Nitric Oxide Induces Cardiac Protection by Preventing Extracellular Matrix Degradation through the Complex Caveolin-3/EMMPRIN in Cardiac Myocytes

Abstract

Inhibition of Extracellular Matrix degradation by nitric oxide (NO) induces cardiac protection against coronary ischemia/reperfusion (IR). Glycosylation of Extracellular Matrix Metalloproteinase Inducer (EMMPRIN) stimulates enzymatic activation of matrix metalloproteinases (MMPs) in the heart, although the mechanisms leading to EMMPRIN glycosylation are poorly understood. We sought to determine if NO may induce cardiac protection by preventing glycosylation of EMMPRIN in a mouse model of IR. Here we found that Caveolin-3 binds to low glycosylated EMMPRIN (LG-EMMPRIN) in cardiac cells and in the hearts of healthy mice, whereas IR disrupted the complex in nitric oxide synthase 2 (NOS2) knockout (KO) mice. By contrast, the binding was partially restored when mice were fed with an NO donor (DEA-NO) in the drinking water, showing a significant reduction on infarct size (NOS2KO: 34.6±5 vs NOS2KO+DEA-NO: 20.7±9), in expression of matrix metalloproteinases, and cardiac performance was improved (left ventricular ejection fraction (LVEF). NOS2KO: 31±4 vs NOS2KO+DEA-NO: 46±6). The role of Caveolin-3/EMMPRIN in NO-mediated cardiac protection was further assayed in Caveolin-3 KO mice, showing no significant improvement on infarct size (Caveolin-3 KO: 34.8±3 vs Caveolin-3 KO+DEA-NO:33.7±5), or in the expression of MMPs, suggesting that stabilization of the complex Caveolin-3/LG-EMMPRIN may play a significant role in the cardioprotective effect of NO against IR.

Fig 1. Ischemia/Reperfusion induces the expression and glycosylation of EMMPRIN in NOS2 KO mice.

Immunoblot detection of EMMPRIN in WT and NOS2 KO mice 48 hours after IR. GAPDH was used as loading control. HG: high glycosylated EMMPRIN. LG: Low glycosylated EMMPRIN. (n = 9 mice, mean ± SD *p<0.05 WT HG 48 vs NOS2 HG 48. #p<0.05 WT LG 48 vs NOS2 LG 48).

Fig 2. Glycosylated EMMPRIN binds to Caveolin-3 in cardiac cells.

Confocal microscopy detection of Caveolin-3 (Cy-3, red) and EMMPRIN (FITC, green) in HL1B resting (upper panels), incubated 8 hours with 100 μM IL-18 (middle panels), or with 100 μM IL-18 plus 10 μM tunicamycin (lower panels). Colocalization of both signals is detected in merged panels (yellow) (n = 9 plus triplicates). Bottom left: Co-localization analysis as detected by calculation of overlapping correlation coefficient ((n = 9 plus triplicates. *p<0.05 IL-18 vs IL-18+tunicamycin). Bottom right: Micrographs corresponding to one representative assay in which overlapping green and red signals are highlighted in white.

Fig 3. EMMPRIN co-localizes with Caveolin-3 at the cell surface of healthy mouse hearts.

Confocal microscopy detection of Caveolin-3 (FITC, green), and EMMPRIN (Cy3, red) in heart sections from healthy WT and NOS KO mice with specific antibodies. Co-localization (yellow) is shown in the merged panels (n = 9 mice/strain/triplicated). Bottom left: Co-localization measurement as detected by calculation of overlapping correlation coefficient ((n = 9 plus triplicates). Bottom right: Micrographs corresponding to one representative assay in which overlapping green and red signals are highlighted in white.

Fig 4. Ischemia/Reperfusion disrupts the complex Caveolin-3/EMMPRIN in mouse hearts.

