ALTERED CALCIUM REGULATION IN CARDIOMYOPATHIES

描述(申请人的逐字描述)：心脏变薄的突变 细丝蛋白肌钙蛋白I(CTnI)已被确定为某些形式的致病因子 家族性肥厚型心肌病(FHC)。这个项目的总体目标是 提案是描述这六个方面的功能后果 CTnI C末端与疾病相关的突变。关于以下方面的具体预测 它们对稳态力-主成分分析关系的影响源于 TnI‘s C末端与N末端结合突变的定位 CTNC(抑制肽和R162W的R145G和R145Q突变)；或 (Ii)抑制肽中的肌动蛋白-TM(R145G和R145Q突变) 肌动蛋白-TM结合位点I的一部分和肌动蛋白-TM结合的一部分的K183 Delta 站点II)。此外，TnI的C末端截断研究预测R162W， K183Delta、G203S和K206Q均可降低cTn的松弛能力 舒张期心肌。重组cTnI结构将改变为 在FHC中发现突变。突变和野生型(WT)结构将是 加入渗透性肌肉制剂中.用于测量 肌节结构--和调节的肌动蛋白细丝--用于测量 单个肌动蛋白细丝的体外运动分析。突变的蛋白质将是 首先通过用突变体完全替代WT进行测试，然后在变化中 WT和突变型AS在病变心肌中的比例，第三 具有模拟Ser22和Ser23或Thr143磷酸化的额外突变 (引入酸性残留物)。对于每个突变，我们将确定 稳态等长力、细丝滑动、 以及张力再发展速度(Ktr；这一参数对于 评估心脏功能)。这一结果将有助于理解正常的钙离子 心脏的调节、肥厚的病理机制(S)和 化验将有助于确定治疗方法。

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.