Marian A J, Zhao G, Seta Y, Roberts R, Yu Q T
Department of Medicine, Baylor College of Medicine, Houston, Tex, USA.
Circ Res. 1997 Jul;81(1):76-85. doi: 10.1161/01.res.81.1.76.
The mechanism(s) by which mutations in sarcomeric proteins cause hypertrophic cardiomyopathy (HCM) remains unknown. A leading hypothesis proposes that mutant sarcomeric proteins impair cardiac myocyte contractility, providing an impetus for compensatory hypertrophy. To test this hypothesis, we determined the impact of expression of a mutant (Arg92Gln) human cardiac troponin T (cTnT), known to cause HCM in humans, on adult cardiac myocyte contractility. A full-length human cTnT cDNA was cloned, and the Arg92Gln mutation was induced. Recombinant adenoviruses Ad5/CMV/cTnT-N and Ad5/CMV/cTnT-Arg92Gln were generated through homologous recombination. Adult feline cardiac myocytes were infected with recombinant adenoviruses or a control viral vector (Ad5 delta E1) at a multiplicity of infection of 100. Expression levels of the full-length normal and mutant cTnT proteins were equal on Western blots. Expression of the exogenous cTnT proteins in cardiac myocytes was also shown by immunocytochemistry and immunofluorescence, and their incorporation into myofibrils was confirmed by Western blotting on myofibrillar extracts. Electron microscopy showed intact sarcomere structure in rod-shaped cardiac myocytes in all groups. Cell fractional shortening and the peak velocity of shortening were not significantly different among the groups 24 hours after transduction. However, 48 hours after transduction, both fractional shortening and the peak velocity of shortening were significantly reduced (24% [P < .001] and 26% [P < .001], respectively) in cardiac myocytes in the Ad5/CMV/cTnT-Arg92Gln compared with the Ad5/CMV/cTnT-N groups. The magnitude of the reductions was greater at 72 hours after transduction (45% and 39%, respectively; P < .001). Our results indicated that expression of the mutant (Arg92Gln) cTnT, known to cause HCM in humans, impaired intact adult cardiac myocyte contractility. Our data also show that both normal and mutant cTnT were incorporated into myofibrils. These results provide a potential mechanism by which mutations in sarcomeric proteins cause HCM.
肌节蛋白突变导致肥厚型心肌病（HCM）的机制仍不清楚。一个主要假说是，突变的肌节蛋白损害心肌细胞收缩力，从而为代偿性肥大提供动力。为了验证这一假说，我们确定了已知会在人类中导致HCM的突变型（Arg92Gln）人心脏肌钙蛋白T（cTnT）的表达对成年心肌细胞收缩力的影响。克隆了全长人cTnT cDNA，并诱导产生了Arg92Gln突变。通过同源重组产生了重组腺病毒Ad5/CMV/cTnT-N和Ad5/CMV/cTnT-Arg92Gln。以感染复数100用重组腺病毒或对照病毒载体（Ad5 delta E1）感染成年猫心肌细胞。在蛋白质印迹上，全长正常和突变cTnT蛋白的表达水平相等。免疫细胞化学和免疫荧光也显示了心肌细胞中外源cTnT蛋白的表达，并且通过对肌原纤维提取物进行蛋白质印迹证实了它们掺入到肌原纤维中。电子显微镜显示所有组的杆状心肌细胞中肌节结构完整。转导后24小时，各组之间的细胞缩短分数和缩短峰值速度没有显著差异。然而，与Ad5/CMV/cTnT-N组相比，转导后48小时，Ad5/CMV/cTnT-Arg92Gln组心肌细胞的缩短分数和缩短峰值速度均显著降低（分别为24%[P <.001]和26%[P <.001]）。转导后72小时降低幅度更大（分别为45%和39%；P <.001）。我们的结果表明，已知会在人类中导致HCM的突变型（Arg92Gln）cTnT的表达损害了成年心肌细胞的完整收缩力。我们的数据还表明正常和突变cTnT都掺入到了肌原纤维中。这些结果提供了肌节蛋白突变导致HCM的一种潜在机制。
