VCOM Research Day Program Book 2023

Undergraduate Student Research Biomedical

03 Acute Effects of Trimethylamine N-Oxide on Cardiomyocytes Isolated from Mouse Heart

Ranya Ridha; Sondos Elnady; Shreya Raj; Ahmed Elywa; Suzanne Tunder; Jia-Qiang He Corresponding author:

Department of Biomedical Sciences and Pathobiology, College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg

The gut microbiota in our gastrointestinal system, especially those in the intestine, play important role in maintaining the homeostasis of our body via direct and indirect interaction pathways. For example, trimethylamine (TMA), a minor amine oxide, is a known metabolite generated by the gut microbiota through digestion of carnitine-, betaine-, and/or choline-rich meals, such as red meat, egg yolk, and dairy products. Following absorption into the blood stream from the intestine, TMA is then converted into trimethylamine N-oxide (TMAO) in the liver. To date, numerous published studies have demonstrated that a high level of the plasma TMAO promotes the development of various disorders, such as high blood pressure, high cholesterol, and heart attack. It is believed that TMAO-induced pathogenesis is associated with hyper-activity platelet, which then trigger inflammation responses, increase the formation of foam cells in the artery, and decrease transportation

of the reverse cholesterol. We hypothesize that acute treatment of TMAO on cultured cardiomyocytes undermine cardiomyocyte contractility. To this end, we first tuned the existing procedures to increase the cell viability of isolated cardiomyocytes for post-cell culture and compound testing. Briefly, after exposing the mouse's heart by cutting off the diaphragm, the lungs were removed to cut the descending aorta and inferior vena cava. Right after, 9 ml of EDTA buffer was injected into the right ventricle and the heart was then extracted and placed in an EDTA buffer dish and followed by an injection of 10 mL of EDTA, 3 mL of perfusion and 25-50 mL of collagenase buffer into the left ventricle. Of note, the amounts of collagenase buffer used in each heart depends on the age and size of the mouse. For example, a 25 mL of the enzyme should be sufficient for a younger and smaller mouse, while 50 mL of the same buffer is needed for an older and larger mouse. Next, the pre

digested heart was separated into smaller parts by gently teasing apart and triturating for 2 min with a pipette in a dish containing fresh collagenase buffer. By following this protocol restrictively, we (even a new undergraduate student) were able to successfully isolate cardiomyocyte with 70–90% viable rod-shaped cells. The ongoing and upcoming experiments are examining the acute effects of TMAO on isolated cardiomyocytes in the terms of cell contraction, reactive oxygen species (ROS) generation, and gene/protein expression associated with myocardial contraction.

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