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Abstract: Mice are extensively used for gene modification research and isolated cardiomyocytes are essential for evaluation of cardiac function without interference from non-myocyte contribution. This study was designed to characterize cardiomyocyte excitation–contraction coupling in FVB/N-C57BL/6 intercrossed brown mice. Mechanical and intracellular Ca2+ properties were evaluated using an IonOptix softedge system including peak shortening (PS), time-to-PS (TPS), time-to-90% relengthening (TR90), maximal velocity of shortening and relengthening (± dL /dt), intracellular Ca2+ rise and decay rate. Resting cell length was longer in age- and gender-matched C57BL/6 and brown mice compared to FVB strain. PS and ± dL /dt were significantly lower in brown mice compared to FVB/N and C57BL/6 groups. TPS was shortened in C57BL/6 mice and TR90 was prolonged in brown mice compared to other groups. Resting intracellular Ca2+ level and single exponential intracellular Ca2+ decay constant were comparable among all three mouse lines. Rise in intracellular Ca2+ in response to electrical stimulus was higher in C57BL/6 mouse myocytes whereas bi-exponential intracellular Ca2+ decay was faster in brown mice. Myocytes from all three groups exhibited similar fashion of reduction in PS in response to increased stimulus frequency. These data suggest that inherent differences in cardiomyocyte excitation–contraction coupling exist between strains, which may warrant caution when comparing data from these mouse lines. [Copyright &y& Elsevier]