Caveolin-1 ablation reduces the adverse cardiovascular effects of N-omega-nitro-L-arginine methyl ester and angiotensin II

Abstract

Caveolae are the major cellular membrane structure through which extracellular mediators transmit information to intracellular signaling pathways. In vascular tissue (but not ventricular myocardium), caveolin-1 (cav-1) is the main component of caveolae; cav-1 modulates enzymes and receptors, such as the endothelial nitric oxide synthase and the angiotensin II (AngII) type 1 receptor. Evidence suggests that AngII and aldosterone (ALDO) are important mediators of ventricular injury. We have described a model of biventricular damage in rodents that relies on treatment with N-omega-nitro-l-arginine methyl ester (L-NAME (nitric oxide synthase inhibitor)) and AngII. This damage initiated at the vascular level and was observed only in the presence of ALDO and an activated mineralocorticoid receptor (MR). We hypothesize that cav-1 modulates the adverse cardiac effects mediated by ALDO in this animal model. To test this hypothesis, we assessed the ventricular damage and measures of inflammation, in wild-type (WT) and cav-1 knockout (KO) mice randomized to either placebo or L-NAME/AngII treatment. Despite displaying cardiac hypertrophy at baseline and higher blood pressure responses to L-NAME/AngII, cav-1 KO mice displayed, as compared with WT, decreased treatment-induced biventricular damage as well as decreased transcript levels of the proinflammatory marker plasminogen activator inhibitor-1. Additionally, L-NAME/AngII induced an increase in cardiac MR levels in WT but not cav-1-ablated mice. Moreover and despite similar circulating ALDO levels in both genotypes, the myocardial damage (as determined histologically and by plasminogen activator inhibitor-1 mRNA levels) was less sensitive to ALDO levels in cav-1 KO vs. WT mice, consistent with decreased MR signaling in the cav-1 KO. Thus, we conclude that the L-NAME/AngII-induced biventricular damage is mediated by a mechanism partially dependent on cav-1 and signaling via MR/ALDO.

Figures

Figure 1

Myocardial damage and inflammatory markers in cav-1 WT and KO mice. A, MDSs were determined in heart tissues obtained from placebo- (control) or L-NAME/AngII-treated animals (n > 18/group). B, Representative H&E staining of myocardial tissue sections from cav-1 WT (left panels) and KO (right panels) animals, treated with either placebo (upper panels) or L-NAME/AngII (lower panels). Magnification, ×400. C and D, Heart tissues were analyzed by real-time RT-PCR for PAI-1 (C, n > 11/group) and CD-68 (D, n > 8/group) levels, as described in Materials and Methods. *, P < 0.01 vs. control animals of the same genotype.

Figure 2

eNOS expression in cav-1 WT and KO mice. Heart tissues from animals treated with placebo (control) or L-NAME/AngII were analyzed by Western blotting under denaturing (A and B) or nondenaturing (C) conditions, as described in Materials and Methods, using total eNOS (A and C) and phospho-eNOS (Ser1177) antibodies (B), respectively. Representative Western blots are depicted under each bar graph. *, P < 0.001 vs. control animals of the same genotype.

Figure 3

A, cav-1 binding motif within MR sequence. A putative cav-1 binding motif is located in the midportion of the N-terminal region of the MR. Sequence alignment between mouse, human, and rat MR shows that this region is highly conserved between species. φ, Aromatic amino residues Trp, Phe, or Try; X, any amino acid. B and C, Coimmunoprecipitation patterns for cav-1, AT1R, and MR in mouse hearts. Representative Western blots (WB) showing reciprocal immunoprecipitation (IP) of cav-1 with the AT1R and MR. Tissue homogenates from WT and cav-1 KO mice were immunoprecipitated with anti-cav-1 (B and C, left panels), anti-AT1R (B, right panel), anti-MR (C, right panel) and then analyzed by Western blotting for the presence of AT1R (B, left panel), cav-1 (B and C, right panel), and MR (C, left panel), respectively, as described in Materials and Methods.

Figure 4

Effect of L-NAME/AngII treatment on AT1R transcript levels and ERK1/2 and PKCδ protein expression in cav-1 WT and KO mouse hearts. Heart tissues from animals treated with placebo (control) or L-NAME/AngII were analyzed by real-time RT-PCR (A) or Western blotting (B and C), as described in Materials and Methods. Representative Western blots are depicted under each bar graph (B and C). *, P < 0.01; #, P < 0.05 vs. control animals of the same genotype.

Figure 5

Effect of L-NAME/AngII treatment on ALDO and MR protein levels in cav-1 WT and KO mouse hearts. Plasma ALDO levels (A) were determined in placebo- and L-NAME/AngII-treated mice, as described in Materials and Methods. Heart tissues from animals treated with placebo (control) or L-NAME/AngII were analyzed by Western blotting for MR protein levels (B), as described in Materials and Methods. Representative Western blots are depicted under the bar graph (B). *, P < 0.001; #, P < 1.E-05 vs. control animals of the same genotype.

Figure 6

Myocardial damage scores and PAI-1 expression are dependent on ALDO tertiles in cav-1 WT and cav-1 KO mice. Myocardial damage scores (A) and cardiac PAI-1 transcript levels (B) were determined in a subset of WT and cav-1 KO mice (after placebo or L-NAME/AngII treatment), for which ALDO levels were also available (C). Data were grouped by plasma ALDO levels tertiles (170 ng/dl). ALDO levels for WT and cav-1 KO within each tertile were not different from each other (C). *, P < 0.0001; #, P < 0.01; §, P < 0.05 vs. animals with the same genotype and ALDO levels in the lowest tertile.

Figure 7

ALDO-induced ERK1/2 phosphorylation in ECs is reduced in the absence of cav-1. A, Primary aortic ECs were isolated from WT and cav-1 KO mice, grown for two to three passages and incubated with vehicle or 10−8 m aldosterone in the presence or absence of 10−6 m MR antagonist canrenoate. Five hours later, cells were lysed and analyzed for the levels of total and phosphorylated ERK1/2. Data were normalized to results obtained in WT cells treated with vehicle. B, Representative Western blots showing pERK1/2 (top panels) and total ERK1/2 levels (bottom panels) in WT (left panels) and cav-1 KO ECs (right panels), treated with vehicle (first lanes) or ALDO in the absence (second lanes) or presence of canrenoate (third lanes). *, P < 0.001 vs. vehicle-treated cells of the same genotype; #, P < 0.01 vs. ALDO-treated cells of the same genotype.