The effects of multiple mitochondrial dysfunction (MMD) on heart, a highly mitochondria-dependent tissue, is still unclear. This study was the first to verify the effect of ISCA1 gene deficiency, which has been shown to cause multiple mitochondrial dysfunction syndromes type 5 (MMDS5), on cardiac development in vivo, that is cardiomyocytes suffer from energy shortage due to abnormal metabolism of iron ion, which leads to oncosis and eventually HF and body death. Subsequently, we determine a new interacting molecule for ISCA1, six-transmembrane epithelial antigen of prostate 3 (STEAP3), which acts as a reductase in the reduction of Fe3+ to Fe2+. Forward and reverse validation experiments demonstrated that STEAP3 plays an important role in iron metabolism and energy generation impairment induced by ISCA1 deficiency. This result provides theoretical basis for understanding of MMDS pathogenesis, especially on heart development and the pathological process of heart diseases, and finally provides new clues for searching of clinical therapeutic targets.