res

Background: Calcium (Ca²⁺) uptake by mitochondria occurs via the mitochondrial Ca²⁺ uniporter (MCU). MCU exists as a complex (MCUc), regulated by three MICU (mitochondrial Ca²⁺ uptake) proteins localized in the intermembrane space, MICU1, 2, and 3. Although MICU3 is present in heart, its role is largely unknown.

Methods: We used CRISPR-Cas9 to generate a mouse with global deletion of MICU3 (MICU3-KO) and an adeno-associated virus (AAV9) to overexpress MICU3 (MICU2-OE) in wild-type (WT) 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 MCUc via Western blot, immunoprecipitation and Blue Native PAGE analysis. Finally we measured MICU3 expression in failing human hearts.

Results: MICU3-KO hearts and cardiomyocytes exhibited a significantly smaller increase in [Ca²⁺]m than WT hearts following acute isoproterenol infusion. In contrast, MICU3-OE hearts exhibited an enhanced increase in [Ca²⁺]m compared to control hearts. Echocardiography analysis showed no significant difference in cardiac function in MICU3-KO mice relative to WT at baseline. However, MICU3-OE animals 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 MICU3-OE hearts compared to controls consistent with hypertrophy. We also found a significant decrease in MICU3 protein and expression in failing human hearts.

Conclusion: 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.