Henze Marcus, Patrick Stacey E, Hinken Aaron, Scruggs Sarah B, Goldspink Paul, de Tombe Pieter P, Kobayashi Minae, Ping Peipei, Kobayashi Tomoyoshi, Solaro R John
Department of Physiology and Biophysics, University of Illinois, Chicago, IL 60612, USA.
Biochim Biophys Acta. 2013 Apr;1833(4):823-32. doi: 10.1016/j.bbamcr.2012.08.012. Epub 2012 Aug 25.
Previous structural studies indicated a special functional role for an acidic region composed of residues 1-10 in the unique N-terminal peptide of cardiac troponin I (cTnI). Employing LC-MS/MS, we determined the presence of phosphorylation sites at S5/S6 in cTnI from wild type mouse hearts as well as in hearts of mice chronically expressing active protein kinase C-ε (PKCε) and exhibiting severe dilated cardiomyopathy (DCM). To determine the functional significance of these phosphorylations, we cloned and expressed wild-type cTnI, (Wt), and cTnI variants expressing pseudo-phosphorylation cTnI-(S5D), cTnI(S6D), as well as cTnI(S5A) and cTnI(S6A). We exchanged native Tn of detergent-extracted (skinned) fiber bundles with Tn reconstituted with the variant cTnIs and measured tension and cross-bridge dynamics. Compared to controls, myofilaments controlled by cTnI with pseudo-phosphorylation (S6D) or Ala substitution (S6A) demonstrated a significant depression in maximum tension, ATPase rate, and ktr, but no change in half-maximally activating Ca(2+). In contrast, pseudo-phosphorylation at position 5 (S5D) had no effects, although S5A induced an increase in Ca(2+)-sensitivity with no change in maximum tension or ktr. We further tested the impact of acidic domain modifications on myofilament function in studies examining the effects of cTnI(A2V), a mutation linked to DCM. This mutation significantly altered the inhibitory activity of cTnI as well as cooperativity of activation of myofilament tension, but not when S23/S24 were pseudo-phosphorylated. Our data indicate a new functional and pathological role of amino acid modifications in the N-terminal acidic domain of cTnI that is modified by phosphorylations at cTnI(S23/S24). This article is part of a Special Issue entitled: Cardiomyocyte Biology: Cardiac Pathways of Differentiation, Metabolism and Contraction.
先前的结构研究表明，心肌肌钙蛋白I（cTnI）独特的N端肽中由1-10位残基组成的酸性区域具有特殊的功能作用。我们采用液相色谱-串联质谱法（LC-MS/MS），确定了野生型小鼠心脏以及长期表达活性蛋白激酶C-ε（PKCε）并表现出严重扩张型心肌病（DCM）的小鼠心脏中cTnI的S5/S6位点存在磷酸化位点。为了确定这些磷酸化的功能意义，我们克隆并表达了野生型cTnI（Wt）以及表达假磷酸化的cTnI变体cTnI-(S5D)、cTnI(S6D)，还有cTnI(S5A)和cTnI(S6A)。我们用变体cTnI重构的肌钙蛋白（Tn）替换了去污剂提取的（去垢）纤维束中的天然Tn，并测量了张力和横桥动力学。与对照组相比，由假磷酸化（S6D）或丙氨酸替代（S6A）的cTnI控制的肌丝在最大张力、ATP酶速率和ktr方面表现出显著降低，但半最大激活Ca(2+)没有变化。相比之下，5位的假磷酸化（S5D）没有影响，尽管S5A诱导了Ca(2+)敏感性增加，而最大张力或ktr没有变化。我们在研究与DCM相关的突变cTnI(A2V)的影响时，进一步测试了酸性结构域修饰对肌丝功能的影响。该突变显著改变了cTnI的抑制活性以及肌丝张力激活的协同性，但当S23/S24假磷酸化时则没有改变。我们的数据表明，cTnI N端酸性结构域中氨基酸修饰具有新的功能和病理作用，该结构域会被cTnI(S23/S24)的磷酸化所修饰。本文是名为：心肌细胞生物学：心脏分化、代谢和收缩途径的特刊的一部分。
