Ca2+ paradox injury mediated through TRPC channels in mouse ventricular myocytes

BACKGROUND AND PURPOSE The Ca2+ paradox is an important phenomenon associated with Ca2+ overload-mediated cellular injury in myocardium. The present study was undertaken to elucidate molecular and cellular mechanisms for the development of the Ca2+ paradox. EXPERIMENTAL APPROACH Fluorescence imaging was performed on fluo-3 loaded quiescent mouse ventricular myocytes using confocal laser scanning microscope. KEY RESULTS The Ca2+ paradox was readily evoked by restoration of the extracellular Ca2+ following 10-20 min of nominally Ca2+-free superfusion. The Ca2+ paradox was significantly reduced by blockers of transient receptor potential canonical (TRPC) channels (2-aminoethoxydiphenyl borate, Gd3+, La3+) and anti-TRPC1 antibody. The sarcoplasmic reticulum (SR) Ca2+ content, assessed by caffeine application, gradually declined during Ca2+-free superfusion, which was further accelerated by metabolic inhibition. Block of SR Ca2+ leak by tetracaine prevented Ca2+ paradox. The Na+/Ca2+ exchange (NCX) blocker KB-R7943 significantly inhibited Ca2+ paradox when applied throughout superfusion period, but had little effect when added for a period of 3 min before and during Ca2+ restoration. The SR Ca2+ content was better preserved during Ca2+ depletion by KB-R7943. Immunocytochemistry confirmed the expression of TRPC1, in addition to TRPC3 and TRPC4, in mouse ventricular myocytes. CONCLUSIONS AND IMPLICATIONS These results provide evidence that (i) the Ca2+ paradox is primarily mediated by Ca2+ entry through TRPC (probably TRPC1) channels that are presumably activated by SR Ca2+ depletion; and (ii) reverse mode NCX contributes little to the Ca2+ paradox, whereas inhibition of NCX during Ca2+ depletion improves SR Ca2+ loading, and is associated with reduced incidence of Ca2+ paradox in mouse ventricular myocytes.