AMPKα2 Overexpression Reduces Cardiomyocyte Ischemia-Reperfusion Injury Through Normalization of Mitochondrial Dynamics

Figures

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  FIGURE 1. AMPK2α overexpression attenuates hypoxia/reoxygenation-mediated cardiomyocyte dysfunction and apoptosis. Adenovirus loaded with AMPKα2 were transfected into cardiomyocytes before H/R injury. (A) H/R injury was established through 1-h hypoxia, 2-h reoxygenation RNA was isolated from cardiomyocyte, and qPCR was used to evaluate the transcription of AMPKα2. (B) Proteins were collected from H/R-treated cardiomyocytes, and then, the expression of AMPKα2 was determined through western blots. (C) MTT assay was used to evaluate cell viability in response to H/R injury. (D,E) ELISA assay was used to measure the levels of troponin T (TnT) and creatine kinase-MB (CK-MB) in H/R-treated cardiomyocytes. (F–H) The cardiomyocytes contractile properties in the context of HI/R injury. The data represent the mean ± SEM. P < 0.05.

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  FIGURE 2. AMPK2α overexpression sustains mitochondrial homeostasis. Adenovirus loaded with AMPKα2 were transfected into cardiomyocytes before H/R injury. (A,B) Mitochondrial membrane potential was measured through JC-1 probe relative fluorescence intensity in H/R cardiomyocytes. (C,D) Intracellular ROS were determined through immunofluorescence. (E,F) H/R-mediated calcium overload was determined through immunofluorescence. (G,H) ELISA was used to measure the activities of caspase-9 and Bax in cardiomyocytes treated with H/R injury. The data represent the mean ± SEM. P < 0.05.

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  FIGURE 3. Mitochondrial dynamics are disrupted by hypoxia/reoxygenation due to decreased AMPK2α. (A–C) Mitochondrial morphology was measured through immunofluorescence. The average length of mitochondria as well as the ratio of cardiomyocyte with fragmented mitochondria was measured. (D–I) RNA was isolated from treated cardiomyocytes, and then the transcription of Drp1, Mff, Fis1, Mfn1, Mfn2, and Opa1 was measured. The data represent the mean ± SEM. P < 0.05.

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  FIGURE 4. Induction of mitochondrial dynamics disorder abolishes AMPK2α overexpression-mediated cardioprotection. (A,B) TUNEL staining was applied to observe cell apoptosis. Cardiomyocytes transfected with AMPKα2 adenovirus were treated with FCCP to induce mitochondrial dynamics disorder. Then, the number of TUNEL-positive cells was recorded. (C) LDH release assay was used to measure cell viability. (D–F) The cardiomyocytes contractile properties in the context of H/R injury. The data represent the mean ± SEM. P < 0.05.

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  FIGURE 5. AMPK2α sustains mitochondrial dynamics through the Sirt3/PGC1α signaling pathway. (A,B) RNA was isolated from cardiomyocyte, and qPCR was used to evaluate the transcription of Sirt3 and PGC1α. (C–E) Immunofluorescence assay for Sirt3 and PGC1α in cardiomyocytes treated with H/R injury. (F–H) Mitochondrial morphology was determined through immunofluorescence. 3-TYP, an inhibitor of the Sirt3/PGC1α signaling pathway, was added into the cardiomyocyte medium before AMPK2α overexpression. The data represent the mean ± SEM. P < 0.05.