The NF-κB subunit c-Rel stimulates cardiac hypertrophy and fibrosis

Abstract

Cardiac remodeling and hypertrophy are the pathological consequences of cardiovascular disease and are correlated with its associated mortality. Activity of the transcription factor NF-κB is increased in the diseased heart; however, our present understanding of how the individual subunits contribute to cardiovascular disease is limited. We assign a new role for the c-Rel subunit as a stimulator of cardiac hypertrophy and fibrosis. We discovered that c-Rel-deficient mice have smaller hearts at birth, as well as during adulthood, and are protected from developing cardiac hypertrophy and fibrosis after chronic angiotensin infusion. Results of both gene expression and cross-linked chromatin immunoprecipitation assay analyses identified transcriptional activators of hypertrophy, myocyte enhancer family, Gata4, and Tbx proteins as Rel gene targets. We suggest that the p50 subunit could limit the prohypertrophic actions of c-Rel in the normal heart, because p50 overexpression in H9c2 cells repressed c-Rel levels and the absence of cardiac p50 was associated with increases in both c-Rel levels and cardiac hypertrophy. We report for the first time that c-Rel is highly expressed and confined to the nuclei of diseased adult human hearts but is restricted to the cytoplasm of normal cardiac tissues. We conclude that c-Rel-dependent signaling is critical for both cardiac remodeling and hypertrophy. Targeting its activities could offer a novel therapeutic strategy to limit the effects of cardiac disease.

Figure 1

c-Rel regulates cardiac growth in mouse and human. A: Human LV tissue from normal and diseased hearts was stained for c-Rel protein. Black arrows indicate nuclear staining; red arrows indicate cytoplasmic staining. Photomicrographs are representative of five normal and five diseased human hearts. H9c2 cells were transiently transfected with RSV-β-gal (control plasmid) or RSV-c-Rel. B: Quantification of mRNA showed that c-Rel positively regulates cardiac expression of transcriptional activators of hypertrophy and hypertrophic markers. Relative level of transcriptional difference RLTD was expressed as mean fold change ± SEM, compared with RSV, of five independent transfections. C: Representative images of hearts isolated from adult WT (left) and Rel^−/− mice (right) show decreased heart size in the knockout mice. Cardiac hypotrophy is further highlighted by a decrease in heart/body weight ratio in mice lacking Rel, compared with WT. Results are expressed as mean ratio change in Rel^−/− mice compared with WT ± SEM; n = 20 mice/group. D: Cardiomyocyte size is reduced in Rel^−/− mice, compared with controls. Photomicrographs show heart sections from WT and Rel^−/− mice, stained with hematoxylin and eosin. Original magnification, ×400. Scale bars: 100 μm. Image analysis was used to calculate mean cytoplasmic/nuclear area ratios. Results are expressed as mean ratio change compared with WT ± SEM; n = 5 mice/group. E: Eight of the 10 genes measured were down-regulated in Rel^−/− mice, compared with WT. Data are expressed as mean RLTD fold change ± SEM, relative to WT; n = 5 mice/genotype. All P values were calculated using a one-way analysis of variance or an unpaired two-tailed Student's t-test. *P = 0.05, **P = 0.01, and ***P = 0.001.

Figure 2

Rel knockout mice are protected from angiotensin-induced cardiac hypertrophy and fibrosis. A: The heart/body weight ratio was calculated in Rel^−/− and WT mice after 4 weeks of saline vehicle or angiotensin (Ang) infusion. B: Mean cytoplasmic/nuclear area ratio was calculated using image analysis of laminin-stained hearts. Data are expressed as mean percentage change, compared with WT. Cardiomyocyte size was increased in WT mice after angiotensin, indicative of a hypertrophic response. By contrast, cardiomyocytes of angiotensin-treated Rel^−/− mice were smaller than in the WT, suggesting a cardioprotective effect. Representative photomicrographs show heart sections from angiotensin-infused WT and Rel^−/− mice immunostained with anti-laminin antibodies. Cardiac remodeling and fibrosis was reduced in angiotensin-infused Rel^−/− mice, compared with WT. Original magnification, ×400. Scale bars: 100 μm. C: Densitometric analysis of Sirius Red-stained heart tissues showed a statistically significant increase in collagen deposition in WT mice after angiotensin infusion; however, less collagen was observed in both the saline and angiotensin-infused Rel^−/− mice. Representative photomicrographs show Sirius Red-stained heart sections from angiotensin-treated WT and Rel^−/− mice. Data are expressed as mean ratio or percentage change ± SEM; n = 5 (Rel^−/−) and n = 6 (WT) mice/group. D: Relative mRNA levels of hypertrophy-associated genes were determined in Rel^−/− and WT mice after angiotensin infusion. Angiotensin infusion induces cardiac expression of transcriptional regulators of hypertrophy; however, this response is attenuated in Rel^−/− mice. The RLTD was calculated between WT and Rel^−/− mice and expressed as a mean fold change ± SEM, relative to WT; n = 4 mice/genotype. All P values were calculated using a one-way analysis of variance. *P = 0.05, **P = 0.01, and ***P = 0.001.