(A) Confocal microscopy detection of Caveolin-3 (FITC, green), and EMMPRIN (Cy3, red) in heart sections from WT and NOS KO mice 48 hours after IR with specific antibodies. Co-localization (yellow) is shown in the merged panels (n = 9 mice/strain/triplicate). Bottom left: Co-localization measurement as detected by calculation of overlapping correlation coefficient ((n = 9 plus triplicates. *p<0.05 Control vs NOS2 KO). Bottom right: Micrographs corresponding to one representative assay in which overlapping green and red signals are highlighted in white. (B) Left. Immunoblot detection of Caveolin-3 from heart lysates after IR at the times indicated. The expression of GAPDH was used as loading control. Right. Densitometric analysis of the bands corresponding to the expression of Caveolin-3 in reference to the levels of GAPDH.

Fig 5. The complex Caveolin-3/EMMPRIN is disrupted in NOS2 KO mice 48 hours after Ischemia/Reperfusion.

(A) Immunoblot (IB) detection of Caveolin-3 from immunoprecipitated extracts (IP) with anti-EMMPRIN antibody, isolated from heart lysates of mice under IR at the times indicated. Immunoblot detection of GAPDH was used as loading control (n = 9 mice, mean ± SD *p<0.05 WT vs NOS2 48h). (B) Upper-Left panel. Immunoblot detection of EMMPRIN from total lysates or immunoprecipitated extracts with anti-Caveolin-3, and isolated from mouse hearts 48 hours after IR. Bottom-left panel. Immunoblot of Caveolin-3 from the same extracts as above. Upper-right panel. Immunoblot detection of Caveolin-3 from immunoprecipitated extracts with anti-EMMPRIN and isolated from WT and NOS2 KO hearts isolated 48 hours after IR. Middle-Right panel. Immunoblot detection of EMMPRIN from immunoprecipitated extracts with anti-Caveolin-3, from the same mice as before. Lower-Right panel. Immunoblot detection of Caveolin-3 from total cell lysates and immunoprecipitated with anti-Caveolin-3 from the same mice as before (n = 9 mice by triplicate). (C) Immunoblot detection of Caveolin-3 in WT and NOS2 KO heart proteins separated by discontinuous sucrose gradient fractionation. Right panel. Densitometric analysis of Caveolin-3 distribution in WT and NOS2 KO fractions. (n = 3 mice by triplicate, mean ± SD). (D) Immunoblot detection of LG-EMMPRIN in buoyant (2, 3, 4) and non buoyant (8, 9, and 10) fractions from WT and NOS2 KO heart proteins separated by discontinuous sucrose gradient fractionation (n = 3 mice by triplicate, mean ± SD *p<0.05 WT vs NOS2 BF; ^#p<0.05 WT vs NOS2 NBF).

Fig 6. NOS2 induces cardiac protection through the binding of Caveolin-3 with LG-EMMPRIN.

(A) Infarct size of WT and Caveolin-3 KO hearts 48 hours after IR, as detected by double Evans Blue/TTC staining (n = 9 mice/group; mean ± SD; *p <0.05) (B) LVEF in the same mice as before (mean ± SD; *p <0.05, WT IR vs Caveolin-3 KO IR). (C) Infarct size 48 hours after IR of NOS2 KO and Caveolin-3 KO mice and infused with saline or DEA-NO (n = 9 mice/group; mean ± SD; *p <0.05 NOS2 KO saline vs NOS2 KO DEA-NO). (D) Upper panel. Immunoblot detection of MMP-9 from total protein lysates isolated from Caveolin-3 KO mice and Caveolin-3 KO mice treated with saline or DEA-NO, 48 hours after IR. The expression of GAPDH was used as loading control. (n = 9 mice/group; mean ± SD). (E) Upper-panel. Immunoblot detection of MMP-9 from total protein lysates isolated from Caveolin-3 KO, NOS2 KO and NOS2 KO mouse hearts treated with DEA-NO. Middle-panel. Immunoblot detection of EMMPRIN from immunoprecipitated extracts with anti-Caveolin-3, from the same mice as before. Lower-panel. Immunoblot of Caveolin-3 from immunoprecipitated extracts with anti-EMMPRIN, from the same mice as before (n = 9 mice by triplicated). (F) Infarct size of WT, NOS2, and Caveolin-3 KO hearts 48 hours after IR, and previously fed with 50 mg/kg doxycycline for 1 week (n = 9 mice/group; mean ± SD; *p <0.05 NOS2 KO vs NOS2 KO + DOX. ^$p <0.05 CAV3 KO vs CAV3 KO + DOX).