Caveolin-3 protects diabetic hearts from acute myocardial infarction/reperfusion injury through β2AR, cAMP/PKA, and BDNF/TrkB signaling pathways

Figures

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  Figure 1. Myocardial ischemia reperfusion (I/R) injury model in diabetes mellitus (DM) rats SD rats were subjected to DM induction and then received coronary artery ligation to induce myocardial infarction (MI). Rats were randomly assigned to three groups: sham surgery group, DM group, I/R group, and I/R + DM group. (A) Representative photos of myocardial infarct size. Four weeks after I/R operation, rats were then examined for (A, C, D, E, and F) LVIDs, LVIDd, LVEF, and LVFS by echocardiography; (B) myocardial infarct size. N=5. **P<0.01, compared to sham group; #P<0.05, ##P<0.01, compared to DM group; && P<0.01, compared to I/R group.

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  Figure 2. cAMP/PKA and β-adrenergic signaling pathways upstream of TrkB are dysregulated in I/R and diabetic I/R group. (A) ADRB2 mRNA expression in MI and MI + DM rats based on online microarray profile (GSE12639). (B) ADRB2 mRNA expression in rat hearts (n=5) from Sham, DM, I/R, or I/R + DM group determined by real-time PCR. (C) The concentration of cAMP in rat hearts from Sham, DM, I/R, or I/R +DM group determined by ELISA. (D, E) The protein levels of ADRB2, p-PKA, and PKA in rat hearts from Sham, DM. I/R, or I/R + DM group determined by Immunoblotting, n=3. **P<0.01, compared to sham group; #P<0.05, ##P<0.01, compared to DM group; && P<0.01, compared to I/R group.

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  Figure 3. An ADRB2 agonist activates cAMP/PKA signaling and the recovery of H9C2 cells from H/R injury under high-glucose (HG) stimulation. H9C2 cells were subjected to H/R injury with or without HG stimulation and ADRB2 agonist ISO treatment and examined for (A, B) the cell apoptosis by Flow cytometry (n=3); (C, D) the protein levels of apoptotic Bax/Bcl-2 signaling factors Bax and Bcl-2 by immunoblotting (n=3); (E) the cell viability by MTT assay (n=5); (F) the cAMP concentrations (n=3); (G, H) the protein levels of p-PKA, PKA, TrkB, p-Akt, and Akt by immunoblotting, (n=3).**P<0.01, compared to control group; #P<0.05, ##P<0.01, compared to H/R group; &&P<0.01, compared to H/R + HG group.

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  Figure 4. Caveolin-3 promotes the localization of β2AR onto the cytomembrane of H9C2 cells. (A, B) H9C2 cells were transfected with pcDNA3.1/Caveolin-3 to conduct Caveolin-3 overexpression. The total protein levels of Caveolin-3 and ADRB2 in Caveolin-3-transfected cells were determined using immunoblotting, (n=3). (C, D) The membrane protein levels of ADRB2 in Caveolin-3-transfected cells were determined using immunoblotting, (n=3). (E) The content and distribution of ADRB2 protein in the control, H/R, H/R + HG, and H/R + HG + Caveolin-3 groups determined by IF staining, (n=3). (F–H) H9C2 cells were transfected with pcDNA3.1/Caveolin-3 under H/R or HG stimulation and examined for cell apoptosis by Flow cytometry (n=3) and cell viability by MTT assay (n=5). *P<0.05, **P<0.01 compared to pcDNA3.1+H/R group; ##P<0.01, compared to pcDNA3.1+H/R group.

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  Figure 5. Caveolin-3 enhances the activity of BDNF/TrkB signaling through ADRB2 and cAMP. (A, B) ADRB2 silencing was conducted in H9C2 cells by transfection of si-ADRB2, as confirmed by immunoblotting. Next, under H/R+HG stimulation, H9C2 cells were co-transfected with pcDNA3.1/Caveolin-3 and si-ADRB2 and examined for (C, D) cAMP, p-PKA, PKA, TrkB, p-Akt, and Akt proteins. N=3. **P<0.01 compared to si-NC group or pcDNA3.1+si-NC group; ##P<0.01, compared to pcDNA3.1+si-ADRB2 group.