Yu Q, Zhao G, Marian A J
Section of Cardiology, Department of Medicine, Baylor College of Medicine, Houston, TX, 77030, USA.
Proc Assoc Am Physicians. 1999 Jan-Feb;111(1):45-56. doi: 10.1046/j.1525-1381.1999.09416.x.
Cardiac myocyte disarray is the pathological hallmark of hypertrophic cardiomyopathy (HCM), a disease of sarcomeric proteins. Mutations in the cardiac troponin T (cTnT), a major gene responsible for HCM, are associated with severe myocyte disarray. To study the pathogenesis of cardiac myocyte disarray, we expressed normal and mutant cTnT proteins in the myocardium of adult rabbits via direct intramyocardial injection of recombinant adenoviruses. Aliquots of 1010 plaque-forming units of normal (Ad/CMV/cTnT-Arg92) and mutant (Ad/CMV/cTnT-Gln92) recombinant viruses or a control vector (Ad/DeltaE) virus were mixed with equal aliquots of a reporter virus (Ad/CMV/Lac-Z) and co-injected into the myocardium of adult rabbits (n = 12). One week following gene transfer, thin myocardial sections were obtained and analyzed for beta-galactosidase, messenger RNA (mRNA) and protein expression, hematoxylin and eosin, Masson's trichrome, immunofluorescence staining, and electron microscopy. The efficiency of gene transfer varied from 2% to 60% of the cells in an area approximately 2.5 mm in length. Northern blotting confirmed expression of the transgenes into mRNA. Immunoblotting of the myofibrillar protein extracts and indirect immunofluorescence staining confirmed expression and incorporation of the transgene proteins into myofibrils. Expression of the mutant cTnT was up to 18% of the endogenous. Light and electron microscopic studies showed normal cardiac myocyte and sarcomere structures. Thus, despite incorporation of the mutant cTnT-Gln92, stable myofibrillar formation and sarcomere assembly proceeded in vivo. The absence of myocyte and sarcomere disarray may reflect the duration, or the level of expression, or the extent of myofibrillar incorporation of the mutant cTnT-Gln92, as well as the site and timing of expression of the transgenes, and interspecies variation in the pathogenesis of HCM.
心肌细胞排列紊乱是肥厚型心肌病（HCM）的病理标志，HCM是一种肌节蛋白疾病。心肌肌钙蛋白T（cTnT）是导致HCM的主要基因，其突变与严重的心肌细胞排列紊乱有关。为了研究心肌细胞排列紊乱的发病机制，我们通过直接心肌内注射重组腺病毒，在成年兔心肌中表达正常和突变的cTnT蛋白。将1010个噬斑形成单位的正常（Ad/CMV/cTnT-Arg92）和突变（Ad/CMV/cTnT-Gln92）重组病毒或对照载体（Ad/DeltaE）病毒的等分试样与等量的报告病毒（Ad/CMV/Lac-Z）混合，并共同注射到成年兔（n = 12）的心肌中。基因转移一周后，获取心肌薄片并分析β-半乳糖苷酶、信使核糖核酸（mRNA）和蛋白质表达、苏木精和伊红染色、Masson三色染色、免疫荧光染色以及电子显微镜检查。基因转移效率在长度约2.5毫米区域内的细胞中为2%至60%。Northern印迹证实转基因表达为mRNA。肌原纤维蛋白提取物的免疫印迹和间接免疫荧光染色证实转基因蛋白表达并掺入肌原纤维。突变型cTnT的表达量高达内源性表达量的18%。光镜和电镜研究显示心肌细胞和肌节结构正常。因此，尽管掺入了突变型cTnT-Gln92，但体内仍进行了稳定的肌原纤维形成和肌节组装。心肌细胞和肌节无排列紊乱可能反映了突变型cTnT-Gln92的持续时间、表达水平、肌原纤维掺入程度，以及转基因的表达位点和时间，还有HCM发病机制中的种间差异。
