Study for detection of intracellular sodium transients and their pathophysiological roles in myocytes

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In cardiac myocytes the Ca^<2+>-induced Ca^<2+> release (CICR) from the sarcoplasmicr reticulum (SR) plays pivotal roles in Ca transients. However, the changes in intracellular Na^+ concentration ([Na^+]_i) also contribute significantly to CICR via Na^+/Ca^<2+> exchange. The negative inotropic effect of Na^+ channel blockers (NCB) is mediated by disturbance of intracellular Ca^<2+> regulation. The Na^+ channel gating could induce intracellular Na^+ accumulation, thereby increasing Ca^<2+> influx via the reverse-mode Na^+/Ca^<2+> exchange (rNCX). The primary aim of this study was to investigate whether the reduction of cellular Na^+ accumulation by NCB actually contributes to the negative inotropic effects through the elimination of Ca^<2+> influx via rNCX. To elucidate the involvement of rNCX in the negative inotropic effects of Na^+ channel blockers, we examined the effects of a pure Na^+ channel blocker, pilsicainide, on the frequency-dependent increases in twitch cell shortenings (C … More S) and Ca^<2+> transients (CaT) in Indo-1 or fluo-3 loaded guinea pig ventricular myocytes. The changes in [Na^+]_i was also evaluated with Sodium-Green and laser scanning confocal microscopy. Na^+/Ca^<2+> exchange current (I_<NCX>) was measured by the whole cell patch clamp method. (1) Pilsicainide (>5 μM) significantly reduced CaT at all stimulation rates, and the reduction was more prominent use-dependently (p<0.05 vs. control, n=7). (2) An inhibitor of rNCX, 1 μM KB-R7943 decreased CS and CaT only at 2 Hz (peak indo-1 ratio ; from 1.02±0.13 to 0.98±0.11 at 0.5 Hz, n.s., 1.96±0.11 to 1.45±0.11 at 2 Hz, p<0.05, n=7). (3) On diminishing CS and CaT by 30 μM plisicainide at 2 Hz, the following addition of 1 μM KB-R7943 had no further effects. (4) 100 μM Pilsicainide did not affect to I_<NCX>. (5) The significant cytosolic Na^+ accumulation was observed only at 2 Hz in control, and Pilsicainide suppressed the accumulation in [Na^+]_i dose-dependently at 2 Hz. (p<0.05 vs. control, n=5). The negative inotropic effects of NCB could involve the reduction of Ca^<2+> influx via rNCX by preventing cytosolic (not only subsarcolemmal) Na^+ accumulation. The mechanism would have a more important implication in failed heart with tachyarrhythmia. Less