Asahi Michio, Otsu Kinya, Nakayama Hiroyuki, Hikoso Shungo, Takeda Toshihiro, Gramolini Anthony O, Trivieri Maria G, Oudit Gavin Y, Morita Takashi, Kusakari Yoichiro, Hirano Shuta, Hongo Kenichi, Hirotani Shinichi, Yamaguchi Osamu, Peterson Alan, Backx Peter H, Kurihara Satoshi, Hori Masatsugu, MacLennan David H
Department of Internal Medicine and Therapeutics, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan.
Proc Natl Acad Sci U S A. 2004 Jun 22;101(25):9199-204. doi: 10.1073/pnas.0402596101. Epub 2004 Jun 16.
Sarcolipin (SLN) inhibits the cardiac sarco(endo)plasmic reticulum Ca(2+) ATPase (SERCA2a) by direct binding and is superinhibitory if it binds through phospholamban (PLN). To determine whether overexpression of SLN in the heart might impair cardiac function, transgenic (TG) mice were generated with cardiac-specific overexpression of NF-SLN (SLN tagged at its N terminus with the FLAG epitope). The level of NF-SLN expression (the NF-SLN/PLN expression ratio) was equivalent to that which induces profound superinhibition when coexpressed with PLN and SERCA2a in HEK-293 cells. In TG hearts, the apparent affinity of SERCA2a for Ca(2+) was decreased compared with non-TG littermate control hearts. Invasive hemodynamic and echocardiographic analyses revealed impaired cardiac contractility and ventricular hypertrophy in TG mice. Basal PLN phosphorylation was reduced. In isolated papillary muscle subjected to isometric tension, peak amplitudes of Ca(2+) transients and peak tensions were reduced, whereas decay times of Ca(2+) transients and relaxation times of tension were increased in TG mice. Isoproterenol largely restored contractility in papillary muscle and stimulated PLN phosphorylation to wild-type levels in intact hearts. No compensatory changes in expression of SERCA2a, PLN, ryanodine receptor, and calsequestrin were observed in TG hearts. Coimmunoprecipitation indicated that overexpressed NF-SLN was bound to both SERCA2a and PLN, forming a ternary complex. These data suggest that NF-SLN overexpression inhibits SERCA2a through stabilization of SERCA2a-PLN interaction in the absence of PLN phosphorylation and through the inhibition of PLN phosphorylation. Inhibition of SERCA2a impairs contractility and calcium cycling, but responsiveness to beta-adrenergic agonists may prevent progression to heart failure.
肌浆球蛋白（SLN）通过直接结合抑制心肌肌浆网Ca(2+)ATP酶（SERCA2a），如果它通过受磷蛋白（PLN）结合，则具有超抑制作用。为了确定心脏中SLN的过表达是否会损害心脏功能，构建了心脏特异性过表达NF-SLN（在其N末端标记有FLAG表位的SLN）的转基因（TG）小鼠。NF-SLN的表达水平（NF-SLN/PLN表达比率）与在HEK-293细胞中与PLN和SERCA2a共表达时诱导深度超抑制的水平相当。在TG心脏中，与非TG同窝对照心脏相比，SERCA2a对Ca(2+)的表观亲和力降低。侵入性血流动力学和超声心动图分析显示TG小鼠的心脏收缩力受损和心室肥大。基础PLN磷酸化降低。在承受等长张力的离体乳头肌中，TG小鼠的Ca(2+)瞬变峰值幅度和峰值张力降低，而Ca(2+)瞬变衰减时间和张力松弛时间增加。异丙肾上腺素在很大程度上恢复了乳头肌的收缩力，并在完整心脏中将PLN磷酸化刺激到野生型水平。在TG心脏中未观察到SERCA2a、PLN、兰尼碱受体和肌集钙蛋白表达的代偿性变化。免疫共沉淀表明过表达的NF-SLN与SERCA2a和PLN都结合，形成三元复合物。这些数据表明，在不存在PLN磷酸化的情况下，NF-SLN过表达通过稳定SERCA2a-PLN相互作用并通过抑制PLN磷酸化来抑制SERCA2a。SERCA2a的抑制会损害收缩力和钙循环，但对β-肾上腺素能激动剂的反应性可能会阻止进展为心力衰竭。
