Alcohol Induced Oxidative Stress in neonatal Cardio myocytes
Authors: Miriyala Sumitra , Horsne Ara , Sharma Sudha , Bhattarai Susmita , Panchatcharam Manikandan 
Published: April 2020

INTRODUCTIONAlcohol consumed at high‐dose in binging, is a major public health concern due to the increase in cardiac morbidity and mortality, in addition to damage to the liver and the central nervous system. The peak blood ethanol concentration of the dose associated with a protective effect is ~ 9.0 mM, which usually can be seen in a man with average body weight (70 kg or 154 Ibs) after ingestion of one and a half American standard drinks (14 grams of ethanol/each). Lysophosphatidic acid (LPA) production involves hydrolysis of lysophosphatidylcholine by the secreted enzyme autotaxin (ATX). In contrast, lipid phosphate phosphatase 3 (LPP3) catalyzes LPA dephosphorylation to generate lipid products that are not receptor active. The present work focused on synthesis and degradation of LPA, to examine their influence on alcohol induced ROS in neonatal cardiomyocytes.METHODSMale seven‐week old mice will be gavage fed with 2.8 g/kg/day ethanol (heavy alcohol consumption) or volume‐matched water once a day for 8 weeks. Neonatal cardiomyocytes will be exposed to 40mM ethanol for 2h/day for a period of 8 weeks for in vitro studies. Mitochondrial respiration and glycolysis were measured through use of Seahorse Bioscience XF24 Extracellular Flux Analyzer. Visualization of superoxide production by the mitochondria was performed by utilizing the MitoSOX.RESULTSThe vehicle group was gavage fed with 10 ml/kg water. The ethanol concentration reduced rapidly and approached to zero at 2 h in the 0.7 g/kg/day ethanol group. In contrast, plasma ethanol concentration declined slowly in the 2.8 g/kg/day ethanol group. Eight‐week feeding produced a significant decrease and a significant increase in plasma ethanol concentration at 15 min (p = 0.013) and 1 h (p = 0.049), respectively, in the 2.8 g/kg/day ethanol group. ATX and miR‐92a mRNA expression levels were significantly upregulated, whereas KLF2 and LPP3 mRNA expressions were significantly downregulated following 2.8 g/kg/day for 8 weeks in ethanol‐induced cardiac injury. We observed a ~3 fold increase in the ATX protein levels, whereas LPP3 protein levels were significantly downregulated as observed through Western blot analysis in these myocardial tissues, these enzymatic changes corresponded to a ~3 fold increase in plasma LPA levels after 8 weeks of 2.8 g/kg/day ethanol treatment as compared to vehicle. Our findings suggest ATX and LPP3 are strongly regulated in myocardium following heavy alcohol consumption (2.8 g/kg/day for 8 weeks). There also was a significant reduction in the basal oxygen consumption rate, mitochondrial ATP production following heavy alcohol consumption. We examined glycolysis in neonatal cardiomyocytes chronically exposed to alcohol by ECAR, and the opposite pattern to OCR was observed for glycolysis rates between chronic alcohol exposure and control cardiomyocytes. Increased basal superoxide production was observed in neonatal cardiomyocytes chronically exposed to alcohol.CONCLUSIONUnderstanding these mechanism by which alcohol‐mediated oxidative stress that regulates LPA‐associated cellular events will identify novel targets for future clinical interventions to treat cardiovascular disease.Support or Funding InformationThis work was supported by 10SDG4190036 to Dr. Panchatcharam and 110101074A to Dr. Miriyala(A) Dynamic change of plasma ethanol concentration in the 0.7 g/kg/day and 2.8 g/kg/day ethanol groups at the beginning and end of an 8‐week feeding period. Values are means ± SD for four mice in each group. *P < 0.05 vs. Beginning of 8‐week. (B) qPCR was used to measure expression of miR‐92a., KLF2., LPP3., and autotaxin foilowing 2.8 g/kg/day ethanol group., end of 8‐week feeding period. (C) Western blot analysis of autotax:in and LPP3 protein expression following vehicle., 0. 7 and 2.8 g/kg/day ethanol groups at the end of 8‐week feeding period. GAPDH was used as a loading control. LPP3 and autotaxin expression was normalized to GAPDH staining (n = 4) and graphed as mean ± SD in arbitrary units in which the density of LPP3 and autotax:in in vehicle mice samples were set to 1. Mean ± SD, *P<0.05. D) Plasma lysophosphatidic levels in mice following vehicle, 0.7 and 2.8 g/kg/day ethanol groups at the end of an 8‐week feeding period.Figure 1The effects of acute and chronic alcohol exposure on OCR in neonatal cardiomyocytes(NC).(A) OCR profile in NCs acutely exposed to EtOH. (B) Acute exposure of 50 mM EtOH significantly inhibited basal (difference between the baseline OCR and the OCR after antimycin A and rotenone), ATP‐linked (difference between the baseline OCR and the OCR after oligomycin), and maximal (difference between the OCR after FCCP and the OCR after antimycin A and rotenone) respiration. Values are means ± SE for 8 wells in each group. *P < 0.05 versus Vehicle group. (C) OCR profile at 24 hours of reoxygenation after a 3‐hour OGD in NCs chronically exposed to EtOH. To avoid the effect of acute alcohol exposure. EtOH was not given on the day of OGD. (D) OGD/re oxygenation‐induced reductions in basal, ATP‐linked, and maximal respiration were significantly lower in NCs chronically exposed to 10 mM EtOH, but significantly greater in NCs chronically exposed to 50 mM EtOH compared to the vehicle group. Values are means ± SE for 8 wells in each group. *P < 0.05 versus Vehicle group.Figure 2