Phosphodiesterases Restrict Spontaneous Beating of Cardiac Pacemaker Cells

兔窦房结细胞（SANCs）的自发跳动是由camp介导的，蛋白激酶依赖的局部肌上皮下ryanodine受体Ca2+释放（LCRs）控制的。lcr激活了向内的Na+/ Ca2+交换电流，增加了舒张末期去极化率，从而增加了自发的SANC跳动率。SANCs的基础cAMP升高，表明磷酸二酯酶（PDEs）对cAMP的降解可能很低。令人惊讶的是，广谱PDE抑制剂3-异丁基甲基黄嘌呤（IBMX）对PDE活性的完全抑制，使cAMP水平增加了9倍，cAMP介导的蛋白激酶依赖的磷蛋白磷酸化增加了一倍，并使SANC发射率增加了55%，这表明sans中PDEs的基础活性很高。特异性PDE1与-5抑制剂的比较显示，特异性PDE3抑制剂米立酮使自发放电加速了47%（其他抑制剂的影响较小），L型Ca2+电流（ICa，L）的振幅增加了46%，表明PDE3是基础状态下主要的组成活性PDE。PDE依赖性对自发SANC放电的控制严重依赖于肌层下LCR，即PDE抑制增加了LCR振幅和大小，缩短了LCR周期，导致LCR Ca2+释放提前和增强，Na+/ Ca2+交换电流增加，放电速率增加。当ryanodine受体被ryanodine禁用时，IBMX和milrinone都不能扩增lcr，加速舒张去极化速率，或增加SANC放电速率，尽管保留了PDE抑制诱导的ICa，L振幅增加。因此，基础组成性PDE激活提供了一种新颖而有力的机制来降低cAMP，限制cAMP介导的，蛋白激酶依赖性的舒张期ryanodine受体Ca2+释放的增加，并限制自发的SANC搏动率。为了研究PDE抑制如何控制SR Ca2+再填充和LCR周期，我们比较了PDE抑制和对照后SR Ca2+再填充的动力学。磷蛋白的磷酸化（PLB）被用作SR泵送速率的指标，SR再填充是通过ap启动的整体胞质Ca2+瞬态（T-90）衰减到90%的时间来估计的。广谱磷酸二酯酶抑制剂（IBMX）、特异性磷酸二酯酶-3抑制剂（milrinone）或特异性PKA抑制剂肽（PKI）对PLB的分级磷酸化与T-90的比例变化平行。同时T-90和LCR周期的变化与自发周期长度的变化高度相关。因此，抑制PDE活性增加camp介导的pka依赖性磷酸化，加速SR Ca2+再填充速率，减少LCR周期，Ca2+时钟，并增加SANC的自发搏动速率。

Spontaneous beating of rabbit sinoatrial node cells (SANCs) is controlled by cAMP-mediated, protein kinase Adependent local subsarcolemmal ryanodine receptor Ca2+ releases (LCRs). LCRs activated an inward Na+/ Ca2+ exchange current that increases the terminal diastolic depolarization rate and, therefore, the spontaneous SANC beating rate. Basal cAMP in SANCs is elevated, suggesting that cAMP degradation by phosphodiesterases (PDEs) may be low. Surprisingly, total suppression of PDE activity with a broad-spectrum PDE inhibitor, 3-isobutylmethylxanthine (IBMX), produced a 9-fold increase in the cAMP level, doubled cAMP-mediated, protein kinase Adependent phospholamban phosphorylation, and increased SANC firing rate by ≈55%, indicating a high basal activity of PDEs in SANCs. A comparison of specific PDE1 to -5 inhibitors revealed that the specific PDE3 inhibitor, milrinone, accelerated spontaneous firing by ≈47% (effects of others were minor) and increased amplitude of L-type Ca2+ current (ICa,L) by ≈46%, indicating that PDE3 was the major constitutively active PDE in the basal state. PDE-dependent control of the spontaneous SANC firing was critically dependent on subsarcolemmal LCRs, ie, PDE inhibition increased LCR amplitude and size and decreased LCR period, leading to earlier and augmented LCR Ca2+ release, Na+/ Ca2+ exchange current, and an increase in the firing rate. When ryanodine receptors were disabled by ryanodine, neither IBMX nor milrinone was able to amplify LCRs, accelerate diastolic depolarization rate, or increase the SANC firing rate, despite preserved PDE inhibitioninduced augmentation of ICa,L amplitude. Thus, basal constitutive PDE activation provides a novel and powerful mechanism to decrease cAMP, limit cAMP-mediated, protein kinase Adependent increase of diastolic ryanodine receptor Ca2+ release, and restrict the spontaneous SANC beating rate. To study how PDE inhibition controls the SR Ca2+ refilling and LCR period we compared kinetics of SR Ca2+ refilling in control and after PDE inhibition. Phosphorylation of phospholamban (PLB) has been used as index of SR pumping rate and SR refilling was estimated by the time to 90% decay of the AP-initiated global cytosolic Ca2+ transient (T-90). Graded PLB phosphorylation by a broad-spectrum phosphodiesterase inhibitor (IBMX), specific phosphodiesterase-3 inhibitor (milrinone), or by specific PKA inhibitor peptide (PKI) were paralleled by proportional changes in T-90. Concomitant changes in T-90 and LCR period were highly correlated with changes in the spontaneous cycle length. Thus, suppression of PDE activity increases cAMP-mediated PKA-dependent phosphorylation, accelerates the SR Ca2+ refilling rate, decreases the LCR period, Ca2+ clock, and increases spontaneous beating rate of SANC.