AIMS: The PDHA1 gene, responsible for regulating the conversion of the glycolytic product pyruvate to acetyl CoA, is significantly reduced in cardiomyocytes of patients with hypertrophic cardiomyopathy. Cardiac-specific PDHA1-deficient mice demonstrate cardiac hypertrophy and heart failure. However, the mechanisms underlying the pathogenesis of PDHA1 deficiency remain unclear. MAIN METHODS: PDHA1 gene in human induced pluripotent stem cell line (iPSC) was knockout (KO) using CRISPR-Cas9 technology and differentiated it into cardiomyocytes (CMs) in vitro. Contractile force was quantified by video analysis, Ca KEY FINDINGS: The PDHA1 KO iPSC-CMs displayed myocardial hypertrophy phenotypes by day 40 post-differentiation, characterized by enlarged cell size, increased contractility, abnormal calcium handling, and progressed to mimic heart failure phenotypes by day 50, including reduced contractility, lower calcium release and increased ROS generation. RNA-seq analysis revealed dysregulated expression of pathways related to cardiac hypertrophy and the calcium signaling pathway in KO iPSC-CMs. Furthermore, KO iPSC-CMs exhibited decreased energy production before the manifestation of myocardial hypertrophic phenotype at day 30, exacerbating intracellular lactate accumulation, leading to increased sodium‑hydrogen and sodium‑calcium exchange, ultimately resulting in elevated diastolic calcium concentration. Augmenting energy production with l-carnitine restored diastolic Ca SIGNIFICANCE: Elevated diastolic Ca