Laboratory of Cardiovascular Science, Intramural Research Program, Gerontology Research Center, National Institute on Aging, National Institutes of Health, Baltimore MD 21224, USA.
J Mol Cell Cardiol. 2011 Nov;51(5):730-9. doi: 10.1016/j.yjmcc.2011.07.028. Epub 2011 Aug 4.
Recent perspectives on sinoatrial nodal cell (SANC)(*) function indicate that spontaneous sarcoplasmic reticulum (SR) Ca(2+) cycling, i.e. an intracellular "Ca(2+) clock," driven by cAMP-mediated, PKA-dependent phosphorylation, interacts with an ensemble of surface membrane electrogenic molecules ("surface membrane clock") to drive SANC normal automaticity. The role of AC-cAMP-PKA-Ca(2+) signaling cascade in mouse, the species most often utilized for genetic manipulations, however, has not been systematically tested. Here we show that Ca(2+) cycling proteins (e.g. RyR2, NCX1, and SERCA2) are abundantly expressed in mouse SAN and that spontaneous, rhythmic SR generated local Ca(2+) releases (LCRs) occur in skinned mouse SANC, clamped at constant physiologic [Ca(2+)]. Mouse SANC also exhibits a high basal level of phospholamban (PLB) phosphorylation at the PKA-dependent site, Serine16. Inhibition of intrinsic PKA activity or inhibition of PDE in SANC, respectively: reduces or increases PLB phosphorylation, and markedly prolongs or reduces the LCR period; and markedly reduces or accelerates SAN spontaneous firing rate. Additionally, the increase in AP firing rate by PKA-dependent phosphorylation by β-adrenergic receptor (β-AR) stimulation requires normal intracellular Ca(2+) cycling, because the β-AR chronotropic effect is markedly blunted when SR Ca(2+) cycling is disrupted. Thus, AC-cAMP-PKA-Ca(2+) signaling cascade is a major mechanism of normal automaticity in mouse SANC.
最近的观点表明，窦房结细胞 (SANC)(*) 的功能，即由 cAMP 介导的、PKA 依赖性磷酸化驱动的自发性肌浆网 (SR) Ca(2+) 循环，与一组细胞膜电活性分子（“细胞膜时钟”）相互作用，驱动 SANC 的正常自律性。然而，AC-cAMP-PKA-Ca(2+) 信号级联在作为遗传操作最常用物种的小鼠中的作用尚未得到系统测试。在这里，我们表明 Ca(2+) 循环蛋白（例如 RyR2、NCX1 和 SERCA2）在小鼠 SAN 中大量表达，并且在 SKINNED 小鼠 SANC 中发生自发的、节律性的 SR 产生局部 Ca(2+) 释放 (LCR)，保持恒定的生理 [Ca(2+)]。小鼠 SANC 还表现出高的 PLB 磷酸化水平（PKA 依赖性位点，丝氨酸 16 位）。内在 PKA 活性的抑制或 PDE 在 SANC 中的抑制分别：降低或增加 PLB 磷酸化，并显著延长或缩短 LCR 周期；并显著降低或加速 SAN 自发性放电率。此外，β-肾上腺素能受体 (β-AR) 刺激的 PKA 依赖性磷酸化增加 AP 放电率需要正常的细胞内 Ca(2+) 循环，因为当 SR Ca(2+) 循环被破坏时，β-AR 变时作用明显减弱。因此，AC-cAMP-PKA-Ca(2+) 信号级联是小鼠 SANC 正常自律性的主要机制。
