ALTERED CALCIUM REGULATION IN CARDIOMYOPATHIES

描述（申请人的逐字描述）：心肌细胞突变 肌丝蛋白肌钙蛋白I（cTnI）已被确定为某些形式的致病因素 家族性肥厚型心肌病（FHC）这个项目的总体目标是 建议是描述这六个功能的后果 cTnI C端的疾病相关突变。具体预测关于 它们对稳态力-pCa关系的影响来自于 TnI的C-末端与以下结合中的突变的定位：（i）N-末端 cTnC（抑制肽中的R145 G和R145 Q突变和R162 W）;或 (ii)肌动蛋白-Tm（抑制肽中的R145 G和R145 Q突变， actin-Tm结合位点I的一部分和actin-Tm结合位点I的一部分K183 δ 站点II）。此外，TnI的C-末端截短研究预测R162 W， K183 δ、G203 S和K206 Q均能损害cTn松弛血管的能力。 心肌缺血。重组cTnI构建体将被改变， FHC中发现的突变。突变体和野生型（WT）构建体将被 掺入透化的肌肉组织中-用于测量 肌节力学-并进入调节肌动蛋白促动剂-用于测量 单肌动蛋白丝使用在体外运动性测定。突变蛋白质将是 测试，首先通过突变体完全取代WT，其次在不同的 在患病心肌中发生的WT和突变体的比例，以及第三， 具有模拟Ser 22和Ser 23或Thr 143磷酸化的额外突变 （引入酸性残基）。对于每个突变，我们将确定 对稳态等长力的Ca 2+敏感性的影响，细丝滑动， 和张力再发展速率（kTR;对于 评价心脏功能）。结果将有助于了解正常的Ca 2 + 心脏的调节、肥大的病理机制以及 测定将可用于鉴定治疗。

DESCRIPTION (the applicant's description verbatim): Mutations in cardiac thin filament protein troponin I (cTnI) have been identified as causal in some forms of familial hypertrophic cardiomyopathy (FHC). The overall goal of this proposal is to characterize functional consequences of these six disease-related mutations in the C-terminus of cTnI. Specific predictions about their effects on steady-state force-pCa relationship derive from the localization of mutations in binding of TnI's C-terminus to: (i) the N-terminus of cTnC (R145G and R145Q mutations in the inhibitory peptide and R162W); or (ii) actin-Tm (R145G and R145Q mutations in the inhibitory peptide which is part of actin-Tm binding site I and K183delta which is part of actin-Tm binding site II). In addition, C-terminal truncation studies of TnI predict that R162W, K183delta, G203S and K206Q could all compromise the ability of cTn to relax the myocardium during diastole. Recombinant cTnI constructs will be altered with mutations found in FHC. Mutant and wild type (WT) constructs will be incorporated into permeabilized muscle preparations-for measurements of sarcomere mechanics-and into regulated actin filaments-for measurements on single actin filaments using in vitro motility assays. Mutant proteins will be tested, first by complete substitution of mutant for WT, secondly in varying proportions of WT and mutant as occurs in the diseased myocardium, and thirdly with additional mutations that mimic phosphorylation of Ser22 & Ser23 or Thr143 (introduction of acidic residues). For each mutation, we will determine the effects on Ca2+ sensitivity of steady-state isometric force, filament sliding, and the rate of tension redevelopment (kTR; a parameter that is important for evaluating cardiac function). The results will aid understanding normal Ca2+ regulation of the heart, pathological mechanism(s) of hypertrophy, and the assays will he useful for identifying treatments.