Abstract Body: Introduction: Myocardial ischemia exposes cardiomyocytes to chronic hypoxia and oxidative stress, generating DNA lesions such as 8-oxoguanine that require repair through genome maintenance pathways including mismatch repair (MMR) and base excision repair (BER). While oxidative DNA damage accumulates in ischemic cardiomyocytes, the contribution of impaired DNA repair to cardiomyocyte dysfunction remains poorly understood. We Aim to evaluate how intermittent hypoxia affects DNA damage and DNA repair pathway in cardiomyocytes; define the mutational landscape generated under ischemic stress; and assess whether MMR deficiency directly contributes to cardiomyocyte dysfunction. Approach: Human induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CMs) were exposed to six weeks of intermittent hypoxia to model chronic ischemic stress. Oxidative DNA damage was quantified by 8-oxoG immunostaining. Expression of MMR (MSH2, MSH6, MLH1, MLH3) and BER (XRCC1, DNA-PKcs) proteins was measured. Duplex sequencing characterized mutational signatures. To establish a causal link between MMR deficiency and cardiomyocyte dysfunction, we generated MSH2-knockout (MSH2KO) in hiPSC-CMs using CRISPR. Results:Intermittent hypoxia increased oxidative DNA damage (~2.5-fold, p<0.01) and reduced MMR and BER proteins. Genome sequencing revealed mutational spectra dominated by C>A and C>T substitutions enriched for oxidative damage and MMR-deficiency signatures. MSH2KO cardiomyocytes displayed impaired calcium handling with reduced Ca transient amplitude, prolonged repolarization, decreased beating frequency, and increased nuclear abnormalities. Conclusion:Chronic hypoxic stress induces oxidative DNA damage while suppressing DNA repair pathways in cardiomyocytes. MMR deficiency alone is sufficient to impair cardiomyocyte function, suggesting defective genome maintenance as a mechanistic contributor to myocardial ischemic injury and a potential therapeutic target in ischemic heart disease.