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

在心肌细胞中，钙诱导的肌浆网钙释放(CICR)在钙瞬变中起着关键作用。然而，细胞内Na~+浓度([Na~^]_i)的变化也通过Na~(2+)/Ca~(2+)交换对CICR有重要作用。Na~+通道阻滞剂(NCB)的负性肌力作用是通过细胞内钙调节紊乱而实现的。Na~+通道门控可诱导细胞内Na~+蓄积，从而通过反向Na~+/Ca~(2+)&gt；交换(RNCX)增加Ca~(2+)内流。本研究的主要目的是探讨NCB减少细胞内Na~+蓄积是否通过rNCX消除Ca~(2+)内流而导致负性肌力作用。为了阐明rNCX参与Na~+通道阻滞剂的负性变力作用，我们研究了纯Na~+通道阻滞剂Pilsicainide对抽动细胞缩短(C…)频率依赖性增加的影响在Indo-1或Fluo-3负载的豚鼠心室肌细胞上有更多的钙瞬变(CAT)和钙瞬变(CAT)。用钠绿和激光扫描共聚焦显微镜观察细胞内[Na^]i的变化。用全细胞膜片钳方法测定Na^/Ca^；2；交换电流(I&lt；NCx&gt；)。(1)在所有刺激率下，吡西卡胺(&gt；5μM)均可显著降低CAT活性，且这种抑制作用呈剂量依赖性(P&lt；0.0 5 vs对照组，n=7)。(2)RNCX抑制剂1μM KB-R7943仅在2赫兹时降低CS和CAT(INDO-1峰值比值：0.5赫兹时由1.02±0.13降至0.98±0.11，2赫兹时由1.96±0.11降至1.45±0.11，p&lt；0.05，n=7)。(3)加入1μM KB-R7943对30μM plisicainide在2 Hz频率下降低CS和CAT的作用无进一步作用。(4)100μM吡西卡胺对I_lt；Ncx&gt；(5)对照组仅在2 Hz时出现明显的胞浆Na~+积聚，而Pilsicainide在2 Hz时呈剂量依赖性地抑制[Na~+]_i的积聚。(P&lt；0.05与对照组，n=5)。NCB的负性肌力作用可能涉及通过rNCX阻止胞浆(不仅仅是肌膜下)Na~+积聚而减少Ca~(2+)内流。这一机制将对快速性心律失常的心力衰竭有更重要的意义。较少

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