Figure 3

c-Rel binds to hypertrophy-related gene promoters. Cross-linked ChIP analysis revealed binding of c-Rel to the promoters of transcriptional activators of hypertrophy (A and B) and to cardioprotective factors (C). In the promoter schematics, blue boxes indicate NF-κB-c-Rel consensus sequences, yellow boxes indicate preferential c-Rel consensus sequences, and purple boxes indicate NF-κB consensus sequences. H9c2 cells were formalin-fixed. Chromatin was then isolated and sheared by sonication, and 100 μg of chromatin was incubated with an anti-c-Rel antibody, anti-p50 antibody, or irrelevant IgG isotype control. Immunoprecipitation reactions were performed, proteins were digested, and cross-links were reversed before purification of genomic DNA and qRT-PCR amplification of promoters of interest. Binding was normalized to total input genomic DNA and is expressed as fold IgG control. Data are representative of at least three separate experiments. All P values were calculated using one way analysis of variance. *P = 0.05, **P = 0.01.

Figure 4

p50 suppresses the prohypertrophic effects of c-Rel. H9c2 cells were transiently transfected with expression plasmids (control RSV-β-gal, RSV-p50, or RSV-c-Rel). RNA was isolated, and cDNA was generated and used as a template for qRT-PCR. The RLTD was calculated and expressed as mean fold change ± SEM, relative to RSV, of five independent transfections. A: Gene expression levels of the individual NF-κB subunits revealed that overexpression of p50 repressed expression of c-Rel. B: RNA was isolated from the hearts of adult WT and Nfkb1^−/− mice, and mRNA levels of the NF-κB subunits RelA, NF-κB2, c-Rel, and RelB were quantified using qRT-PCR. c-Rel was the only NF-κB subunit to be up-regulated in Nfkb1^−/− mice. The RLTD between WT and Nfkb1^−/− mice was calculated and expressed as a mean fold change ± SEM relative to WT; n = 5 mice/genotype. *P = 0.05, **P = 0.01. C: Representative photographs of hearts isolated from adult WT and Nfkb1^−/− mice show increased heart size in Nfkb1^−/− mice. D: The heart/body weight ratio was calculated in Nfkb1^−/− and WT control mice on a pure C57Bl/6 background; data are expressed as mean percentage change ± SEM, compared with WT; n = 17 mice/genotype. An increase in heart/body weight ratio was observed in male Nfkb1^−/− mice, compared with WT controls, in both backgrounds. ***P < 0.001.

Figure 5

Features of cardiac hypertrophy and fibrosis are observed in Nfkb1 knockout mice. A: Heart sections were stained for α-sarcomeric actin and sarcomeric units were counted. The mean number (Ave) of sarcomeric units in Nfkb1^−/− mice was significantly increased, compared with WT. *P = <0.05. B: Representative photomicrographs of laminin-stained WT and Nfkb1^−/− mouse hearts. Mean cardiomyocyte cytoplasmic/nuclear area ratios were calculated using image analysis software. The ratio was significantly increased in Nfkb1^−/− hearts, compared with WT. **P < 0.01. C: Representative photomicrographs of Sirius Red-stained heart sections from WT and Nfkb1^−/− mice. Densitometric analysis revealed a statistically significant increase in collagen deposition (red fibers) in Nfkb1^−/− mice. *P = <0.05. Data are expressed as means ± SEM of eight WT and six Nfkb1^−/− mice. +ve, positive. D: Relative mRNA levels of the transcriptional regulators of hypertrophy myocyte enhancer factor 2 (Mef2) A, C, and D (but not Mef2B), Gata4, and Foxm1b were elevated in Nfkb1^−/− mice, compared with WT. Deletion of Nfkb1 was associated with an increase in gene expression of Nkx2-5 and of Tbx20. Cardiac mRNA levels of the cardioprotective proteins brain natriuretic peptide (BNP) and atrial natriuretic peptide (ANP) were quantified. BNP expression was increased in the Nfkb1^−/− mice, compared with WT; however, expression of ANP was barely detectable in Nfkb1^−/− mice. RLTD was calculated between WT and Nfkb1^−/− mice and expressed as mean fold change ± SEM, relative to WT; n = 5 mice/genotype. Original magnification, ×400. Scale bars: 100 μm.