Brittsan Angela G, Ginsburg Kenneth S, Chu Guoxiang, Yatani Atsuko, Wolska Beata M, Schmidt Albrecht G, Asahi Michio, MacLennan David H, Bers Donald M, Kranias Evangelia G
Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, 231 Albert Sabin Way, PO Box 670575, Cincinnati, Ohio 45267-0575, USA.
Circ Res. 2003 Apr 18;92(7):769-76. doi: 10.1161/01.RES.0000066661.49920.59. Epub 2003 Mar 13.
Phospholamban, the critical regulator of the cardiac SERCA2a Ca2+ affinity, is phosphorylated at Ser16 and Thr17 during beta-adrenergic stimulation (eg, isoproterenol). To assess the impact of nonphosphorylatable phospholamban, a S16A, T17A double-mutant (DM) was introduced into phospholamban knockout mouse hearts. Transgenic lines expressing DM phospholamban at levels similar to wild types (WT) were identified. In vitro phosphorylation confirmed that DM phospholamban could not be phosphorylated, but produced the same shift in EC50 of SERCA2a for Ca2+ as unphosphorylated WT phospholamban. Rates of basal twitch [Ca2+]i decline were not different in DM versus WT cardiomyocytes. Isoproterenol increased the rates of twitch [Ca2+]i decline in WT, but not DM myocytes, confirming the prominent role of phospholamban phosphorylation in this response. Increased L-type Ca2+ current (ICa) density, with unaltered characteristics, was the major compensation in DM myocytes. Consequently, the normal beta-adrenergic-induced increase in ICa caused larger dynamic changes in absolute ICa density. Isoproterenol increased Ca2+ transients to a comparable amplitude in DM and WT. There were no changes in myofilament Ca2+ sensitivity, or the expression levels and Ca2+ removal activities of other Ca2+-handling proteins. Nor was there evidence of cardiac remodeling up to 10 months of age. Thus, chronic inhibition of SERCA2a by ablation of phospholamban phosphorylation (abolishing its adrenergic regulation) results in a unique cellular adaptation involving greater dynamic ICa modulation. This ICa modulation may partly compensate for the loss in SERCA2a responsiveness and thereby partially normalize beta-adrenergic inotropy in DM phospholamban mice.
受磷蛋白是心脏肌浆网Ca2+-ATP酶（SERCA2a）钙亲和力的关键调节因子，在β-肾上腺素能刺激（如异丙肾上腺素）过程中，其丝氨酸16（Ser16）和苏氨酸17（Thr17）位点会发生磷酸化。为了评估不可磷酸化的受磷蛋白的影响，将S16A、T17A双突变体（DM）导入受磷蛋白基因敲除小鼠心脏。鉴定出表达水平与野生型（WT）相似的DM受磷蛋白转基因品系。体外磷酸化证实DM受磷蛋白不能被磷酸化，但与未磷酸化的WT受磷蛋白一样，可使SERCA2a对Ca2+的半数有效浓度（EC50）发生相同的变化。DM与WT心肌细胞的基础收缩期[Ca2+]i下降速率并无差异。异丙肾上腺素可增加WT心肌细胞的收缩期[Ca2+]i下降速率，但对DM心肌细胞无此作用，这证实了受磷蛋白磷酸化在此反应中的重要作用。DM心肌细胞的主要代偿机制是L型钙电流（ICa）密度增加，且特性未改变。因此，正常的β-肾上腺素能诱导的ICa增加导致绝对ICa密度发生更大的动态变化。异丙肾上腺素使DM和WT心肌细胞的Ca2+瞬变增加至相当的幅度。肌丝对Ca2+的敏感性、其他钙处理蛋白的表达水平和Ca2+清除活性均无变化。在10月龄前也没有心脏重塑的证据。因此，通过消除受磷蛋白磷酸化（消除其肾上腺素能调节）来慢性抑制SERCA2a会导致一种独特的细胞适应性变化，涉及更大的动态ICa调节。这种ICa调节可能部分补偿SERCA2a反应性丧失，从而部分使DM受磷蛋白小鼠的β-肾上腺素能正性肌力作用正常化。
