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. 2005 May 10;111(18):2339-46.
doi: 10.1161/01.CIR.0000164233.09448.B1. Epub 2005 May 2.

Forced expression of alpha-myosin heavy chain in the rabbit ventricle results in cardioprotection under cardiomyopathic conditions

Jeanne James  1 , Lisa Martin, Maike Krenz, Carmen Quatman, Fred Jones, Raisa Klevitsky, James Gulick, Jeffrey Robbins
Affiliations

Forced expression of alpha-myosin heavy chain in the rabbit ventricle results in cardioprotection under cardiomyopathic conditions

Jeanne James et al. Circulation. .

Abstract

Background: The biochemical differences between the 2 mammalian cardiac myosin heavy chains (MHCs), alpha-MHC and beta-MHC, are well described, but the physiological consequences of basal isoform expression and isoform shifts in response to altered cardiac load are not clearly understood. Mature human ventricle contains primarily the beta-MHC isoform. However, the alpha-MHC isoform can be detected in healthy human ventricle and appears to be significantly downregulated in failing hearts. The unique biochemical properties of the alpha-MHC isoform might offer functional advantages in a failing heart that is expressing only the beta-MHC isoform. This hypothesis cannot be tested in mice or rats because both species express alpha-MHC as the predominant isoform.

Methods and results: To test the effects of persistent alpha-MHC expression on the background of beta-MHC, we made transgenic (TG) rabbits that expressed rabbit alpha-MHC cDNA in the ventricle so that the endogenous myosin was partially replaced by the transgenically encoded species. Molecular, histological, and functional analyses showed no significant baseline effects in the TG rabbits compared with nontransgenic (NTG) littermates. To determine whether alpha-MHC expression afforded any advantages to stressed myocardium, a cohort of TG and NTG rabbits was subjected to rapid ventricular pacing. Although both the TG and NTG rabbits developed dilated cardiomyopathy, the TG rabbits had a higher shortening fraction, less septal thinning, and more normal +/-dP/dt than paced NTG rabbits.

Conclusions: Transgenic expression of alpha-MHC does not have any apparent detrimental effects under basal conditions and is cardioprotective in experimental tachycardia-induced cardiomyopathy.

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Figures

Figure 1
Figure 1
Protein levels and basic characteristics of the α-MHC TG rabbits. A, TG protein replacement and separation of the MHC isoforms via PAGE. The LV NTG sample used in this gel has no detectable α-MHC. LV samples from the four TG lines show varying amounts of replacement, with 40% α-MHC in line 18, 43% in line 45, 30% in line 49 and 15% in line 69. The amounts varied ± 3% between individuals from the same line. B, H&E staining of LV myocardium in an 18 month, line 18 TG rabbit. C, NTG littermate of B also stained with H&E. 20X original magnification. D, RNA levels of selected markers for hypertrophy (ANF) and failure (SERCA, PLN). E, Actin-activated ATPase activity of myosin purified from adult NTG atrium (100% α-MHC, ▴), NTG ventricle (approximately 97% β-MHC, ♦) and TG line 18 ventricle (40% α-MHC, □).
Figure 2
Figure 2
Timeline of the rapid ventricular pacing protocol. Cardiac function was assessed by echocardiography under isoflurane anesthesia. LV dimensions and shortening fraction were measured by M-mode. VCFc was calculated from M-mode measurements and LV ejection time (pulse wave Doppler of the ascending aorta).
Figure 3
Figure 3
Relative RNA levels in unpaced and paced atria and ventricles. RNAs were collected at the end of the protocol and blotted onto nitrocellulose. Transcript levels were determined via hybridization to transcript specific oligonucleotides and those data used to generate the histograms.
Figure 4
Figure 4
Post-pacing characteristics of TG and NTG rabbits. A, PAGE of ventricular myosin showing the α- and β-MHC isoforms after completion of the pacing protocol. The ratio of α-MHC:β-MHC increased slightly in the paced TG ventricles while no α-MHC is detected in paced NTG ventricle. Wt/V = non-paced NTG ventricle with predominately β-MHC and a small amount of α-MHC; Wt/A = non-paced NTG atrium with 100% α-MHC; TG = paced TG ventricle; NTG = paced NTG ventricle. Note the lack of any α-MHC in paced NTG hearts as compared to the non-paced NTG ventricle. B, Histology. H&E and trichrome staining of the LV of NTG unpaced control, TG paced and NTG paced rabbits shows variation in cardiomyocyte size in the paced animals. 20X original magnification. C, Quantification of cardiomyocyte cross-sectional area in NTG unpaced control, paced TG and paced NTG. 192, 933 and 662 cells were measured for NTG unpaced control, paced TG and paced NTG, respectively. Values are mean ± standard deviation. *P<.001 compared to NTG control. D–F, M-mode tracings of the left ventricle in the parasternal long axis view of (D) NTG non-paced, (E) NTG paced and (F) TG paced.
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