Burton David, Abdulrazzak Hassan, Knott Adam, Elliott Kathryn, Redwood Charles, Watkins Hugh, Marston Steven, Ashley Chris
University Laboratory of Physiology, University of Oxford, Parks Road, Oxford OX1 3PT, UK.
Biochem J. 2002 Mar 1;362(Pt 2):443-51. doi: 10.1042/0264-6021:3620443.
We investigated the effects of two mutations in human cardiac troponin I, Arg(145)-->Gly and Gly(203)-->Ser, that are reported to cause familial hypertrophic cardiomyopathy. Mutant and wild-type troponin I, overexpressed in Escherichia coli, were used to reconstitute troponin complexes in vanadate-treated guinea pig cardiac trabeculae skinned fibres, and thin filaments were reconstituted with human cardiac troponin and tropomyosin along with rabbit skeletal muscle actin for in vitro motility and actomyosin ATPase assays. Troponin containing the Arg(145)-->Gly mutation inhibited force in skinned trabeculae less than did the wild-type, and had almost no inhibitory function in the in vitro motility assay. There was an enhanced inhibitory function with mixtures of 10-30% [Gly(145)]troponin I with the wild-type protein. Skinned trabeculae reconstituted with troponin I containing the Gly(203)-->Ser mutation and troponin C produced less Ca(2+)-activated force (64+/-8% of wild-type) and demonstrated lower Ca(2+) sensitivity [Delta(p)Ca(50) (log of the Ca(2+) concentration that gave 50% of maximal activation) 0.25 unit (P<0.05)] compared with wild-type troponin I, but thin filaments containing [Ser(203)]-troponin I were indistinguishable from those containing the wild-type protein in in vitro motility and ATPase assays. Thus these two mutations each result in hypertrophic cardiomyopathy, but have opposite effects on the overall contractility of the muscle in the systems we investigated, indicating either that we have not yet identified the relevant alteration in contractility for the Gly(203)->Ser mutation, or that the disease does not result directly from any particular alteration in contractility.
我们研究了人类心肌肌钙蛋白I中的两种突变，即精氨酸（145）→甘氨酸和甘氨酸（203）→丝氨酸，据报道这两种突变会导致家族性肥厚型心肌病。在大肠杆菌中过表达的突变型和野生型肌钙蛋白I，被用于在经钒酸盐处理的豚鼠心脏小梁去表皮纤维中重构肌钙蛋白复合物，并且用人心肌肌钙蛋白、原肌球蛋白以及兔骨骼肌肌动蛋白重构细肌丝，用于体外运动性和肌动球蛋白ATP酶分析。含有精氨酸（145）→甘氨酸突变的肌钙蛋白对去表皮小梁中力的抑制作用比野生型小，并且在体外运动性分析中几乎没有抑制功能。10 - 30%的[甘氨酸（145）]肌钙蛋白I与野生型蛋白的混合物具有增强的抑制功能。用含有甘氨酸（2）→丝氨酸突变的肌钙蛋白I和肌钙蛋白C重构的去表皮小梁产生的钙激活力较小（为野生型的64±8%），并且与野生型肌钙蛋白I相比，表现出较低的钙敏感性[Δ（p）Ca50（产生最大激活50%的钙浓度的对数）为0.25个单位（P<0.05）]，但是在体外运动性和ATP酶分析中，含有[丝氨酸（203）] - 肌钙蛋白I的细肌丝与含有野生型蛋白的细肌丝没有区别。因此，这两种突变均会导致肥厚型心肌病，但在我们研究的系统中，它们对肌肉的整体收缩性具有相反的影响，这表明要么我们尚未确定甘氨酸（203）→丝氨酸突变在收缩性方面相关的改变，要么该疾病并非直接由收缩性的任何特定改变引起。
