Abstract
Background Cardiovascular diseases remain the leading cause of death worldwide, accounting for approximately 19.41 million deaths annually. Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) hold great promise for disease modeling and therapy. However, variability in differentiation efficiency and maturation state remains a significant challenge. Calcium signaling plays a pivotal role in cardiac differentiation and maturation, with mitochondrial calcium dynamics closely linked to transcriptional activation. Recent evidence suggests that closure of the mitochondrial permeability transition pore promotes cardiomyocyte differentiation by stabilizing calcium homeostasis and reducing oxidative stress. Methods We investigated the effect of 7-aminoindole (7-AI), a mitochondrial calcium uniporter inhibitor, on cardiomyocyte differentiation. Mouse embryonic stem cells and hiPSCs were treated with 7-AI during the cardiac progenitor stage (day 4). Nuclear calcium levels, Ca2+/calmodulin-dependent protein kinase (CaMK) activation, CREB phosphorylation, and cardiac marker expression were assessed. Results 7-AI increased nuclear calcium levels, activated CaMK and CREB, and upregulated cardiac-specific markers, including cTnT, α-SA, and MYH6/7. The resulting cardiomyocytes exhibited improved sarcomere organization and enhanced contractility. Moreover, CREB-deficient cells failed to exhibit these effects, confirming that CREB activation is essential for 7-AI-mediated cardiac maturation. Conclusions Collectively, our findings demonstrate that inhibition of mitochondrial calcium influx redistributes calcium to the nucleus, thereby activating the CaMK-CREB signaling axis and promoting cardiomyocyte differentiation. Targeting mitochondrial calcium handling at the cardiac progenitor stage provides a mechanistic and pharmacological approach to enhance the structural and functional maturation of cardiomyocytes for therapeutic application.