Abstract Heart degenerative diseases pose a significant challenge due to the limited ability of native heart to restore lost cardiomyocytes. Direct cellular reprogramming technology, particularly the use of small molecules, has emerged as a promising solution to prepare functional cardiomyocyte through faster and safer processes without genetic modification. However, current methods of direct reprogramming often exhibit low conversion efficiencies and immature characteristics of the generated cardiomyocytes, limiting their use in regenerative medicine. This study proposes the use of mitochondrial delivery to metabolically reprogram chemically induced cardiomyocyte‐like cells (CiCMs), fostering enhanced maturity and functionality. Our findings show that mitochondria sourced from high‐energy‐demand organs (liver, brain, and heart) can enhance structural maturation and metabolic functions. Notably, heart‐derived mitochondria resulted in CiCMs with a higher oxygen consumption rate capacity, enhanced electrical functionality, and higher sensitivity to hypoxic condition. These results are related to metabolic changes caused by increased number and size of mitochondria and activated mitochondrial fusion after mitochondrial treatment. In conclusion, our study suggests that mitochondrial delivery into CiCMs can be an effective strategy to promote cellular maturation, potentially contributing to the advancement of regenerative medicine and disease modeling.
