Pyrazole Reduces the Effects of Oxidative Stress in Cardiomyocytes Induced by Rotenone and Doxorubicin

Abstract

BackgroundIn the United States, cardiovascular disease accounts for approximately 1 in every 4 deaths. One factor that has been identified as a source for the development of cardiovascular disease is oxidative stress in the mitochondria of cardiomyocytes. Oxidative stress is a condition where the number of reactive oxygen species (ROS) is greater than the number of antioxidants. The consequences of oxidative stress in cardiomyocytes include tissue damage through cell death and the formation of 4‐hydroxynenal (4‐HNE) aggresomes through lipid peroxidation. These known effects have led to this study in which we determined the antioxidant function of a synthetic drug called pyrazole. We specifically tested the antioxidant property of two pyrazoles known as S9 and S10 in the HL‐1 cardiomyocyte cell line.MethodsOxidative stress was induced by using a compound known as rotenone and a chemotherapy agent known as doxorubicin (DOX). Both have been found to cause cardiotoxicity partly due to their ability to induce oxidative stress within cardiomyocytes. MitoSOX®and high pressure liquid chromatography (HPLC) were used to measure mitochondrial superoxide production. Immunocytochemistry (ICC) was used to measure the formation of 4‐HNE aggresomes. Western blot analysis was used to measure the expression of the antioxidant enzyme manganese‐dependent superoxide dismutase (SOD2). Seahorse XF24 analyzer was used to measure oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) to determine mitochondrial function.ResultsDOX and rotenone induced mitochondrial superoxide production was found to be reduced in cells treated with S9 or S10. From ICC, S9 and S10 treatments showed reduced formation of 4‐HNE aggreosomes compared to the level of 4‐HNE aggresome formation following treatment with DOX or rotenone alone. Western blot analysis results confirmed that both S9 and S10 were capable of restoring expression of SOD2 which was reduced by rotenone or DOX in HL‐1 cells. Seahorse analysis showed that cells treated with either DOX or rotenone and either S9 or S10 resulted in increased OCR, ATP turnover, reserve capacity, and glycolysis & glycolytic reserve capacity compared to the cells treated with rotenone or DOX alone.ConclusionThe results from our study indicate that pyrazoles S9 and S10 have the ability to reduce the oxidative stress in cardiomyocytes and therefore have the potential to serve as therapeutics for cardiovascular disease.