Myocardial remodeling in diabetic cardiomyopathy associated with cardiac mast cell activation

Abstract

Diabetic cardiomyopathy is a specific disease process distinct from coronary artery disease and hypertension. The disease features cardiac remodeling stimulated by hyperglycemia of the left ventricle wall and disrupts contractile functions. Cardiac mast cells may be activated by metabolic byproducts resulted from hyperglycermia and then participate in the remodeling process by releasing a multitude of cytokines and bioactive enzymes. Nedocromil, a pharmacologic stabilizer of mast cells, has been shown to normalize cytokine levels and attenuate cardiac remodeling. In this study, we describe the activation of cardiac mast cells by inducing diabetes in normal mice using streptozotocin (STZ). Next, we treated the diabetic mice with nedocromil for 12 weeks and then examined their hearts for signs of cardiac remodeling and quantified contractile function. We observed significantly impaired heart function in diabetic mice, as well as increased cardiac mast cell density and elevated mast cell secretions that correlated with gene expression and aberrant cytokine levels associated with cardiac remodeling. Nedocromil treatment halted contractile dysfunction in diabetic mice and reduced cardiac mast cell density, which correlated with reduced bioactive enzyme secretions, reduced expression of extracellular matrix remodeling factors and collagen synthesis, and normalized cytokine levels. However, the results showed nedocromil treatments did not return diabetic mice to a normal state. We concluded that manipulation of cardiac mast cell function is sufficient to attenuate cardiomyopathy stimulated by diabetes, but other cellular pathways also contribute to the disease process.

Figure 1. The blood glucose level and body weight of normal and STZ-induced diabetic mice were monitored for 12 weeks after nedocromil (Ned) treatment, as described in Methods.

(A) The blood glucose levels of the different treatment groups at the indicated time points. (B) The body weights of the different treatment groups at the indicated time points. (n = 15 per group).

Figure 2. Cardiac function of isolated and perfused hearts from normal and STZ-induced diabetic mice after nedocromil (Ned) treatments.

Contractile function of the heart was determined using a multi-channel physiological recorder. Changes in heart work (A), heart rate (B), maximal rate of contraction (C), and relaxation (D) are presented. Quantitative data are shown as means ± SD (n = 15 per group). *P<0.05 vs. untreated normal group; ^#P<0.05 vs. untreated diabetic group.

Figure 3. Characterization of cardiac mast cell activation in normal or STZ-induced diabetic mice after nedocromil (Ned) treatments.

(A) Quantification of chymase-positive mast cells. Chymase-positive cells from immunohistochemically stained cardiac tissues were counted in 100 random fields at 400X magnification. (B) Mast cell density in sample tissues was determined by dividing the number of chymase-positive cells by the total number of cells in the visual fields. (C) mRNA expression levels of chymase in sample tissues were normalized to GAPDH expression and depicted as fold-change relative to the untreated normal group. (D) Representative results of protein expression analysis for chymase, tryptase, and histamine in sample tissues using western blot. GAPDH expression is shown as loading control. Quantitative data are displayed as means ± SD (n = 15 per group). ^*P<0.05 vs. untreated normal group; ^#P<0.05 vs. untreated diabetic group.

Figure 4. Collagen deposits in the left ventricle (LV) of normal and STZ-induced diabetic mice after nedocromil (Ned) treatments.

(A) Representative images of LV sections stained with Masson's trichrome at 100X magnification. Blue color indicates collagen deposits. (B) Quantification of LV collagen volume fraction (CVF). CVF is represented as the ratio of collagen area/total area. (C) The mRNA expression levels of type I and type III collagen are normalized to GAPDH expression and depicted as fold-change relative to the untreated normal group. (D) Representative results of protein expression analysis for collagen I, collagen III, MMP-2, and MMP-9 in sample tissues using western blot. GAPDH expression is shown as loading control and used for normalization. (E) Quantification of type I and type III collagen, MMP-2, and MMP-9 protein levels normalized to GAPDH. Quantitative data are displayed as means ± SD (n = 15 per group). ^*P<0.05 vs. untreated normal group; ^#P<0.05 vs. untreated diabetic group.

Figure 5. ELISA assay for cytokine expression in cardiac tissues of normal and STZ-induced diabetic mice treated with nedocromil (Ned) or controls.

The graphs show the quantitative levels of (A) TNF-α, (B) IFN-γ, (C) IL-4, (D) IL-10, and (E) Angiotensin II (Ang II) of the indicated sample groups. Quantitative data are displayed as means ± SD (n = 15 per group). ^*P<0.05 vs. untreated normal group; ^#P<0.05 vs. untreated diabetic group.