Left Ventricular and Myocardial Function in Mice Expressing Constitutively Pseudophosphorylated Cardiac Troponin I
Left Ventricular and Myocardial Function in Mice Expressing Constitutively Pseudophosphorylated Cardiac Troponin I
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DOI：
10.1161/circresaha.109.205427
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发表时间：
2009-12-04
影响因子：
20.1
通讯作者：
Shroff, Sanjeev G.
中科院分区：
文献类型：
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作者：
Kirk, Jonathan A.;MacGowan, Guy A.;Shroff, Sanjeev G.
基本原理：蛋白激酶C（PKC）诱导的心肌肌钙蛋白I（cTnI）磷酸化可调节心肌收缩。目的：利用转基因小鼠模型，研究PKC诱导的cTnI磷酸化的功能效应，并探讨其机制（cTnI（PKC-P））表达突变型cTnI方法和结果：二维凝胶分析显示突变体形式对内源性cTnI的替代为7.2+/-0.5%。实验包括：机械测量（灌注的离体心脏、离体乳头肌和皮肤纤维制备物）、生物化学和分子生物学测量以及用于综合解释的基于数学模型的分析。与野生型小鼠相比，cTnI（PKC-P）小鼠在离体心脏（峰值发展压力降低14%）、乳头肌（最大发展力降低53%）和皮肤纤维（最大激活力，F-max降低14%）中表现出负性肌力。此外，cTnI（PKC-P）小鼠在离体心脏和完整乳头肌中均表现出舒张减慢。cTnI（PKC-P）小鼠在钙敏感性、协同性、稳态力-MgATP酶关系、钙瞬变（振幅和松弛）或其他肌营养蛋白的基线磷酸化方面无差异。基于模型的分析表明，在cTnI（PKC-P）小鼠中的实验观察结果可以通过2种同时发生的扰动再现：跨桥形成速率的降低和肌丝活性状态的钙非依赖性持续性的增加。（约7%）可显著改变心肌收缩：通过减少横桥形成的负性肌力和通过持续肌丝活性状态的负性肌力。根据我们的数据和文献数据，我们推测PKC介导的cTnI磷酸化作用具有位点特异性（S43/S45 vs T144）。(Circ Res. 2009; 105：1232-1239）。
Rationale: Protein kinase (PK)C-induced phosphorylation of cardiac troponin (cTn)I has been shown to regulate cardiac contraction.Objective: Characterize functional effects of increased PKC-induced cTnI phosphorylation and identify underlying mechanisms using a transgenic mouse model (cTnI(PKC-P)) expressing mutant cTnI (S43E, S45E, T144E).Methods and Results: Two-dimensional gel analysis showed 7.2+/-0.5% replacement of endogenous cTnI with the mutant form. Experiments included: mechanical measurements (perfused isolated hearts, isolated papillary muscles, and skinned fiber preparations), biochemical and molecular biological measurements, and a mathematical model-based analysis for integrative interpretation. Compared to wild-type mice, cTnI(PKC-P) mice exhibited negative inotropy in isolated hearts (14% decrease in peak developed pressure), papillary muscles (53% decrease in maximum developed force), and skinned fibers (14% decrease in maximally activated force, F-max). Additionally, cTnI(PKC-P) mice exhibited slowed relaxation in both isolated hearts and intact papillary muscles. The cTnI(PKC-P) mice showed no differences in calcium sensitivity, cooperativity, steady-state force-MgATPase relationship, calcium transient (amplitude and relaxation), or baseline phosphorylation of other myofilamental proteins. The model-based analysis revealed that experimental observations in cTnI(PKC-P) mice could be reproduced by 2 simultaneous perturbations: a decrease in the rate of cross-bridge formation and an increase in calcium-independent persistence of the myofilament active state.Conclusions: A modest increase in PKC-induced cTnI phosphorylation (approximate to 7%) can significantly alter cardiac muscle contraction: negative inotropy via decreased cross-bridge formation and negative lusitropy via persistence of myofilament active state. Based on our data and data from the literature we speculate that effects of PKC-mediated cTnI phosphorylation are site-specific (S43/S45 versus T144). (Circ Res. 2009; 105: 1232-1239.)