Effects of Pyrazoles on Hypoxia/Reoxygenation‐Induced Oxidative Stress in Neonatal Cardiomyocytes
Authors: Bess Ezar , Sharma Sudha , Bhattarai Susmita , Ara Hosne , Panchatcharam Manikandan , Murru Siva , Miriyala Sumitra 
Published: April 2020

INTRODUCTION

Despite the major advancements in cardiovascular medicine, heart disease and stroke remain the number one public health concern and primary causes of death today. The development of effective medicines that control blood pressure and cholesterol has significantly reduce deaths from heart disease. Under normal physiological conditions, the mitochondria maintain a balance of naturally produced ROS and its subsequent neutralization through various antioxidant mechanisms. The accumulation of ROS can result in increased 4‐hydroxynonenal (4‐HNE), a product of lipid peroxidation and biomarker of oxidative stress. Numerous studies have shown that inhibiting these pathways by pharmacological strategies exhibit beneficial effects in animal or in vitro experiments. Accordingly, the identification of small molecules that positively modulate the mitochondrial respiratory function has broad applications in fundamental research and drug discovery. Heterocycles have been frequently found as a key structural unit in a wide variety of drugs, such as vitamins, natural products, and biologically active compounds. Pyrazoles and triazole compounds are gaining more attention for drug discovery research. On continuation of our interest in synthesizing small molecule heterocyclic compounds and evaluating their biological activities, the present work focused on synthesis of some novel pyrazole and triazole derivatives to examine their influence on hypoxia/reoxygenation induced ROS in neonatal cardiomyocytes.

METHODS

The neonatal cardiomyocytes were maintained in gelatin‐coated cell culture plates with a supplemented Claycomb Medium and subjected to hypoxia/reperfusion. For immunoblotting, the blots were treated with mouse monoclonal primary antibodies for either MnSOD2, PERK/ERK, or 4‐HNE. 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 MitoSOX.

RESULTS

Protein immunoblotting revealed that with the addition of pyrazoles KT‐01‐8A and KT‐01‐10B, there is a relatively higher level of MnSOD2 and lower level of 4‐HNE when compared to doxorubicin treatment alone. Higher level of pERK was also shown on protein immunoblotting with the addition of KT‐01‐14B. The oxygen consumption rate and extracellular acidification rate increased and decreased, respectively, within each pyrazole treatment groups, which suggests a rescue effect on mitochondrial functions. MitoSOX staining revealed decreased production of superoxide radicals with the addition of pyrazoles KT‐01‐8A and KT‐01‐10B.

CONCLUSION

According to the data, our research study shows the inhibitory effects of small molecules heterocycles on the pathophysiology processes mentioned above in order to prevent and/or treat cardiovascular diseases.

Support or Funding Information

NIH‐HL141998; HL14199801S1, AA023610; AA025744; AA02574402S1; COBRE1P20GM121307; AAA‐FGAP‐5148

Figure 1