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. Author manuscript; available in PMC: 2019 Mar 2.
Published in final edited form as: Circ Res. 2017 Dec 28;122(5):712–729. doi: 10.1161/CIRCRESAHA.117.312317

Figure 5. Overexpression of AMPKα2 prevents the phenylephedrine (PE)-induced impairment of mitophagy in CMs.

Figure 5

A. HL-1 mouse CMs were infected with an adenovirus encoding AMPKα2 and then subjected to PE (50 μmol/L) stimulation, followed by the induction of mitophagy. Mitophagy induction was performed by treatment with 20 μmol/L CCCP 24 h after PE stimulation. Representative images of TEM assay are shown. B. Mitophagy levels in CMs. Bar graph indicates the number of autophagosomes containing mitochondria (black arrows in [A]) per total number of mitochondria from a cross-sectional assessment of the CMs in TEM. *P < 0.05 vs. Ad-LacZ group; #P < 0.05 vs. PE+Ad-LacZ group. Blue arrows in (A) indicate prolonged mitochondria. C. Mitochondrial membrane potential (ΔΨm) was examined by flow cytometry with JC-1 probe treatment. The excitation ratio (JC-1 aggregates, red; monomer, green) indicates ΔΨm. Bar graphs indicate the quantification of ΔΨm. *P < 0.05 vs. Ad-LacZ group; #P < 0.05 vs. PE+Ad-LacZ group. D. Representative immunoblots of PINK1, p-Parkin S65, and Parkin in the presence or absence of CCCP in HL-1 CMs are shown. E. Quantitative analysis of PINK1/VDAC1, Parkin/VDAC1, and p-AMPKα2/AMPKα2 in the presence of CCCP. F. Isolated mouse adult CMs were infected with an adenovirus encoding AMPKα2 and then subjected to PE (50 μmol/L) stimulation, followed by the induction of mitophagy or treatment with 0.1 μmol/L bafilomycin A1 (Baf A1). Representative contour plots of CMs stained with MitoTracker are shown. G. Mean fluorescence intensity (MFI) of MitoTracker Deep Red. *P < 0.05 vs. Ad-LacZ group; #P < 0.05 vs. corresponding PE+Ad-LacZ group. H. Expression levels of p-AMPKα2 and AMPKα2 in (F). I. Bar graph indicates the mean number of autophagosomes (yellow) and autolysosomes (red) per cell. *1P < 0.05, *3P < 0.05 vs. Ad-LacZ group; #1P < 0.05, #3P < 0.05 vs. corresponding PE+Ad-LacZ group; *2P < 0.05, *4P < 0.05 vs. Baf A1+Ad-LacZ group; #2P < 0.05, #4P < 0.05 vs. Baf A1+PE+Ad-LacZ group. Lines in yellow indicate statistical comparisons for autophagosomes; Lines in red indicate statistical comparisons for autolysosomes; J. Representative immunoblots of p-AMPKα2, AMPKα2, LC3, SQSTM1, Beclin1, autophagy protein 5 (Atg5), and anti-ubiquitin in CMs are shown. All data represent the mean ± SEM from at least four independent experiments.