PDE3, PDE4 and PKC regulate local Ca2+ releases and cardiac pacemaker firing

PDE3、PDE4 和 PKC 调节局部 Ca2 释放和心脏起搏器放电

• 批准号: 8736511
• 负责人: Edward Lakatta
• 金额: $ 20.67万
• 依托单位: NATIONAL INSTITUTE ON AGING
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8736511
• 关键词: Acceleration; Artificial cardiac pacemaker; Cells; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Disabled Persons; Ensure; Goals; Length; Mediating; Oryctolagus cuniculus; Phosphodiesterase Inhibitors; Phosphorylation; Pump; Regulation; Role; Rolipram; Ryanodine; Ryanodine Receptors; Signal Transduction; Sinoatrial Node; Site; Testing; cilostamide; inhibitor/antagonist; nodal myocyte; patch clamp; phospholamban; phosphoric diester hydrolase; synergism

To test the hypothesis that combined activity of (PDE3+PDE4) impacts on basal spontaneous SANC firing via regulation of cAMP/PKA signaling, we used phosphorylation of phospholamban (PLB) at Ser16 site as a marker of cAMP/PKA-dependent protein phosphorylation in SANC. Specific PDE3 inhibitor, cilostamide (Cil, 0.3 mkmol/L), or a PDE4 inhibitor, rolipram (Rol, 2 mkmol/L), increased PLB phosphorylation by 20%, but the combination of Cil+Rol increased PLB phosphorylation by 110%, an effect similar to that (140%) produced by broad spectrum PDE inhibitor IBMX. L-type Ca2+ current (ICa,L) ensures LCR existence, providing Ca2+ available for pumping in SR. Cil or Rol alone increased the amplitude of ICa,L by 60% and 4%, respectively, while (Cil+Rol) or IBMX increased ICa,L by 100%. Cilostamide increased the spontaneous SANC firing rate (perforated patch-clamp) by 20% (from 14813 to 17513 beat/min, n=8), while rolipram produced no acceleration of spontaneous firing at 2, 20 or 100 mkmol/L. Similar to IBMX, (Cil+Rol) increased the spontaneous SANC beating rate by 50% (from 1348 to 19711 beat/min, n=9), the effect was due to a marked increase in the LCR number, size and decrease in the LCR period that predicted the concomitant decrease in the spontaneous cycle length. When RyR were disabled by ryanodine and LCRs were abolished, both IBMX and (Cil+Rol) failed to accelerate DD rate or increase SANC firing rate indicating key role of Ca2+ cycling for PDE-dependent control of spontaneous beating. We conclude that both PDE3 and PDE4 regulate spontaneous SANC firing, and a crucial role of PDE4 is revealed only when PDE3 and PDE4 are concurrently inhibited. Thus, synergism of (PDE3+PDE4) suppresses basal cAMP/PKA-dependent PLB phosphorylation and reduces ICa,L amplitude to decrease RyR Ca2+ release, prolong the LCR period and limit the spontaneous SANC firing rate.

为了验证(PDE3+PDE4)的联合活动通过调节cAMP/PKA信号而影响基础自发放电的假说，我们使用Ser16位点的磷蛋白(PLB)磷酸化作为SANC中cAMP/PKA依赖的蛋白磷酸化的标志。特异性PDE3抑制剂西洛胺(Cil，0.3mkmol/L)或PDE4抑制剂罗利普兰(Rolipram，2mkmoL/L)可使PLB磷酸化增加20%，但Cil+Rol联合应用可使PLB磷酸化增加110%，与广谱PDE抑制剂IBMX的作用相似(140%)。L型钙电流(ICa，L)保证了LCR的存在，为SR提供了可供泵入的钙离子。CIL或Rol使ICa、L的波幅分别增加60%和4%，而(CIL+Rol)或IBMX使ICa、L的波幅增加100%。西洛司胺使自发性放电频率(穿孔膜片钳)增加20%(从14813次/分增加到17513次/分，n=8)，而罗利普兰在2、20和100mkmol/L时对自发放电无加速作用。与IBMX相似，(CIL+Rol)使自发性搏动频率增加50%(从1,348次/分增加到19711次/分，n=9)，这是由于LCR的数目、大小和LCR期的减少预测了伴随的自发周期长度的减少。当RyR被ryanodine禁用和LCRs被取消时，IBMX和(Cil+Rol)都不能加速DD率或增加SANC放电率，这表明钙循环在PDE依赖的自发搏动控制中起关键作用。我们的结论是，PDE3和PDE4都调节自发放电，只有当PDE3和PDE4同时被抑制时，PDE4才显示出关键作用。因此，(PdE_3+PdE_4)协同作用抑制基础cAMP/PKA依赖的原球蛋白的磷酸化，降低ICa、L的幅度，从而减少RyRCa~(2+)释放，延长LCR周期，限制自发放电频率。