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Cardiac Mitochondrial PTEN-L determines cell fate between apoptosis and survival during chronic alcohol consumption

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Abstract

Chronic alcohol consumption induces myocardial damage and a type of non-ischemic cardiomyopathy termed alcoholic cardiomyopathy, where mitochondrial ultrastructural damages and suppressed fusion activity promote cardiomyocyte apoptosis. The aim of the present study is to determine the role of mitochondrial fission proteins and/or other proteins that localise on cardiac mitochondria for apoptosis upon ethanol consumption. In vivo and in vitro chronic alcohol exposure increased mitochondrial Drp1 levels but knockdown of the same did not confer cardioprotection in H9c2 cells. These cells displayed downregulated expression of MFN2 and OPA1 for Bak-mediated cytochrome c release and apoptosis. Dysregulated PTEN/AKT cell survival signal in both ethanol treated and Drp1 knockdown cells augmented oxidative stress by promoting  mitochondrial PTEN-L and MFN1 interaction. Inhibiting this interaction with VO–OHpic, a reversible PTEN inhibitor, prevented Bak insertion into the mitochondria and release of cytochrome c to cytoplasm. Thus, our study provides evidence that Drp1-mediated mitochondrial fission is dispensable for ethanol-induced cardiotoxicity and that stress signals induce mitochondrial PTEN-L accumulation for structural and functional dyshomeostasis. Our in vivo results also demonstrates the therapeutic potential of VO–OHpic for habitual alcoholics developing myocardial dysfunction.

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Acknowledgements

Science & Engineering Research Board (SERB), Government of India, supported this study [Grant Number: EMR/2014/000892]. The authors also acknowledge DBT, UGC-NRCBS, and –CAS, DST-PURSE, Government of India for instrumental support.

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  1. Cardiac Hypertrophy Laboratory, Department of Molecular Biology, School of Biological Sciences, Madurai Kamaraj University, Madurai, 625 021, Tamil Nadu, India

    Anusha Sivakumar, Suresh Shanmugarajan, Ramasamy Subbiah & Rekha Balakrishnan

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  1. Anusha Sivakumar
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  2. Suresh Shanmugarajan
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  3. Ramasamy Subbiah
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  4. Rekha Balakrishnan
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RS, AS—concept, design of experiments; AS, SS—execution of experiments, AS, RB—in vivo experiments; AS—data analysis and manuscript writing; RS,AS,SS,RB-Manuscript corrections and review.

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Correspondence to Ramasamy Subbiah.

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Supplementary Fig. 1. Chronic ethanol ingestion induces cardiac oxidative stress, in vivo. (A)

Animals consuming alcohol have increased serum CK-MB levels compared to control. (B) Chronic alcohol exposure increased myocardial lipid peroxidation. (C) Alcohol consumption did not alter total antioxidant levels between control and treated. Data are represented as mean ± SEM where ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05, ns not significant versus the control group. Supplementary Fig. 2. Silencing of Drp1 augmented ethanol induced oxidative stress, in vitro. (A) Ethanol treatment increased lipid peroxidation in Drp1 silenced cells. (B) Total antioxidant levels decreased on ethanol treatment to Drp1 depleted cells. (C) Pre-treatment with z-vad ameliorated Drp1 silencing-induced loss in cell viability upon ethanol exposure. Data are represented as mean ± SEM where ***P < 0.001, **P < 0.01, *P < 0.05, ns not significant versus the respective control group, $$P < 0.01, $ < 0.05 versus scramble control and ### P < 0.001, ##P < 0.01, #P < 0.05, ns- not significant versus ethanol treated group. Supplementary Fig. 3. Ethanol-induced mitochondrial dysfunction in Drp1 silenced H9c2 cells is abrogated with Resveratrol pre-treatment. Mitochondrial superoxide levels are reduced on RES pre-treatment in Drp1 depleted cells exposed to ethanol. Supplementary Fig. 4. Pre-treatment with VO-OHpic improves cell survival response to ethanol-induced cytotoxicity in Drp1 depleted cells. (A) VO increased p-Akt1 levels at 20 min and normalised to control at 24 h and 48 h. (B) Quantitative representation of increase in p-Akt1 levels compared to control at 20 min. (C) VO pre-treatment improved ATP production in Drp1 depleted cells. (D) & (E) VO significantly reduced mitochondrial superoxide levels upon ethanol exposure. (F) Lipid peroxidation was significantly reduced upon VO pre-treatment in Drp1 depleted cells. Data are represented as mean ± SEM where ***P < 0.001, **P < 0.01, *P < 0.05, ns not significant versus the respective control group, $$$P < 0.001, $$P < 0.01, $P < 0.05 versus scramble control and ###P < 0.001, ##P < 0.01, #P < 0.05, ns not significant versus scramble siRNA transfected and ethanol treated group. Supplementary Fig. 5. Mdivi-1 increases oxidative stress upon ethanol exposure. (A) Pre-treatment with Mdivi-1 does not improve cell viability on ethanol exposure. (B) Mdivi-1 does not reduce ethanol-induced lipid peroxidation of H9c2 cells. (C) Mdivi-1 supresses antioxidant levels in ethanol treated cells. (D) Ethanol treatment of Mdivi-1 pre-treated cells reduced ATP producing capacity of the cells. Data are represented as mean ± SEM where ***P < 0.001, **P < 0.01, *P < 0.05, ns not significant versus the respective control group, ###P < 0.001, ##P < 0.01, #P < 0.05, ns not significant versus Mdivi1 + ethanol treated group. Supplementary Fig. 6. VO pre-treatment reduced alcohol-induced cardiac oxidative stress. (A) Ethanol exposure did not alter cardiac total antioxidant levels during VO pre-treatment. (B) VO treatment prevented lipid peroxidation of heart tissue on ethanol exposure. Data are represented as mean ± SEM (n = 6) where **P < 0.01, *P < 0.05, ns not significant versus the control group and ##P < 0.01 versus EtOH treated animals.

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Sivakumar, A., Shanmugarajan, S., Subbiah, R. et al. Cardiac Mitochondrial PTEN-L determines cell fate between apoptosis and survival during chronic alcohol consumption. Apoptosis 25, 590–604 (2020). https://doi.org/10.1007/s10495-020-01616-2

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