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 3

The levels of autophagy and apoptosis are critically dependent on BAG3 levels.

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The levels of autophagy and apoptosis are critically dependent on BAG3 l...
To determine the effects of decreased BAG3 levels on autophagy, neonatal mouse ventricular cardiomyocytes (NMVCs) were transfected with an autophagy reporter system consisting of double-labeled Red fluorescent protein-GFP-microtubule-associated protein 1 light chain 3 (RFP-GFP-LC3-I). Both RFP (red fluorescence) and GFP (green fluorescence) can be identified in autophagosomes as yellow puncta; however, when autophagosomes fuse with lysosomes, the acidity of the autolysosome quenches the GFP fluorescence, resulting in predominantly red puncta. Independent cultures of NMVCs were cultured under normal conditions (Control, n = 9), exposed to hypoxia/reoxygenation (H/R; n = 9), infected with an adenovirus encapsulated with a siRNA for BAG3 (Ad-siBAG3; n = 9) or exposed to H/R as well as infection with an adenovirus encapsulated with BAG3 (Ad-BAG3; n = 9). As shown in representative confocal images in (A), yellow fluorescence was more prominent in NMVCs in which BAG3 was reduced by either H/R or Ad- siBAG3. Despite H/R incubation, which should result in decreased BAG3 levels, RFP signals were more prominent in BAG3-overexpressed NMVCs after H/R (A), suggesting that increased incorporation of LC3 into autolysosomes is consistent with an increased level of autophagy. Scale bars: 10 μM. (B) The subjective evaluations of the confocal images were analyzed by counting the number of yellow and red puncta in each group (number of cells counted: n = 10, control and H/R; n = 9, siBAG3; n = 7, H/R plus Ad-BAG3). Red puncta are indicated by red dots and yellow puncta are indicated by green dots. (C) The amount of autophagy was expressed by calculating the ratio of autolysosomes (red puncta) to autophagosome (yellow puncta) per cell. Data are presented as mean ± SEM for continuous variables. Two-way ANOVA with Bonferroni multiple comparisons adjustments was used to assess differences across the 4 investigational groups. Autophagy in NMVCs was significantly reduced after H/R, which was restored to normal levels by BAG3 overexpression. Reduction of BAG3 levels by Ad-siBAG3 also resulted in decreased autophagy. To provide a second assessment of autophagy flux we assessed LC-3 levels in normal control cells exposed to Ad-GFP or Ad-BAG3 and similarly treated cells exposed to H/R. (D) A representative Western blot and (E) quantification of several experiments (n = 6).