Exendin-4 ameliorates cardiac ischemia/reperfusion injury via caveolae and caveolins-3

Abstract

Background: Exendin-4, an exogenous glucagon-like peptide-1 receptor (GLP-1R) agonist, protects the heart from ischemia/reperfusion injury. However, the mechanisms for this protection are poorly understood. Caveolae, sarcolemmal invaginations, and caveolins, scaffolding proteins in caveolae, localize molecules involved in cardiac protection. We tested the hypothesis that caveolae and caveolins are essential for exendin-4 induced cardiac protection using in vitro and in vivo studies in control and caveolin-3 (Cav-3) knockout mice (Cav-3 KO).

Methods: Myocytes were treated with exendin-4 and then incubated with methyl-β-cyclodextrin (MβCD) to disrupt caveolae formation. This was then followed by simulated ischemia/reperfusion (SI/R). In addition, cardiac protection in vivo was assessed by measuring infarct size and cardiac troponin levels.

Results: Exendin-4 protected cardiac myocytes (CM) from SI/R [35.6 ± 12.6% vs. 64.4 ± 18.0% cell death, P = 0.034] and apoptosis but this protection was abolished by MβCD (71.8 ± 10.8% cell death, P = 0.004). Furthermore, Cav-3/GLP-1R co-localization was observed and membrane fractionation by sucrose density gradient centrifugation of CM treated with MβCD + exendin-4 revealed that buoyant (caveolae enriched) fractions decreased Cav-3 compared to CM treated with exendin-4 exclusively. Furthermore, exendin-4 induced a reduction in infarct size and cardiac troponin relative to control (infarct size: 25.1 ± 8.2% vs. 41.4 ± 4.1%, P < 0.001; troponin: 36.9 ± 14.2 vs. 101.1 ± 22.3 ng/ml, P < 0.001). However, exendin-4 induced cardiac protection was abolished in Cav-3 KO mice (infarct size: 43.0 ± 6.4%, P < 0.001; troponin: 96.8 ± 26.6 ng/ml, P = 0.001).

Conclusions: We conclude that caveolae and caveolin-3 are critical for exendin-4 induced protection of the heart from ischemia/reperfusion injury.

Figure 1

Effects of exendin-4 on simulated ischemia/reperfusion (SI/R) of cardiac myocytes. (A) Cardiac myocytes were rod-shaped and treated with various concentration of exendin-4 prior to exposure to SI/R. (B) Cell death was determined by trypan blue staining. Optimal protection was observed at 3.0 nM exendin-4. Group sizes are indicated on the individual bars in parentheses.

Figure 2

In vitro assessment of the role of caveolae in exendin-4 (Ex-4) induced cardiac protection. (A) Summary illustration of in vitro experimental groups. (B) Cardiac myocytes exposed to simulated ischemia/reperfusion (SI/R) were exposed to experimental procedures outlined in A. Cell death was determined by trypan blue staining. Cardiac myocytes under control conditions (Control) had minimal cell death. Methyl-β-cyclodextrin (MβCD) abolished the Ex-4 induced cardiac protection effect. Group sizes are indicated on the individual bars in parentheses. (C) Apoptotic changes were measured by investigating mitochondrial membrane potential using JC-1 after SI/R. The excitation rate (red/green) indicates changes within the mitochondrial membrane potential. *P < 0.001 vs. SI/R, SIR + Ex-4, SI/R with MβCD, and SI/R + Ex-4 with MβCD. #P < 0.05 vs. Control, SI/R + Ex-4, and Control with MβCD. n = 4 per each group. (D) Real-time polymerase chain reaction analysis of pro-apoptotic and anti-apoptotic gene expression after re-oxygenation. n = 4 per each group.

Figure 3

Glucagon-like peptide-1 receptor (GLP-1R) localization with caveolins or caveolae. (A) Immunofluorescence analysis of the expression and colocalization of caveolin-3 (Cav-3) and GLP-1R in cardiac myocytes. Fluorescent secondary antibodies were used to determine Cav-3 (green) and GLP-1R (red) localization, and strong colocalization (merged images, yellow) were observed on the cell surface membrane. Bar length = 10 μm. (B) The colocalization was confirmed by immunoprecipitation (IP). Immunoblot (IB) analysis detected Cav-3 and GLP-1R in Cav-3 and GLP-1R IP of cell lysates. Supernatants (SUP) from which the immunoprecipitates were generated were stained for GAPDH as loading controls.

Figure 4

Lysed and fractionated hearts on sucrose density gradient. Fractions were collected and probed for caveolin-3 (Cav-3). We defined fractions 4–6 as buoyant membrane fractions enriched in caveolae and proteins associated with caveolae; fractions 9–12 were defined as nonbuoyant fractions, noncaveolar membranes. Fraction 7–8 were considered a transition zone and were not analyzed. Significant localization of Cav-3 in buoyant fractions was observed in the groups treated with exendin-4 (Ex-4), whereas control and methyl-β-cyclodextrin (MβCD)-treated with Ex-4 cells showed no effects on Cav-3 localization. Group sizes are indicated on the individual bars in parentheses.

Figure 5

Caveolin-3 expression and reduction in infarct size. Mice underwent 30-min coronary artery occlusion followed by 2-h reperfusion after 24-h recovery from pretreatment with oxygen (Control) or exendin-4 (Ex-4) in wild-type and caveolin-3 knockout (Cav-3 KO) mice. (A) In vivo Ex-4 induced cardiac protection protocol. (B) Area at risk was calculated as a percentage of the left ventricle and revealed no significant differences between all groups. Ex-4 induced cardiac protection was abolished Cav-3 KO mice, as shown by no significant decrease in percent infarct size / area at risk when compared to control Cav-3 KO; however, a significant decrease in infarct size was observed between wild-type Ex-4 and Cav-3 KO Ex-4. (C) Cardiac troponin I, a marker of myocardial damage also revealed a significant decrease in Ex-4 treated control mice, but no effect in Cav-3 KO mice. Group sizes are indicated on the individual bars in parentheses. *P < 0.001 compared with Cav-3 KO pretreated with Ex-4.