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 beta-myosin heavy chain (MyHC) gene, R400Q. Wild-type and mutant human beta-MyHC cDNAs were cloned 3' to a 7-kb murine beta-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 beta-MyHC-Q(403) in transgenic rabbits induced hypertrophy, myocyte and myofibrillar disarray, interstitial fibrosis, and premature death, phenotypes observed in humans patients with HCM due to beta-MyHC-Q(403).

Figure 1

Schematic illustration of wild-type (β-MyHC-R⁴⁰³) and mutant (β-MyHC-Q⁴⁰³) transgene constructs. Wild-type and mutant human cDNAs were cloned 3′ to a 7-kb murine mutant β-MyHC promoter. A 600-bp 3′ untranslated fragment (UT) of human growth hormone (hGH) was also placed downstream to the cDNAs.

Figure 2

Expression of the transgene mRNA in cardiac and noncardiac tissues. (a) Expression of the full-length transgene mRNA in the heart detected by Northern blotting. Lane 1 represents a positive control (human heart), lane 2 represents a negative control (nontransgenic rabbit), lanes 2 and 3 represent 2 lines of wild-type mutant transgenic rabbits, and lanes 4 and 5 represent 2 lines of mutant transgenic rabbits. As shown, a 6-kb band was detected in lanes 1 (positive control) and 2–6 (transgenic rabbits), but it was absent in lane 2 (a negative control). The lower blot represents GAPDH mRNA, which was used to control for loading conditions. (b) Expression pattern of the endogenous β-MyHC mRNA in noncardiac tissues in a nontransgenic littermate (NLM) rabbit detected by RT-PCR. RT controls (RT–), genomic DNA (labeled as DNA), and a PCR negative control (–control) are also included. As shown, a 368-bp RT product was present in lanes representing left ventricle (LV), left atrium (LA), and skeletal muscles (SK) after RT, but was absent in lanes representing lungs and aorta. (c) Expression pattern of the transgene mRNA in noncardiac tissues. Transgene construct and a human heart (HH) sample were included as positive controls and RT– and PCR negative controls were included as negative controls. As shown, a 340-bp band was present in lanes representing transgene (positive control), HH (positive control), LV, LA, and SK after RT. It was absent in lanes representing lungs and aorta.

Figure 3

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.

Figure 4

Left ventricular wall thickness, dimensions, and function. (a) Mean interventricular septal thickness (ST), posterior wall thickness (PWT), and left ventricular mass (LV Mass) in nontransgenic (NLM), wild-type (R⁴⁰³), and mutant (Q⁴⁰³) transgenic rabbits. (b) Left ventricular end diastolic diameter (LVEDD), end systolic diameter (LVESD), and percent fractional shortening (FS) in the 3 groups. Values are mean ± SD.

Figure 5

Histological phenotypes: H&E staining of myocardial sections are shown in the upper panels, picrosirius red staining in the middle panels, and immunofluorescence staining in the lower panels. NLM represents nontransgenic, β-MyHC-R⁴⁰³ represents wild-type, and β-MyHC-Q^403r represents mutant transgenic rabbits. As shown in the upper panels, in β-MyHC-Q⁴⁰³ rabbits, myocytes appear like tangle masses oriented perpendicularly and obliquely to each other, giving a swirl appearance. Pleotrophic and hyperchromatic nuclei are also noted. Myocyte organization was normal in NLM and β-MyHC-Q⁴⁰³ rabbits. The middle panels show increased interstitial collagen (red staining) in β-MyHC-Q⁴⁰³ compared with NLM and β-MyHC-R⁴⁰³. Lower panels represent myofibrillar structure after staining with antimyosin mAb MF-20. Although myofibrils are registered along the long axis of the myocytes in the NLM and β-MyHC-R⁴⁰³ transgenic rabbits, they are disorganized and interspersed in multiple directions within the myocytes in the β-MyHC-Q⁴⁰³ rabbits.

Figure 6

Kaplan-Meier survival curves: 2-year total survival rate of all rabbits in the transgenic lines and nontransgenic littermates (NLM) are depicted. As shown, 2-year survival rate was lower (53%) in β-MyHC-Q⁴⁰³ compared with β-MyHC-R⁴⁰³ (80%) and NLM (90%) (P = 0.0004). The difference in the survival rates of the wild-type and NLM was not statistically significant.