Department of Pharmacology and Cell Biophysics, University of Cincinnati, College of Medicine, Cincinnati, OH 45267-0575, USA.
J Mol Cell Cardiol. 2012 Mar;52(3):773-82. doi: 10.1016/j.yjmcc.2011.11.012. Epub 2011 Dec 1.
Depressed Ca-handling in cardiomyocytes is frequently attributed to impaired sarcoplasmic reticulum (SR) function in human and experimental heart failure. Phospholamban (PLN) is a key regulator of SR and cardiac function, and PLN mutations in humans have been associated with dilated cardiomyopathy (DCM). We previously reported the deletion of the highly conserved amino acid residue arginine 14 (nucleic acids 39, 40 and 41) in DCM patients. This basic amino acid is important in maintaining the upstream consensus sequence for PKA phosphorylation of Ser 16 in PLN. To assess the function of this mutant PLN, we introduced the PLN-R14Del in cardiac myocytes of the PLN null mouse. Transgenic lines expressing mutant PLN-R14Del at similar protein levels to wild types exhibited no inhibition of the initial rates of oxalate-facilitated SR Ca uptake compared to PLN-knockouts (PLN-KO). The contractile parameters and Ca-kinetics also remained highly stimulated in PLN-R14Del cardiomyocytes, similar to PLN-KO, and isoproterenol did not further stimulate these hyper-contractile basal parameters. Consistent with the lack of inhibition on SR Ca-transport and contractility, confocal microscopy indicated that the PLN-R14Del failed to co-localize with SERCA2a. Moreover, PLN-R14Del did not co-immunoprecipitate with SERCA2a (as did WT-PLN), but rather co-immunoprecipitated with the sarcolemmal Na/K-ATPase (NKA) and stimulated NKA activity. In addition, studies in HEK cells indicated significant fluorescence resonance energy transfer between PLN-R14Del-YFP and NKAα1-CFP, but not with the NKA regulator phospholemman. Despite the enhanced cardiac function in PLN-R14Del hearts (as in PLN-knockouts), there was cardiac hypertrophy (unlike PLN-KO) coupled with activation of Akt and the MAPK pathways. Thus, human PLN-R14Del is misrouted to the sarcolemma, in the absence of endogenous PLN, and alters NKA activity, leading to cardiac remodeling.
心肌细胞中钙处理能力下降通常归因于人类和实验性心力衰竭中肌浆网（SR）功能受损。磷蛋白（PLN）是 SR 和心脏功能的关键调节剂，人类的 PLN 突变与扩张型心肌病（DCM）有关。我们之前报道了 DCM 患者中高度保守的精氨酸 14 （核酸 39、40 和 41）的缺失。这种碱性氨基酸对于维持 PLN 上丝氨酸 16 的 PKA 磷酸化的上游保守序列很重要。为了评估这种突变 PLN 的功能，我们在 PLN 缺失小鼠的心肌细胞中引入了 PLN-R14Del。与 PLN 敲除（PLN-KO）相比，表达突变型 PLN-R14Del 的转基因系的初始草酸促进的 SR Ca 摄取率没有受到抑制（PLN-R14Del）。PLN-R14Del 心肌细胞的收缩参数和 Ca 动力学也保持高度刺激，与 PLN-KO 相似，异丙肾上腺素也没有进一步刺激这些高收缩的基础参数。与缺乏对 SR Ca 转运和收缩的抑制一致，共聚焦显微镜表明 PLN-R14Del 未能与 SERCA2a 共定位。此外，PLN-R14Del 没有与 SERCA2a 共免疫沉淀（如 WT-PLN 所做的那样），而是与肌浆网 Na/K-ATP 酶（NKA）共免疫沉淀，并刺激 NKA 活性。此外，HEK 细胞研究表明 PLN-R14Del-YFP 和 NKAα1-CFP 之间存在显著的荧光共振能量转移，但与 NKA 调节剂磷蛋白无关。尽管 PLN-R14Del 心脏（如 PLN 敲除）的心脏功能增强，但存在心肌肥大（与 PLN-KO 不同），同时激活 Akt 和 MAPK 途径。因此，人类 PLN-R14Del 在没有内源性 PLN 的情况下被错误地定向到肌浆网，改变 NKA 活性，导致心脏重构。
