Secondary coronary artery vasospasm promotes cardiomyopathy progression

Abstract

Genetic defects in the plasma membrane-associated sarcoglycan complex produce cardiomyopathy characterized by focal degeneration. The infarct-like pattern of cardiac degeneration has led to the hypothesis that coronary artery vasospasm underlies cardiomyopathy in this disorder. We evaluated the coronary vasculature of gamma-sarcoglycan mutant mice and found microvascular filling defects consistent with arterial vasospasm. However, the vascular smooth muscle sarcoglycan complex was intact in the coronary arteries of gamma-sarcoglycan hearts with perturbation of the sarcoglycan complex only within the adjacent myocytes. Thus, in this model, coronary artery vasospasm derives from a vascular smooth muscle-cell extrinsic process. To reduce this secondary vasospasm, we treated gamma-sarcoglycan-deficient mice with the calcium channel antagonist verapamil. Verapamil treatment eliminated evidence of vasospasm and ameliorated histological and functional evidence of cardiomyopathic progression. Echocardiography of verapamil-treated, gamma-sarcoglycan-null mice showed an improvement in left ventricular fractional shortening (44.3 +/- 13.3% treated versus 37.4 +/- 15.3% untreated), maximal velocity at the aortic outflow tract (114.9 +/- 27.9 cm/second versus 92.8 +/- 22.7 cm/second), and cardiac index (1.06 +/- 0.30 ml/minute/g versus 0.67 +/- 0.16 ml/minute/g, P < 0.05). These data indicate that secondary vasospasm contributes to the development of cardiomyopathy and is an important therapeutic target to limit cardiomyopathy progression.

Figure 1

A: The coronary vascular smooth muscle sarcoglycan complex. Sections from normal (top) or gsg^−/− hearts, including coronary vessels with surrounding cardiomyocytes, were stained with antibodies specific to β-, γ-, δ-, ε-, and ζ-sarcoglycan (anti-β, anti-γ, anti-δ, anti-ε, and anti-ζ, respectively). Vascular smooth muscle expresses β-, δ-, ε-, and ζ-sarcoglycan in both normal and gsg^−/− hearts. In gsg^−/− hearts, the cardiomyocytes surrounding the coronary vessels have no γ-sarcoglycan and reduced β- and δ-sarcoglycan. γ-Sarcoglycan is not present in vascular smooth muscle. B: Immunoblots of δ-sarcoglycan and γ-sarcoglycan in smooth muscle-containing tissues. Aorta, bladder, and uterus whole protein extracts were made from wild-type (WT), gsg^−/− and dsg^−/− tissues. Skeletal muscle microsomes from wild-type animals (SM) were used as a positive control. γ-Sarcoglycan is striated muscle-specific, and δ-sarcoglycan is expressed in gsg^−/− smooth muscle indicating no disruption of the smooth muscle sarcoglycan complex in gsg^−/−. An anti-smooth muscle actin (sm actin) antibody was used to demonstrate loading for the lanes containing smooth muscle-derived lysates (bottom).

Figure 2

Microvascular filling defects in γ-sarcoglycan-null mice. Microfil microvascular filling was used to examine the coronary arterial tree of normal (wt), dsg^−/−, and gsg^−/− hearts. In normal hearts, vascular trees show no evidence of focal narrowing or constriction and generally have smoothly tapered vessels and vessel junctions. In contrast, gsg^−/− and dsg^−/− hearts show evidence of vasospasm with focal narrowings (arrows). Representative images of gsg^−/− microvascular filling typical of the abnormalities seen in both young and old animals (8 and 26 weeks).

Figure 3

Verapamil treatment ameliorates but does not eliminate evidence of cardiomyocyte damage. A, C, and E: Representative images of hearts from untreated 26-week-old gsg^−/− mice. B, D, and F: Representative images from hearts of gsg^−/− mice treated with oral verapamil for 5 months. A–D: Masson trichrome staining of 26-week-old heart sections showing focal necrosis, fibrosis, and inflammatory infiltrate. A reduction in focal necrosis is seen with verapamil treatment. Perivascular inflammation is also reduced with verapamil treatment. E and F: Evans blue dye staining (red) shows areas of cardiomyocyte membrane damage. The plasma membranes of cardiomyocytes are stained with anti-dystrophin antibody (green). Scale bar, 10 μm.

Figure 4

Verapamil treatment improves cardiac function. A–C: Measurements from echocardiography of 6-month-old normal mice (gray triangles), gsg^−/− mice (open circles), and verapamil-treated gsg^−/− mice (filled circles) are shown. Means ± SDs are represented for normal (gray diamond), untreated gsg^−/− (open diamond), and verapamil-treated gsg^−/− (filled diamond) cohorts. Fractional shortening (A), maximal developed velocity at the aortic outflow tract (B), and cardiac index (C) all show functional improvement with verapamil treatment of gsg^−/− mice. *, P < 0.05 versus normal; ^†, P < 0.05 versus untreated gsg^−/−.

Figure 5

Verapamil treatment does not improve expression of the remaining sarcoglycan at the plasma membrane. The major sarcoglycan complex of cardiomyocytes is α-, β-, γ-, and δ-sarcoglycan. Antibodies specific to α-sarcoglycan, β-sarcoglycan, and δ-sarcoglycan were used on normal control and γ-sarcoglycan-null mice (gsg−/−) that were treated and untreated with verapamil. Scale bar, 10 μm.