To elucidate the molecular mechanism by which SSB accumulation in cardiomyocytes exacerbates pressure overload-induced heart failure, we established an in vitro model of cardiomyocytes with SSB accumulation by using alkylating agent methyl methanesulfonate (MMS), which induces only DNA SSB20, and small interfering RNA (siRNA)-mediated knockdown of Xrcc1. To optimize the experimental condition of the comet assays and ISOL staining in in vitro cultured cardiomyocytes, we used irradiation to induce DNA DSB and confirmed that DNA DSB can be detected in both assays (Supplementary Fig. 6a,b). The gene discussed is XRCC1; the disease is heart failure.