Resolution of established cardiac hypertrophy and fibrosis and prevention of systolic dysfunction in a transgenic rabbit model of human cardiomyopathy through thiol-sensitive mechanisms

Abstract

Background: Cardiac hypertrophy, the clinical hallmark of hypertrophic cardiomyopathy (HCM), is a major determinant of morbidity and mortality not only in HCM but also in a number of cardiovascular diseases. There is no effective therapy for HCM and generally for cardiac hypertrophy. Myocardial oxidative stress and thiol-sensitive signaling molecules are implicated in pathogenesis of hypertrophy and fibrosis. We posit that treatment with N-acetylcysteine, a precursor of glutathione, the largest intracellular thiol pool against oxidative stress, could reverse cardiac hypertrophy and fibrosis in HCM.

Methods and results: We treated 2-year-old beta-myosin heavy-chain Q403 transgenic rabbits with established cardiac hypertrophy and preserved systolic function with N-acetylcysteine or a placebo for 12 months (n=10 per group). Transgenic rabbits in the placebo group had cardiac hypertrophy, fibrosis, systolic dysfunction, increased oxidized to total glutathione ratio, higher levels of activated thiol-sensitive active protein kinase G, dephosphorylated nuclear factor of activated T cells (NFATc1) and phospho-p38, and reduced levels of glutathiolated cardiac alpha-actin. Treatment with N-acetylcysteine restored oxidized to total glutathione ratio, normalized levels of glutathiolated cardiac alpha-actin, reversed cardiac and myocyte hypertrophy and interstitial fibrosis, reduced the propensity for ventricular arrhythmias, prevented cardiac dysfunction, restored myocardial levels of active protein kinase G, and dephosphorylated NFATc1 and phospho-p38.

Conclusions: Treatment with N-acetylcysteine, a safe prodrug against oxidation, reversed established cardiac phenotype in a transgenic rabbit model of human HCM. Because there is no effective pharmacological therapy for HCM and given that hypertrophy, fibrosis, and cardiac dysfunction are common and major predictors of clinical outcomes, the findings could have implications in various cardiovascular disorders.

Figure 1

Relative levels of oxidized to total glutathione (GSSG/GSH+GSSG). A and B show the ratio of oxidized to total glutathione in the blood (n = 6) and ventricular tissues (n = 3), respectively. GSSG/GSH+GSSG ratio was increased significantly in the blood and in the heart tissues in the β-MyHC-Q403 transgenic rabbits in the placebo group compared with nontransgenic (NTG) rabbits. In contrast, the ratio was normal in transgenic rabbits treated with NAC.

Figure 2

Histological phenotype of myocyte hypertrophy (top panels), interstitial fibrosis (middle panels), and myocyte disarray (bottom panels). Representative high-magnification (×400) fields are shown (A). B through E show quantitative values of myocyte cross-sectional area (CSA), number of myocytes per each high-magnification (×400) microscopic field, CVF (% of myocardium), and extent of myocyte disarray (% of myocardium), respectively. NTG indicates nontransgenic.

Figure 3

Myofiber α-helical orientation as determined by diffusion-weighted (tensor) MRI. A, Representative cross-sectional images at mid ventricular levels are shown. Significant differences in myofiber orientation between transgenic rabbits in the placebo group and nontransgenic (NTG) rabbits at the endocardium and between the transgenic rabbits in the placebo and NAC groups at the epicardium are noted. B, Diagrammatic representation of myofiber α-helical orientation from epicardium to endocardium. C, Quantitative fiber orientation at mid ventricle is plotted against transmural depth from endocardium (Endo-) to epicardium (Epi-) to illustrate the differences among the 3 groups. WT indicates wild-type; TG, transgenic. D, Overall mean myofiber α-helical angles among the 3 groups.

Figure 4

Effects of NAC on myocardial levels of selective thio-sensitive molecules. A shows relative myocardial levels of active (dephosphorylated) NFATc1 normalized to that in the nontransgenic (NTG) rabbits. B shows myocardial levels of thiol-sensitive active PKG in the experimental groups. The upper blot in C shows phosphorylated p38 (p-p38) levels in the myocardium and the lower blot levels of total p38 levels, detected by immunoblotting. D shows quantitative levels of phospho-p38 as determined by densitometry and normalized to that in the nontransgenic group. In all panels, means and SDs are shown. The probability values at the top of each panel represent differences among the 3 groups. The Bonferroni adjusted probability values for paired groups are shown (n = 4 rabbits per group in each set of experiments).

Figure 5

Immunoblots showing levels of glutathiolated cardiac α-actin, detection of G-actin by immunofluorescence, and subcellular localization of α-actin. A, Coimmunoprecipitation of cardiac α-actin by a GSH antibody. B, Quantitative values of glutathiolated cardiac α-actin. C, Immunofluorescence staining of thin myocardial sections with DNase 1, a binding partner to G-actin, costained with DAPI (overlay pictures). D and E, Immunoblots of cardiac α-actin detected in subcellular compartments cytosolic (C), nuclear (N), membrane (M), and cytoskeletal (Cs). Panels showing expression levels of α-tubulin are shown to reflect comparability of loading conditions as well as purity of the extraction of subcellular proteins. F shows quantitative levels of cardiac α-actin in different cellular compartments. Means and SDs are shown. Comparisons among the 3 groups are shown at the top along with adjusted probability values for pairwise comparisons. NTG indicates nontransgenic.

Figure 6

Effects on vulnerable window and period. A through C show lower and upper limits of vulnerability (ULV) and the vulnerability window (VW) in nontransgenic (NTG), transgenic-placebo, and transgenic-NAC groups. LLV indicates lower limits of vulnerability; VP, vulnerable period; and APD, action potential duration. D, The mean values of combined right ventricular (RV) and LV ULV among the 3 groups. ULV in the placebo group is increased compared with nontransgenic rabbits. ULV and the vulnerable period were significantly lower in the NAC group (compared with the placebo group), indicating reduced susceptibility to shock-induced arrhythmias.