Song Jianliang, Zhang Xue-Qian, Wang JuFang, Cheskis Ellina, Chan Tung O, Feldman Arthur M, Tucker Amy L, Cheung Joseph Y
Division of Nephrology, Department of Medicine, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
Am J Physiol Heart Circ Physiol. 2008 Oct;295(4):H1615-25. doi: 10.1152/ajpheart.00287.2008. Epub 2008 Aug 15.
Phospholemman (PLM) regulates cardiac Na(+)/Ca(2+) exchanger (NCX1) and Na(+)-K(+)-ATPase in cardiac myocytes. PLM, when phosphorylated at Ser(68), disinhibits Na(+)-K(+)-ATPase but inhibits NCX1. PLM regulates cardiac contractility by modulating Na(+)-K(+)-ATPase and/or NCX1. In this study, we first demonstrated that adult mouse cardiac myocytes cultured for 48 h had normal surface membrane areas, t-tubules, and NCX1 and sarco(endo)plasmic reticulum Ca(2+)-ATPase levels, and retained near normal contractility, but alpha(1)-subunit of Na(+)-K(+)-ATPase was slightly decreased. Differences in contractility between myocytes isolated from wild-type (WT) and PLM knockout (KO) hearts were preserved after 48 h of culture. Infection with adenovirus expressing green fluorescent protein (GFP) did not affect contractility at 48 h. When WT PLM was overexpressed in PLM KO myocytes, contractility and cytosolic Ca(2+) concentration (Ca(2+)) transients reverted back to those observed in cultured WT myocytes. Both Na(+)-K(+)-ATPase current (I(pump)) and Na(+)/Ca(2+) exchange current (I(NaCa)) in PLM KO myocytes rescued with WT PLM were depressed compared with PLM KO myocytes. Overexpressing the PLMS68E mutant (phosphomimetic) in PLM KO myocytes resulted in the suppression of I(NaCa) but had no effect on I(pump). Contractility, Ca(2+) transient amplitudes, and sarcoplasmic reticulum Ca(2+) contents in PLM KO myocytes overexpressing the PLMS68E mutant were depressed compared with PLM KO myocytes overexpressing GFP. Overexpressing the PLMS68A mutant (mimicking unphosphorylated PLM) in PLM KO myocytes had no effect on I(NaCa) but decreased I(pump). Contractility, Ca(2+) transient amplitudes, and sarcoplasmic reticulum Ca(2+) contents in PLM KO myocytes overexpressing the S68A mutant were similar to PLM KO myocytes overexpressing GFP. We conclude that at the single-myocyte level, PLM affects cardiac contractility and Ca(2+) homeostasis primarily by its direct inhibitory effects on Na(+)/Ca(2+) exchange.
磷肌膜蛋白（PLM）调节心肌细胞中的心脏钠/钙交换体（NCX1）和钠钾ATP酶。PLM在丝氨酸68（Ser68）位点磷酸化时，会解除对钠钾ATP酶的抑制，但会抑制NCX1。PLM通过调节钠钾ATP酶和/或NCX1来调节心脏收缩力。在本研究中，我们首先证明，培养48小时的成年小鼠心肌细胞具有正常的表面膜面积、横管，以及NCX1和肌质（内质）网钙ATP酶水平，并保留了接近正常的收缩力，但钠钾ATP酶的α1亚基略有下降。从野生型（WT）和PLM基因敲除（KO）心脏分离的心肌细胞在培养48小时后，收缩力的差异得以保留。感染表达绿色荧光蛋白（GFP）的腺病毒在48小时时不影响收缩力。当WT PLM在PLM KO心肌细胞中过表达时，收缩力和胞质钙浓度（[Ca2+]i）瞬变恢复到培养的WT心肌细胞中观察到的水平。与PLM KO心肌细胞相比，用WT PLM挽救的PLM KO心肌细胞中的钠钾ATP酶电流（I泵）和钠/钙交换电流（I NaCa）均降低。在PLM KO心肌细胞中过表达PLM S68E突变体（模拟磷酸化）导致I NaCa受到抑制，但对I泵没有影响。与过表达GFP的PLM KO心肌细胞相比，过表达PLM S68E突变体的PLM KO心肌细胞的收缩力、[Ca2+]i瞬变幅度和肌质网钙含量均降低。在PLM KO心肌细胞中过表达PLM S68A突变体（模拟未磷酸化的PLM）对I NaCa没有影响，但降低了I泵。过表达S68A突变体的PLM KO心肌细胞的收缩力、[Ca2+]i瞬变幅度和肌质网钙含量与过表达GFP的PLM KO心肌细胞相似。我们得出结论，在单细胞水平上，PLM主要通过其对钠/钙交换的直接抑制作用来影响心脏收缩力和[Ca2+]i稳态。
