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. 2015 Apr 1;308(7):H749-58.
doi: 10.1152/ajpheart.00414.2014. Epub 2015 Jan 23.

The beneficial effects of AMP kinase activation against oxidative stress are associated with prevention of PPARα-cyclophilin D interaction in cardiomyocytes

Giselle Barreto-Torres  1 , Jessica Soto Hernandez  1 , Sehwan Jang  1 , Adlín R Rodríguez-Muñoz  1 , Carlos A Torres-Ramos  1 , Alexei G Basnakian  2 , Sabzali Javadov  3
Affiliations

The beneficial effects of AMP kinase activation against oxidative stress are associated with prevention of PPARα-cyclophilin D interaction in cardiomyocytes

Giselle Barreto-Torres et al. Am J Physiol Heart Circ Physiol. .

Abstract

AMP kinase (AMPK) plays an important role in the regulation of energy metabolism in cardiac cells. Furthermore, activation of AMPK protects the heart from myocardial infarction and heart failure. The present study examines whether or not AMPK affects the peroxisome proliferator-activated receptor-α (PPARα)/mitochondria pathway in response to acute oxidative stress in cultured cardiomyocytes. Cultured H9c2 rat embryonic cardioblasts were exposed to H2O2-induced acute oxidative stress in the presence or absence of metformin, compound C (AMPK inhibitor), GW6471 (PPARα inhibitor), or A-769662 (AMPK activator). Results showed that AMPK activation by metformin reverted oxidative stress-induced inactivation of AMPK and prevented oxidative stress-induced cell death. In addition, metformin attenuated reactive oxygen species generation and depolarization of the inner mitochondrial membrane. The antioxidative effects of metformin were associated with the prevention of mitochondrial DNA damage in cardiomyocytes. Coimmunoprecipitation studies revealed that metformin abolished oxidative stress-induced physical interactions between PPARα and cyclophilin D (CypD), and the abolishment of these interactions was associated with inhibition of permeability transition pore formation. The beneficial effects of metformin were not due to acetylation or phosphorylation of PPARα in response to oxidative stress. In conclusion, this study demonstrates that the protective effects of metformin-induced AMPK activation against oxidative stress converge on mitochondria and are mediated, at least in part, through the dissociation of PPARα-CypD interactions, independent of phosphorylation and acetylation of PPARα and CypD.

Keywords: AMPK; H9c2 cardiomyocytes; PPARα; metformin; mitochondria; oxidative stress.

