Mast cell inhibition attenuates myocardial damage, adverse remodeling, and dysfunction during fulminant myocarditis in the rat

Abstract

Background: Myocarditis is a life-threatening heart disease characterized by myocardial inflammation, necrosis, and chronic fibrosis. While mast cell inhibition has been suggested to prevent fibrosis in rat myocarditis, little is known about its effectiveness in attenuating cardiac remodeling and dysfunction in myocarditis. Thus, we sought to test the hypothesis that mast cell inhibition will attenuate the inflammatory reaction and associated left ventricular (LV) remodeling and dysfunction after fulminant autoimmune myocarditis. Methods and

Results: To induce experimental autoimmune myocarditis, we immunized 30 rats with porcine cardiac myosin (PCM) twice at a 7-day interval. On day 8 animals were randomized into treatment with either an intraperitoneal (IP) injection of 25mg/kg of cromolyn sodium (n = 13) or an equivalent volume (∼0.5 mL IP) of normal saline (n = 11). All animals were scanned by serial echocardiography studies before treatment (baseline echocardiogram) and after 20 days of cromolyn sodium (28 days after immunization). Furthermore, serial cardiac magnetic resonance was performed in a subgroup of 12 animals. After 20 days of treatment (28 days from first immunization), hearts were harvested for histopathological analysis. By echocardiography, cromolyn sodium prevented LV dilatation and attenuated LV dysfunction, compared with controls. Postmortem analysis of hearts showed that cromolyn sodium reduced myocardial fibrosis, as well as the number and size of cardiac mast cells in the inflamed myocardium, compared with controls.

Conclusions: Our study suggests that mast cell inhibition with cromolyn sodium attenuates adverse LV remodeling and dysfunction in myocarditis. This mechanism-based therapy is clinically relevant and could improve the outcome of patients at risk for inflammatory cardiomyopathy and heart failure.

Figure 1. Cromolyn sodium attenuates LV dilatation compared to controls, 28 days after induction of EAM by CMR analysis

(a) LV dilatation attenuated in rats treated with cromolyn sodium compared with control group - lower % of change in LVSA (LV end systolic area). (b–e) Representative end-systolic short-axis images from CMR function movies at baseline (b–c) and after 28 days (d–e). Notice the extensive dilatation of both LV and RV (right ventricle) in the control rat (e), which is attenuated in the cromolyn-treated rat (d).

Figure 2. Cromolyn sodium preserves LV wall thickness and mass by postmortem morphometry, 28 days after EAM induction

Representative sections of heart treated with cromolyn sodium (a) or saline (b) in Masson Trichrome staining. Morphometry shows that cromolyn treatment preserves LV wall thickness (c) and area (d) compared to control.

Figure 3. Cromolyn sodium reduces fibrosis by postmortem morphometry, 28 days after EAM induction

Representative sections of the heart treated with cromolyn sodium (a) or saline (b) in Masson Trichrome staining. Morphometry shows that cromolyn treatment reduces proportion of fibrosis (c).

Figure 4. Cromolyn sodium reduces cardiac mast cell density and size, 28 days after EAM induction

Representative sections of the heart treated with cromolyn sodium (a) or saline (b) using a monoclonal antibody to mast-cell tryptase (brown). Arrows indicate mast cells containing tryptase. Analysis based on average density and size shows that cromolyn sodium treatment significantly reduced cardiac mast cell density (c) and size (d).

Figure 5. Cromolyn sodium improves angiogenesis 28 days after EAM induction

Representative sections of the heart treated with cromolyn sodium (a) or saline (b) using antibodies to α-smooth muscle actin. Arrows indicate positively stained small-medium sized blood vessels. Analysis based on average density of blood vessels (c) shows that cromolyn treatment significantly improved angiogenesis.