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 2

Overexpression of BAG3 ameliorates the changes in markers of apoptosis and autophagy in neonatal mouse ventricular cardiomyocytes after hypoxia/reoxygenation.

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Overexpression of BAG3 ameliorates the changes in markers of apoptosis a...
In order to increase levels of BAG3, neonatal mouse ventricular cardiomyocytes (NMVCs) were infected with an adenovirus coupled to BAG3 (Ad-BAG3) or GFP control (Ad-GFP) and cultured for 3 days. NMVCs were then exposed to either hypoxic conditions (5% CO2 and 95% nitrogen at 3 l/min) in the absence of glucose for 14 hours at 37°C followed by reoxygenated for 4 hours with 5% CO2 and 95% humidified air in medium containing glucose or normoxic conditions (5% CO2 and 95% humidified air). Myocytes were then harvested, and cellular lysates were immunoblotted for BAG3, cleaved caspase-3, B cell lymphoma 2 (Bcl-2), lysosomal-associated protein 2 (LAMP-2), and the c-jun N-terminal kinase (JNK) and its phosphorylated form (p-JNK). Three independent cultures of NMVCs were exposed to either hypoxia/reoxygenation or normoxia (control) in each experiment. Each experiment was then repeated 3 times in order to have n = 9 for each intervention. (A) Representative Western blot (n = 3 for each group) demonstrating that BAG3 overexpression in normoxic NMVCs resulted in increased levels of BAG3 (B) but did not significantly change levels of p-JNK (n = 9) (C), LAMP-2 (n = 9) (D), Bcl-2 (n = 9) (E), or cleaved caspase-3 (n = 9) (F). By contrast, in NMVCs after hypoxia/reoxygenation, overexpression of BAG3 significantly increased BAG3 (n = 9) (B), LAMP-2 (n = 9) (D), and Bcl-2 (n = 9) (E) but decreased p-JNK (n = 9) (C) and cleaved caspase-3 (n = 9) (F). Data were normalized to the protein levels measured in NMVCs infected with control Ad-GFP and incubated under normoxic conditions. Data are presented as mean ± SEM for continuous variables. Two-way ANOVA with Bonferroni multiple comparisons adjustments were used to assess differences across the 4 investigational groups.