Troponin I Mutations R146G and R21C Alter Cardiac Troponin Function, Contractile Properties, and Modulation by Protein Kinase A (PKA)-mediated Phosphorylation
Troponin I Mutations R146G and R21C Alter Cardiac Troponin Function, Contractile Properties, and Modulation by Protein Kinase A (PKA)-mediated Phosphorylation
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DOI：
10.1074/jbc.m115.683045
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发表时间：
2015-11-13
影响因子：
4.8
通讯作者：
Regnier, Michael
中科院分区：
文献类型：
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作者：
Cheng, Yuanhua;Rao, Vijay;Regnier, Michael
两个肥厚性心肌病相关的心肌肌钙蛋白I （cTnI）突变R146G和R21C位于cTnI、抑制肽和心脏特异性N端的不同区域。我们最近报道，当Ser-23/Ser-24被磷酸化时，这些区域可能相互作用，削弱cTnI与心脏TnC的相互作用。对于这些突变如何影响心脏TnC对cTnI （KC-I）的亲和力或β -肾上腺素能刺激时的收缩动力学，我们知之甚少。在这里，我们测试了cTnI（R146G）或cTnI（R21C）如何影响收缩激活和松弛以及它们对蛋白激酶A （PKA）的反应。这两种突变都显著增加了Ca2+与cTn （K-Ca）和KC-I的结合亲和力。PKA磷酸化导致所有复合物中K-Ca的相似减少，但KC-I仅与cTnI（WT）一起减少。将cTnI（WT）、cTnI（R146G）和cTnI（R21C）配合成心肌肌钙蛋白并交换到大鼠心室肌原纤维中，测量PKA磷酸化+/- PKA磷酸化后收缩/舒张动力学。cTnI（R146G）和cTnI（R21C）交换的肌原纤维保持最大张力（T-max），张力（pCa(50)）增加。PKA磷酸化降低了cTnI（WT）交换肌原纤维的pCa(50)，但对两种突变均无影响。PKA磷酸化加速了cTnI（WT）肌原纤维的早期慢相松弛，特别是在心脏体内运行的Ca2+水平下。重要的是，这种作用在cTnI（R146G）和cTnI（R21C）交换的肌原纤维中被减弱。分子动力学模拟表明，这两种突变都抑制了N端和cTnI抑制肽之间亚基内接触的形成，这种接触通常在PKA磷酸化后的WT-cTn中可见。总之，我们的研究结果表明，cTnI（R146G）和cTnI（R21C）通过禁止心脏特异性n端与cTnI抑制肽的相互作用，减弱了PKA对激活和松弛动力学的调节。
Two hypertrophic cardiomyopathy-associated cardiac troponin I (cTnI) mutations, R146G and R21C, are located in different regions of cTnI, the inhibitory peptide and the cardiac-specific N terminus. We recently reported that these regions may interact when Ser-23/Ser-24 are phosphorylated, weakening the interaction of cTnI with cardiac TnC. Little is known about how these mutations influence the affinity of cardiac TnC for cTnI (KC-I) or contractile kinetics during beta-adrenergic stimulation. Here, we tested how cTnI(R146G) or cTnI(R21C) influences contractile activation and relaxation and their response to protein kinase A (PKA). Both mutations significantly increased Ca2+ binding affinity to cTn (K-Ca) and KC-I. PKA phosphorylation resulted in a similar reduction of K-Ca for all complexes, but KC-I was reduced only with cTnI(WT). cTnI(WT), cTnI(R146G), and cTnI(R21C) were complexed into cardiac troponin and exchanged into rat ventricular myofibrils, and contraction/relaxation kinetics were measured +/- PKA phosphorylation. Maximal tension (T-max) was maintained for cTnI(R146G)- and cTnI(R21C)-exchanged myofibrils, and Ca2+ sensitivity of tension (pCa(50)) was increased. PKA phosphorylation decreased pCa(50) for cTnI(WT)-exchanged myofibrils but not for either mutation. PKA phosphorylation accelerated the early slow phase relaxation for cTnI(WT) myofibrils, especially at Ca2+ levels that the heart operates in vivo. Importantly, this effect was blunted for cTnI(R146G)- and cTnI(R21C)-exchanged myofibrils. Molecular dynamics simulations suggest both mutations inhibit formation of intra-subunit contacts between the N terminus and the inhibitory peptide of cTnI that is normally seen with WT-cTn upon PKA phosphorylation. Together, our results suggest that cTnI(R146G) and cTnI(R21C) blunt PKA modulation of activation and relaxation kinetics by prohibiting cardiac-specific N-terminal interaction with the cTnI inhibitory peptide.