Temperature and transmural region influence functional measurements in unloaded left ventricular cardiomyocytes

Intact cardiomyocytes are increasingly being used to investigate the molecular mechanisms of contraction and to screen new therapeutic compounds. The function of the cardiomyocytes is often measured from the calcium transients and sarcomere length profiles. We studied the role of experimental temperature and transmural region on indices of function in freshly isolated, unloaded cardiomyocytes. Intact cardiomyocytes were isolated from the subendocardium, midmyocardium, and subepicardium of 3-month-old Sprague-Dawley rats. Myocytes from each region were studied at 25°C, 31°C, and 37°C. Cytosolic calcium transients were measured using Fura-2 fluorescence, whereas sarcomere length shortening and relengthening profiles were measured using high-speed video capture. For both the calcium transients and sarcomere length profiles, the time to peak and the time to half relaxation decreased significantly with increasing temperature. Increasing temperature also raised the minimum and maximum calcium levels of all cells. Of note, there was a reduced coefficient of variation (standard deviation divided by the mean) at higher temperatures for calcium fluorescence amplitudes, time to peak calcium, and rates of sarcomeric shortening and relengthening. The amplitudes and minimum of the calcium transients were significantly dependent on transmural region, and several sarcomere length parameters exhibited statistical interactions between temperature and transmural region. Together, these results show that biological variability can be reduced by performing experiments at 37°C rather than at room temperature, and by isolating cells from a specific transmural region. Adopting these procedures will improve the statistical power of subsequent analyses and increase the efficiency of future experiments.