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Figures

Fig. 1.
Fig. 1.
The effects of metformin (Met) and A-769662 on cell death induced by 75 (A) and 100 (B) H2O2 in H9c2 cells. Cell death was assessed by the trypan blue exclusion test and shown as a percentage of live cells compared with the control group. The control group included cells that were treated with corresponding agonist/inhibitor in the absence of H2O2. *P < 0.01, H2O2 vs. control; +P < 0.05 and ++P < 0.01, H2O2 + Met or H2O2 + A-769662 vs. H2O2; n = 3–5 per each group.
Fig. 2.
Fig. 2.
The effects of H2O2 on AMP kinase (AMPK) activation in the presence or absence of Met. A: concentration-dependent effects of H2O2 on phospho (P)-AMPKα1Thr172 levels. B: concentration-dependent effects of Met on P-AMPKα1Thr172 levels. C: effects of oxidative stress on P-AMPKThr172 in cardiomyocytes pretreated with 2, 5, and 10 mM Met. A–C, top: representative Western blot images of the phosphorylated and total levels of AMPKα. A–C, bottom: quantitative data of AMPKα phosphorylation were normalized to total (t)-AMPK and expressed as percent change relative to the control group (C). *P < 0.05 and **P < 0.01 vs. C; +P < 0.05 and ++P < 0.01 vs. H2O2.; n = 6–8 per group.
Fig. 3.
Fig. 3.
Effects of H2O2 on total reactive oxygen species (ROS) levels and mitochondrial membrane potential (ΔΨm) in Met-treated cells. A: representative images of 5,5′,6,6′-tetraethyl-benzimidazolylcarbocyanine iodide (JC-1) fluorescence. The images were obtained using a Zeiss LSM510 META (Carl Zeiss) microscope from H9c2 cells after incubation with JC-1. The cells pretreated with Met were subjected to 100 μM H2O2. B: quantitative results of JC-1 fluorescence intensity, measured using a Spectramax M3 microplate reader (Molecular Devices, Sunnyvale, CA). Data were normalized as the ratio of red fluorescence of dye aggregates (J-aggregates, high ΔΨm) to green fluorescence of dye monomers (JC-1 monomers, low ΔΨm) and expressed as a percentage of the control group (C). C: effects of 100 μM H2O2 on JC-1 fluorescence in cardiomyocytes pretreated with Met, 10 μM compound C, and/or 10 μM GW6471 (GW). D: electron transport chain complex I activity. Cells were pretreated with 5 mM Met, 10 μM GW, and/or 25 μM A-769662 before 100 μM H2O2 exposure. E: concentration-dependent effects of H2O2 on total ROS levels. F: effects of 100 μM H2O2 on total ROS levels in the cells pretreated with Met. ROS levels were determined by incubation of the cells with the ROS-specific probe 2′, 7′-dichlorofluorescein diacetate (DCFDA). Data are expressed as percent change compared with control. *P < 0.05 and **P < 0.01 vs. C; +P < 0.05 vs. H2O2; #P < 0.05 vs. Met; n = 8–14 per each group.
Fig. 4.
Fig. 4.
Effects of H2O2 on permeability transition pore opening in cells pretreated with Met or A-769662. A: representative images of calcein fluorescence. Cells treated with 100 μM H2O2 in the presence or absence of Met (5 mM) or A-769662 (25 μM) were coloaded with cobalt chloride and calcein-AM and then imaged using an Olympus IX73 (Center Valley, PA) inverted fluorescence microscope. B: quantitative results of calcein fluorescence normalized to individual cells and expressed as a percentage of the control group (C). *P < 0.01 vs. C; +P < 0.01 vs. H2O2; #P < 0.01 vs. Met; n = 3 per each group.
Fig. 5.
Fig. 5.
Effects of Met on H2O2-induced cyclophilin D (CypD)-peroxisome proliferator-activated receptor-α (PPARα) interaction. Cell lysates from each group were immunoprecipitated (IP) with anti-CypD (A) or anti-PPARα (B) antibodies. The complexes were subjected to SDS-PAGE followed by immunoblotting (IB) with CypD, PPARα, adenine nucleotide translocator (ANT), and voltage-dependent anion channel (VDAC). Representative immunoblots (A and B, top) show the effects of oxidative stress on the interaction between PPARα and CypD in the presence and absence of Met. Quantitative results (A and B, bottom) were expressed as percent change compared with control. *P < 0.05 vs. C; +P < 0.05 vs. H2O2; n = 3 to 4 per group.
Fig. 6.
Fig. 6.
Expression of mitochondrial transcriptional network proteins and time-dependent changes of phosphorylation and acetylation (Ac-PPARα) of PPARγ coactivator 1-α (PGC-1α) and PPARα in response to H2O2. A: PGC-1α and PPARα acetylation (Ac-PGC-1α and Ac-PPARα). B: PGC-1α and PPARα phosphorylation (P-PGC-1α and P-PPARαSer21). C: effects of H2O2 on PGC-1α and PPARα phosphorylation in the presence of Met. D: effects of H2O2 on protein expression of PGC-1α, nuclear respiratory factor (NRF) 1 and 2, and mitochondrial transcription factor A (TFAM). Protein levels were normalized to total PGC-1α or PPARα (A–C) and actin (D) and expressed as a percentage of the control (untreated) group. *P < 0.05 vs. C; n = 3–6 per group.
Fig. 7.
Fig. 7.
Time-dependent changes of acetylation and phosphorylation of CypD in response to oxidative stress. A: CypD acetylation. B: CypD phosphorylation. Cell were treated with 100 μM H2O2 and taken for protein analysis at indicated time points. *P < 0.05 vs. C; n = 3 to 4 per group.
Fig. 8.
Fig. 8.
Effects of oxidative stress on mtDNA abundance and damage in H9c2 cardiomyocytes in the presence or absence of Met. Cells were treated with varying concentrations of H2O2 for 1 h followed by DNA isolation and quantitative PCR analysis. A: mtDNA abundance. B: mtDNA damage. C: effect of Met on H2O2-induced mtDNA damage. Graphs represent the average of two PCR reactions performed in duplicate. AU, arbitrary units. *P < 0.05 and **P < 0.01 vs. C; +P < 0.05 vs. H2O2.
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References

    1. Algire C, Moiseeva O, Deschenes-Simard X, Amrein L, Petruccelli L, Birman E, Viollet B, Ferbeyre G, Pollak MN. Metformin reduces endogenous reactive oxygen species and associated DNA damage. Cancer Prev Res (Phila) 5: 536–543, 2012. - PubMed
    1. Barger PM, Brandt JM, Leone TC, Weinheimer CJ, Kelly DP. Deactivation of peroxisome proliferator-activated receptor-α during cardiac hypertrophic growth. J Clin Invest 105: 1723–1730, 2000. - PMC - PubMed
    1. Barger PM, Browning AC, Garner AN, Kelly DP. p38 mitogen-activated protein kinase activates peroxisome proliferator-activated receptor alpha: a potential role in the cardiac metabolic stress response. J Biol Chem 276: 44495–44501, 2001. - PubMed
    1. Barreto-Torres G, Parodi-Rullán R, Javadov S. The role of PPARalpha in metformin-induced attenuation of mitochondrial dysfunction in acute cardiac ischemia/reperfusion in rats. Int J Mol Sci 13: 7694–7709, 2012. - PMC - PubMed
    1. Bernardi P. The mitochondrial permeability transition pore: a mystery solved? Front Physiol 4: 95, 2013. - PMC - PubMed

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