Targeted cardiac expression of soluble Fas prevents the development of heart failure in mice with cardiac-specific expression of MCP-1

Abstract

Monocyte chemoattractant protein-1 (MCP-1) plays a crucial role in initiating coronary heart disease by recruiting monocytes/macrophages to the vessel wall. Transgenic mice with cardiac-specific expression of MCP-1 manifest cardiac inflammation and develop heart failure. The pathways mediating the detrimental effects of MCP-1 expression have not been defined. We postulate that the Fas ligand (FasL) derived from the infiltrating mononuclear cells causes death of cardiac cells resulting in the development of heart failure. Here, we tested this hypothesis by determining whether inhibition of FasL function through cardiac-specific expression of soluble Fas (sFas) would rescue the MCP-1 transgenic mice from developing heart failure. We generated mice with cardiac-specific expression of sFas and double homozygous transgenic mice that express both MCP-1 and sFas. Cardiac-specific expression of sFas in MCP mice, in fact, inhibited apoptosis of infiltrating mononuclear cells, normalized circulating C-reactive protein (CRP) levels, and prevented macrophage activation as well as production of proinflammatory cytokines, tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, and IL-6 in the hearts. sFas expression resulted in restoration of cardiac structure, preservation of cardiac function, and a significant prolongation of survival of MCP mice. These results demonstrate that FasL released from infiltrating mononuclear cells plays a critical role in the detrimental effects of MCP-1 expression, and suggest that Fas/FasL signaling represents a novel therapeutic target for heart failure.

Fig. 1

Characterization of mice with cardiac-targeted expression of both MCP-1 and sFas. (A) Molecular strategy used to express sFas gene with murine α-MHC promoter. (B, C) Real-time PCR demonstrates transgene mRNA expression in the hearts of wild-type and age-matched transgenic mice of 4 months of age (n = 3 per group). Equal expression of transgene was found in mice expressing only one transgene versus mice expressing both transgenes. ND, not detectable. (D) Circulating levels of sFas protein in mice at 6 months of age assayed by ELISA (n = 3 per group). ND, not detectable.

Fig. 2

Cardiac-targeted expression of sFas in MCP mice prevents cardiac deterioration. (A) Representative photographs showing the whole hearts from the wild-type, MCP, MCP/sFas, and sFas mouse at 6 months of age. (B) Representative photomicrographs demonstrating morphology of heart sections from the 6-month-old wild-type and age-matched transgenic mice stained with H&E (upper panel) and Masson's trichrome (lower panel). Blue staining represents collagen deposition. (C) Heart weight to body weight (HW/BW) ratio in the wild-type and transgenic mice was depicted from 2 to 6 months of age. *P < 0.001, ^#P < 0.05 versus age-matched wild-type, sFas, and MCP/sFas mice, respectively. n = 6 per group per time point. (D) Quantitation analysis of myocardial collagen volume fraction in the 6-month-old wild-type and age-matched transgenic mice. *P < 0.001 versus wild-type, sFas, and MCP/sFas mice. n = 5 per group.

Fig. 3

Effect of cardiac-expression of sFas on cellular infiltration in the hearts of wild-type and age-matched transgenic mice. (A) Representative photomicrographs demonstrating immunohistochemical staining for CD45, Mac-1, and Mac-3 in the heart sections from 6-month-old wild-type and age-matched transgenic mice. Positive-stained cells were visualized with diaminobenzidine (brown). (B–D) Histograms showing the number of CD45-, Mac-1-, and Mac-3-positive cells in the hearts of wild-type and transgenic mice. *P < 0.001 versus age-matched wild-type and sFas mice; ^#P < 0.001 versus age-matched wild-type, sFas, and MCP/sFas mice, n = 5 per group per time point. (E) Circulating levels of CRP assayed by ELISA. n = 6 per group per time point. *P < 0.05 versus age-matched wild-type, sFas, and MCP/sFas mice.

Fig. 4

Effect of cardiac-expression of sFas on proinflammatory cytokines mRNA expression in the hearts of wild-type and age-matched transgenic mice. Relative levels of TNF-α (A), IL-1β (B), and IL-6 (C) mRNA expression in the hearts of wild-type and age-matched transgenic mice were determined by real-time PCR, and were normalized by mouse β-actin mRNA. n = 5 per group per time point. *P < 0.001 versus age-matched wild-type, sFas, and MCP/sFas mice; ^#P < 0.05 versus age-matched wild-type and sFas mice.

Fig. 5

Effect of cardiac-expression of sFas on apoptotic cell death in the hearts of wild-type and age-matched transgenic mice. Representative photomicrographs demonstrating TUNEL staining in the heart sections from 6-month-old wild-type (A) and age-matched MCP (B), MCP/sFas (C), and sFas (D) transgenic mice. Blue staining indicates that TUNEL-positive cells and the TUNEL-positive cardiac myocytes are indicated by arrows in panel B. The insert in panel E shows TUNEL-positive vascular cells magnified from the rectangle area from the heart of MCP mouse. The insert in panel F shows no TUNEL-positive vascular cells in the heart of age-matched MCP/sFas mouse. (G, H) Histograms showing the number of TUNEL-positive infiltrating cells and TUNEL-positive cardiomyocytes in the hearts of 6-month-old wild-type and age-matched transgenic mice. n = 5 per group per time point. *P < 0.001 versus wild-type, sFas, and MCP/sFas mice.

Fig. 6

Effect of cardiac-expression of sFas on left ventricle (LV) morphology and function. (A) Representative M-mode echocardiograms from the 6-month-old wild-type and age-matched transgenic mice showing changes in LV dimensions. EDD and ESD indicate LV end-diastolic dimension and end-systolic dimension, respectively. (B, C) LVend-diastolic dimension (LVEDD) and percent functional shortening (FS) were depicted from 2 to 6 months of age. MCP mice had progressive LV dilatation and cardiac dysfunction compared to age-matched wild-type and sFas mice, whereas the MCP/sFas mice had slightly increased LVEDD and reduced FS, but were not significantly different from LVEDD and FS in the age-matched wild-type and sFas mice. n = 6 per group per time point, *P < 0.001 versus age-matched wild-type, sFas, and MCP/sFas mice, ^#P < 0.05 versus age-matched wild-type, sFas, and MCP/sFas mice.

Fig. 7

Kaplan–Meier survival analysis of wild-type (n = 24), MCP (n = 32), MCP/sFas (n = 26), and sFas (n = 28) mice showing drastic improvement in survival profile of the MCP/sFas mice compared with MCP mice. *P < 0.001 versus age-matched wild-type, sFas, and MCP/sFas mice.