Myosin Rod Hypophosphorylation and CB Kinetics in Papillary Muscles from a TnC-A8V KI Mouse Model.
Myosin Rod Hypophosphorylation and CB Kinetics in Papillary Muscles from a TnC-A8V KI Mouse Model.
复制标题
TnC-A8V KI 小鼠模型乳头肌中的肌球蛋白杆低磷酸化和 CB 动力学。
DOI：
10.1016/j.bpj.2017.02.045
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发表时间：
2017
影响因子：
3.4
通讯作者：
Pinto,JoseR
中科院分区：
文献类型：
--
作者：
Kawai,Masataka;Johnston,JamieR;Karam,Tarek;Wang,Li;Singh,RakeshK;Pinto,JoseR
心肌肌钙蛋白C（TnC）-A8 V突变与人类和小鼠的肥厚型和限制型心肌病（HCM和RCM）相关。受影响的残基位于N-螺旋中，该区域已知影响N-末端结构域的Ca 2+结合亲和力。在这里，我们报告的功能影响，这种突变在皮肤乳头肌纤维纯合子敲入TnC-A8 V小鼠。来自左心室的肌纤维在25°C下在工作心肌细胞的离子条件下活化。pCa-张力关系显示突变纤维的Ca 2+敏感性增加3倍，协同性（nH）降低（0.8倍）。通过正弦分析研究了跨桥（CB）循环的基本步骤。ATP研究表明，ATP（K1）对肌球蛋白头的亲和力没有显著变化。在TnC-A8 V突变纤维中，CB脱离速率（k2）及其平衡常数（K2）增加（1.5倍）。磷酸盐研究表明，力产生步骤（k4）的速率常数降低（0.5倍），逆转步骤（k−4）增加（2倍），磷酸盐释放步骤（1/K5）增加（2倍）。带皮纤维样品的Pro-Q Diamond染色显示多种肌节蛋白的总磷酸化没有显著变化。使用液相色谱-串联质谱法的进一步研究显示，与野生型相比，TnC-A8 V突变体纤维中肌球蛋白重链的杆结构域磷酸化不足。免疫印迹证实了质谱分析中观察到的结果。结果表明，CB动力学的扰动-可能是由α-肌球蛋白重链磷酸化谱的变化引起的-作为一种新的机制，据我们所知，TnC突变可以在肌丝中产生涟漪效应，并有助于HCM/RCM的发病机制。
The cardiac troponin C (TnC)-A8V mutation is associated with hypertrophic and restrictive cardiomyopathy (HCM and RCM) in human and mice. The residue affected lies in the N-helix, a region known to affect Ca2+-binding affinity to the N-terminal domain. Here we report on the functional effects of this mutation in skinned papillary muscle fibers from homozygous knock-in TnC-A8V mice. Muscle fibers from left ventricle were activated at 25°C under the ionic conditions of working cardiomyocytes. The pCa-tension relationship showed a 3× increase in Ca2+-sensitivity and a decrease (0.8×) in cooperativity (nH) in mutant fibers. The elementary steps of the cross-bridge (CB) cycle were investigated by sinusoidal analysis. The ATP study revealed that there is no significant change in the affinity of ATP (K1) for the myosin head. In TnC-A8V mutant fibers, the CB detachment rate (k2) and its equilibrium constant (K2) increased (1.5×). The phosphate study revealed that rate constant of the force-generation step (k4) decreased (0.5×), reversal step (k−4) increased (2×), and the phosphate-release step (1/K5) increased (2×). Pro-Q Diamond staining of the skinned fibers samples revealed no significant changes in total phosphorylation of multiple sarcomeric proteins. Further investigation using liquid chromatography-tandem mass spectrometry revealed hypophosphorylation of the rod domain of myosin heavy chain in TnC-A8V mutant fibers compared to wild-type. Immunoblotting confirmed the results observed in the mass spectrometry analysis. The results suggest perturbed CB kinetics—possibly caused by changes in theα-myosin heavy chain phosphorylation profile—as a novel mechanism, to our knowledge, by which a mutation in TnC can have rippling effects in the myofilament and contribute to the pathogenesis of HCM/RCM.