In vivo short-term expression of a hypertrophic cardiomyopathy mutation in adult rabbit myocardium: myofibrillar incorporation without early disarray.
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.
Yu
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Section of Cardiology, Department of Medicine, Baylor College of Medicine, Houston, TX, 77030, USA.
Zhao
G
G
Marian
A J
AJ
D000256:Adenoviridae; D000818:Animals; D002312:Cardiomyopathy, Hypertrophic / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D004195:Disease Models, Animal; D005822:Genetic Vectors; D006801:Humans; D009154:Mutation; D009206:Myocardium / Q000378:metabolism / Q000473:pathology*; D009210:Myofibrils / Q000473:pathology; D011817:Rabbits; D014162:Transfection; D020107:Troponin T / Q000235:genetics*
D020107:Troponin T