Antioxidant Effects of Pyrazoles in Ischemia/Reperfusion Injury in Cardiomyocytes

Abstract

Since the turn of the 21st century, cardiovascular diseases (CVDs) have remained the leading cause of death worldwide. In the United States alone, approximately 1.5 million people annually will have a myocardial infarction (MI), or more commonly known as a heart attack. The most common cause of MI is coronary artery disease (CAD), also known as atherosclerosis, where a significant occlusion in the coronary artery leads to an acute mismatch of oxygen supply and demand in the myocardium, leading to myocardial ischemia. Removing the occlusion is critical to salvaging ischemic tissues; however, reperfusion and restoring blood flow to previously anoxic/hypoxic tissues may cause a paradoxical worsening of cardiac cell dysfunction and death. This phenomenon is known as ischemia/reperfusion injury (IRI). Multiple studies have shown that oxidative stress plays a crucial role in the pathophysiology of IRI, and therapeutic studies are now being conducted to find an agent that can counteract the sudden rise in reactive oxygen species (ROS). Pyrazoles are small, heterocyclic molecules found in many biologically active compounds and have been identified as potential therapeutic agents against CVD due to their anti‐inflammatory effects. Most importantly, they are relatively easy to synthesize and can be fine‐tuned to achieve desired electronic and steric effects. Thus, pyrazoles are gaining more attention in the field of drug discovery research. In our present study, the function of pyrazole KT‐01‐14B as an antioxidant was determined by evaluating its effect on the expression of biomarkers indicative of ischemia/reperfusion (I/R)‐induced oxidative stress, which includes manganese‐dependent superoxide dismutase (SOD2), acetylated tubulin, and 4‐hydroxynonenal (4‐HNE) aggresomes. I/R was induced in vivo by using mice models and in vitro by subjecting HL‐1 cardiomyocytes to hypoxia/reoxygenation (H/R). Histologic evaluation of the cardiac tissue in our in vivo models was accomplished using TTC staining, which revealed that pyrazoles significantly reduce infarction size. To measure the mitochondrial superoxide levels in the HL‐1 cardiomyocytes, MitoSOX staining was used, which indicated that superoxide levels were significantly reduced with pyrazole treatment. Further investigation of pyrazole‐treated HL‐1 cardiomyocytes with immunoblotting analysis and immunocytochemistry revealed increased expression of SOD2, increased expression of acetylated tubulin, and decreased 4‐HNE aggresome formation. In addition to studying the effects of pyrazole on the biomarkers, a cell viability assay was performed to determine the effects of pyrazole on cardiomyocyte cell death. These results conclude that the antioxidant function of pyrazoles can prevent cellular damage caused by I/R.