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

在心肌细胞中，Ca^<2+>-诱导的肌浆网（SR） Ca^<2+>释放（CICR）在Ca瞬态中起关键作用。然而，胞内Na^+浓度（[Na^+]_i）的变化也通过Na^+/Ca^<2+>交换对CICR有显著贡献。Na^+通道阻滞剂（NCB）的负性肌力效应是通过干扰细胞内Ca^<2+>调节介导的。Na^+通道门控可诱导细胞内Na^+积累，从而通过Na^+/Ca^<2+>反向交换（rNCX）增加Ca^<2+>内流。本研究的主要目的是调查NCB通过rNCX消除Ca^<2+>内流而减少细胞Na^+积累是否实际上有助于负性肌力效应。为了阐明rNCX在Na^+通道阻滞剂的负性肌力作用中的作用，我们检测了纯Na^+通道阻滞剂匹西卡因（pilsicainide）对Indo-1或fluo-3负荷豚鼠心室肌细胞抽动细胞缩短（C…More S）和Ca^<2+>瞬变（CaT）的频率依赖性增加的影响。用钠绿显微镜和激光扫描共聚焦显微镜观察[Na^+]_i的变化。采用全细胞膜片钳法测定Na^+/Ca^<2+>交换电流（I_<NCX>）。(1)在所有刺激速率下，匹西卡因胺（bbb50 μM）均能显著降低CaT，且对使用的依赖性更为显著（p<0.05，与对照组相比，n=7）。(2) rNCX抑制剂1 μM KB-R7943仅在2 Hz时降低CS和CaT（峰值indo-1比值从0.5 Hz时的1.02±0.13降至0.98±0.11,n.s， 2 Hz时的1.96±0.11降至1.45±0.11,p<0.05, n=7）。(3)用30 μM plisicainide在2 Hz下降低CS和CaT，随后加入1 μM KB-R7943没有进一步的影响。(4) 100 μM匹西卡因胺对I_<NCX>无影响。(5)对照组仅在2hz时胞内Na^+积累显著，而匹西卡奈在2hz时抑制Na^+积累呈剂量依赖性。（p<0.05与对照组比较，n=5）。NCB的负性肌力效应可能包括通过阻止胞质（不仅是肌层下）Na^+积累，通过rNCX减少Ca^<2+>内流。该机制可能对心力衰竭伴速性心律失常有更重要的意义。少

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