Using Action Potential Clamp Data to Determine the Calcium Fluxes and Contributions in Excitation-Contraction Coupling in Vivo in Cardiomyocytes

Abstract

Aim: The significance of the different calcium influx pathways like L-type Ca2+-channels (LTCC), reverse Na+/Ca2+-exchange (NCXrev) or Ca2+-induced Ca2+-release (CICR) varies with species and the state of a cell. For instance, in case of heart failure, CICR decreases and influx via NCXrev increases. The aim of this study is to develop a method to quantify calcium fluxes in cardiac excitation-contraction coupling under physiological conditions.Methods: We used action potential clamp to measure ILTCC and INCX currents in rainbow trout ventricular myocytes. Fluorescence changes induced by Ca2+-transients were recorded using 1μM Fluo-4 AM. To measure ILTCC and/or INCX, we used 10μM nifedipine and 1 or 5mM NiCl2 in extracellular solution. Ryanodine receptors and SR calcium ATPase were inhibited by incubating cells in the presence of 10μM ryanodine and 2μM thapsigargin. We used 240μg/ml amphotericin B in pipette solution for the perforated-patch clamp configuration. Also, we composed a simplified mathematical model of Ca2+-dynamics. By fitting model solution to the recorded experimental data, we determined contribution of LTCC, NCX and CICR currents in trout.Results: From experiments where CICR was inhibited we determined the calcium buffering capacity in the cells that is 55μM (+/- 8 μM). With this method we quantify the contribution and kinetics of LTCC (10-20%), NCX (20-40%) and CICR (50-70%).Conclusions: We have developed a method to quantify calcium fluxes under physiological conditions in cardiomyocytes. Furthermore, we found that SR plays a significant role in trout EC coupling. This is in sharp contrast with previous estimates. Also, the method is not specific to trout and is easily applicable to different species.