Bcl-2–associated athanogene 3 protects the heart from ischemia/reperfusion injury
Feifei Su, Valerie D. Myers, Tijana Knezevic, JuFang Wang, Erhe Gao, Muniswamy Madesh, Farzaneh G. Tahrir, Manish K. Gupta, Jennifer Gordon, Joseph Rabinowitz, Frederick V. Ramsey, Douglas G. Tilley, Kamel Khalili, Joseph Y. Cheung, Arthur M. Feldman
Feifei Su, Valerie D. Myers, Tijana Knezevic, JuFang Wang, Erhe Gao, Muniswamy Madesh, Farzaneh G. Tahrir, Manish K. Gupta, Jennifer Gordon, Joseph Rabinowitz, Frederick V. Ramsey, Douglas G. Tilley, Kamel Khalili, Joseph Y. Cheung, Arthur M. Feldman
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Research Article Cardiology Cell biology

Bcl-2–associated athanogene 3 protects the heart from ischemia/reperfusion injury

Abstract

Bcl-2–associated athanogene 3 (BAG3) is an evolutionarily conserved protein expressed at high levels in the heart and the vasculature and in many cancers. While altered BAG3 expression has been associated with cardiac dysfunction, its role in ischemia/reperfusion (I/R) is unknown. To test the hypothesis that BAG3 protects the heart from reperfusion injury, in vivo cardiac function was measured in hearts infected with either recombinant adeno-associated virus serotype 9–expressing (rAAV9-expressing) BAG3 or GFP and subjected to I/R. To elucidate molecular mechanisms by which BAG3 protects against I/R injury, neonatal mouse ventricular cardiomyocytes (NMVCs) in which BAG3 levels were modified by adenovirus expressing (Ad-expressing) BAG3 or siBAG3 were exposed to hypoxia/reoxygenation (H/R). H/R significantly reduced NMVC BAG3 levels, which were associated with enhanced expression of apoptosis markers, decreased expression of autophagy markers, and reduced autophagy flux. The deleterious effects of H/R on apoptosis and autophagy were recapitulated by knockdown of BAG3 with Ad-siBAG3 and were rescued by Ad-BAG3. In vivo, treatment of mice with rAAV9-BAG3 prior to I/R significantly decreased infarct size and improved left ventricular function when compared with mice receiving rAAV9-GFP and improved markers of autophagy and apoptosis. These findings suggest that BAG3 may provide a therapeutic target in patients undergoing reperfusion after myocardial infarction.

Authors

Feifei Su, Valerie D. Myers, Tijana Knezevic, JuFang Wang, Erhe Gao, Muniswamy Madesh, Farzaneh G. Tahrir, Manish K. Gupta, Jennifer Gordon, Joseph Rabinowitz, Frederick V. Ramsey, Douglas G. Tilley, Kamel Khalili, Joseph Y. Cheung, Arthur M. Feldman

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Figure 6

BAG3 preserves function and limits infarct size after ischemia/reperfusion.

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BAG3 preserves function and limits infarct size after ischemia/reperfusi...
Male FVB mice (8–10 weeks old) underwent retro-orbital injection with an adeno-associated virus serotype 9 (rAAV9) expressing either GFP or BAG3. Three weeks later, mice were randomly assigned to coronary ligation for 30 minutes followed by reperfusion or sham surgery. Seventy-two hours later, mice underwent echocardiography and sacrifice. Forty-three mice were randomly assigned to Sham-GFP (n = 3), Sham-rAAV9-BAG3 (n = 3), I/R-rAAV9-GFP (n = 16), or I/R-rAAV9-BAG3 (n = 21) in 3 different studies and underwent initial echocardiography. Echocardiographic data represented aggregate information from Western blot analysis of BAG3 in mice injected with rAAV9-BAG3 or rAAV9-GFP prior to ischemia/reperfusion (I/R) or sham (C, n = 19); determination of infarct size by triphenyltetrazolium staining in mice injected with either rAAV9-GFP or rAAV9-BAG3 prior to I/R (D, n = 10); and Western blot analysis of markers of autophagy and apoptosis in mice injected with rAAV9-GFP or rAAV9-BAG3 prior to I/R (Figure 7; n = 9). The difference between the number of animals undergoing echocardiography and the analysis reported in the figures is due to loss of animals prior to subsequent analysis, as detailed in Methods. (A) Representative M-mode echocardiograms of hearts expressing either GFP or BAG3 after I/R or sham. (B) Left ventricular ejection fraction (LVEF) of sham-operated or I/R hearts expressing either GFP or BAG3; Sham-GFP (n = 3), Sham-BAG3 (n = 3), I/R-GFP (n = 16), I/R-BAG3 (n = 21). (C) BAG3 levels from heart homogenates from sham-operated and I/R hearts expressing GFP or BAG3; Sham-GFP (n = 3), Sham-BAG3 (n = 3), I/R-GFP (n = 6), I/R-BAG3 (n = 7) (D) In a separate experiment, 72 hours after I/R, Evans Blue dye was injected into hearts, and they were excised and stained with triphenyltetrazolium. The Evans Blue–stained area (area not at risk), triphenyltetrazolium-negative area (infarcted myocardium [IF]), and area at risk ([AAR], triphenyltetrazolium-negative and -positive areas) were measured. AAR was expressed as percentage of total LV (%AAR/LV). Infarcted myocardium (triphenyltetrazolium-negative area) was expressed as percentage of AAR (%IF/AAR). (E) There were no differences in AAR (AAR/LV%) between after I/R hearts expressing GFP (n = 4) or BAG3 (n = 4), (F) although infarct size (IF/AAR%) was significantly smaller in after I/R hearts infected with rAAV9-BAG3 (n = 4) when compared with hearts infected with rAAV9-GFP (n = 4). Two-way ANOVA with Bonferroni multiple comparisons adjustments assessed differences across the 4 investigational groups. A Student’s 2-tailed t test was used to analyze data comparing two groups.