Li L, Chu G, Kranias E G, Bers D M
Department of Physiology, Stritch School of Medicine, Loyola University Chicago, Maywood, Illinois 60153, USA.
Am J Physiol. 1998 Apr;274(4):H1335-47. doi: 10.1152/ajpheart.1998.274.4.H1335.
Increases in heart rate are accompanied by acceleration of relaxation. This effect is apparent at the single myocyte level and depends on sarcoplasmic reticulum (SR) Ca transport and Ca/calmodulin dependent protein kinase [CaMKII; see R. A. Bassani, A. Mattiazzi, and D. M. Bers. Am. J. Physiol. 268 (Heart Circ. Physiol. 37): H703-H712, 1995]. Because phosphorylation of phospholamban (PLB) by CaMKII can stimulate SR Ca transport, it is a plausible candidate mechanism. We examined this issue using ventricular myocytes isolated from wild-type (WT) mice and those in which the PLB gene was ablated by gene targeting (PLB-KO). During steady-state (SS) stimulation, twitch relaxation and intracellular Ca concentration ([Ca]i) decline were significantly faster than after a rest in both WT and PLB-KO myocytes. Furthermore, the CaMKII inhibitor KN-93 (1 microM) abolished the stimulation-dependent acceleration of twitch [Ca]i decline in PLB-KO. This indicates that neither PLB nor its phosphorylation are required for the CaMKII-dependent acceleration of the SS twitch [Ca]i decline and relaxation. Other quantitative aspects of Ca transport in WT and PLB-KO myocytes were also examined. As expected, the time constant (tau) of [Ca]i decline during the SS twitch is much faster in PLB-KO than in WT myocytes (112 +/- 6 vs. 188 +/- 14 ms, P < 0.0001). There was also an increase in SS SR Ca load, based on the change of [Ca]i during rapid caffeine-induced contractures (CafC) with Na/Ca exchange blocked (565 +/- 74 nM for WT, 1118 +/- 133 nM for PLB-KO, P < 0.01). Accounting for cytosolic Ca buffering, this implies a 37% increase in SR Ca content. The tau for [Ca]i decline of the cafC with Na present indicated slower extrusion by Na/Ca exchange in the PLB-KO mouse (2.2 +/- 0.2 s in WT vs. 3.2 +/- 0.2 in PLB-KO, P < 0.01), although exchanger protein expression was unchanged. Integrated Ca flux analysis in WT and PLB-KO myocytes, respectively, shows that 90 and 96% of Ca during twitch relaxation is removed by the SR Ca-ATPase, 9 and 3.4% by Na/Ca exchange, and 0.5 and 0.1% by slow mechanisms (mitochondria Ca uniporter and sarcolemmal Ca-ATPase). We conclude that the PLB-KO myocytes retain a CaMKII-dependent acceleration of SS twitch [Ca]i decline. The PLB-KO (vs. WT) myocytes also have higher SR Ca pump activity, higher SR Ca load, and reduced Na/Ca exchange activity.
心率增加伴随着舒张加速。这种效应在单个心肌细胞水平上很明显，并且依赖于肌浆网（SR）钙转运和钙/钙调蛋白依赖性蛋白激酶[CaMKII；见R.A.巴萨尼、A.马蒂亚齐和D.M.贝尔斯。《美国生理学杂志》268卷（心脏循环生理学37）：H703 - H712，1995年]。由于CaMKII对受磷蛋白（PLB）的磷酸化可刺激SR钙转运，所以它是一个合理的候选机制。我们使用从野生型（WT）小鼠分离的心室肌细胞以及通过基因靶向使PLB基因缺失的小鼠（PLB - KO）的心室肌细胞来研究这个问题。在稳态（SS）刺激期间，WT和PLB - KO心肌细胞的单收缩舒张和细胞内钙浓度（[Ca]i）下降都比休息后明显更快。此外，CaMKII抑制剂KN - 93（1微摩尔）消除了PLB - KO中刺激依赖性的单收缩[Ca]i下降加速。这表明对于CaMKII依赖性的SS单收缩[Ca]i下降和舒张加速，PLB及其磷酸化都不是必需的。还研究了WT和PLB - KO心肌细胞中钙转运的其他定量方面。正如预期的那样，SS单收缩期间[Ca]i下降的时间常数（tau）在PLB - KO中比WT心肌细胞快得多（112±6对188±14毫秒，P < 0.0001）。基于快速咖啡因诱导的挛缩（CafC）期间[Ca]i的变化且钠/钙交换被阻断，SS SR钙负荷也增加了（WT为565±74纳摩尔，PLB - KO为1118±133纳摩尔，P < 0.01）。考虑到胞质钙缓冲，这意味着SR钙含量增加了37%。存在钠时CafC的[Ca]i下降的tau表明PLB - KO小鼠中钠/钙交换的钙外排较慢（WT中为2.2±0.2秒，PLB - KO中为3.2±0.2秒，P < 0.01），尽管交换蛋白表达没有变化。分别对WT和PLB - KO心肌细胞进行的综合钙通量分析表明，单收缩舒张期间90%和96%的钙通过SR钙ATP酶去除，9%和3.4%通过钠/钙交换去除，0.5%和0.1%通过缓慢机制（线粒体钙单向转运体和肌膜钙ATP酶）去除。我们得出结论，PLB - KO心肌细胞保留了CaMKII依赖性的SS单收缩[Ca]i下降加速。PLB - KO（与WT相比）心肌细胞还具有更高的SR钙泵活性、更高的SR钙负荷和降低的钠/钙交换活性。
