A transgenic rabbit model for human hypertrophic cardiomyopathy
Ali J. Marian, … , Miguel Quinones, Robert Roberts
Ali J. Marian, … , Miguel Quinones, Robert Roberts
Published December 15, 1999
Citation Information: J Clin Invest. 1999;104(12):1683-1692. https://doi.org/10.1172/JCI7956.
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A transgenic rabbit model for human hypertrophic cardiomyopathy

Abstract

Certain mutations in genes for sarcomeric proteins cause hypertrophic cardiomyopathy (HCM). We have developed a transgenic rabbit model for HCM caused by a common point mutation in the β-myosin heavy chain (MyHC) gene, R400Q. Wild-type and mutant human β-MyHC cDNAs were cloned 3′ to a 7-kb murine β-MyHC promoter. We injected purified transgenes into fertilized zygotes to generate two lines each of the wild-type and mutant transgenic rabbits. Expression of transgene mRNA and protein were confirmed by Northern blotting and 2-dimensional gel electrophoresis followed by immunoblotting, respectively. Animals carrying the mutant transgene showed substantial myocyte disarray and a 3-fold increase in interstitial collagen expression in their myocardia. Mean septal thicknesses were comparable between rabbits carrying the wild type transgene and their nontransgenic littermates (NLMs) but were significantly increased in the mutant transgenic animals. Posterior wall thickness and left ventricular mass were also increased, but dimensions and systolic function were normal. Premature death was more common in mutant than in wild-type transgenic rabbits or in NLMs. Thus, cardiac expression of β-MyHC-Q403 in transgenic rabbits induced hypertrophy, myocyte and myofibrillar disarray, interstitial fibrosis, and premature death, phenotypes observed in humans patients with HCM due to β-MyHC-Q403.

Authors

Ali J. Marian, Yun Wu, Do-Sun Lim, Meghan McCluggage, Keith Youker, Qun-tao Yu, Ramon Brugada, Francesco DeMayo, Miguel Quinones, Robert Roberts

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Detection of expression of transgene protein in the heart. (a) Detection...
Detection of expression of transgene protein in the heart. (a) Detection of expression of the transgene and endogenous β-MyHC proteins by high-resolution 2-dimensional gel electrophoresis in conjunction with immunoblotting using pan-specific antisarcomeric myosin mAb MF20. Myofibrillar protein extracts from human (labeled as human heart) and nontransgenic rabbits (labeled as NLM) hearts were included as controls. Myofibrillar protein extracts from a wild-type transgenic rabbit (β-MyHC-R403) and a mutant transgenic rabbit (β-MyHC-Q403) were used to separate transgene and endogenous β-MyHC proteins by IEF and to detect by immunoblotting. As shown, a single 220-kDa band is present in panels representing myofibrillar protein extracts from human and NLM hearts. In contrast, in wild-type and mutant transgenic rabbits, 2 bands, both 220 kDa in size, representing endogenous and human β-MyHC proteins were detected by immunoblotting. The proximal band represents expression of the endogenous β-MyHC protein and the distal band expression of the transgene protein. As shown, expression level of the transgene protein was higher than the endogenous in the wild-type transgenic (55% of the total β-MyHC pool) and lower than the endogenous in the mutant transgenic (41% of the total β-MyHC-pool) rabbits. (b) Immunoblot of total MyHC protein in the heart detected using mAb MF20. Human heart is included as a positive control and a nontransgenic rabbit (NLM) as a negative control. As shown, the total MyHC protein pool was not significantly different among NLM, wild-type (β-MyHC-R403), and mutant (β-MyHC-Q403) transgenic rabbits.