Abstract Thu089: Pannexin-2 Deficiency Exacerbates Thapsigargin-induced Cell Injury in Cardiomyocytes

Background: Pannexins are a family of channel proteins involved in a variety of biological processes, including cell death signaling and immune functions. Pannexin 2 has been proposed as a large pore ATP-permeable channel with critical roles in various physiological processes. However, its exact role in the regulation of cardiomyocyte still poses a question. A prior study showed that Panx2 deficiency sensitizes pancreatic beta cells to cytokine-induced apoptosis. The aim of this study was to investigate how does Panx2 deficiency affects cardiomyocyte function followed by its mechanistic pathway. Methods: Cardiomyocytes were stained with sarcoplasmic reticulum (SR) and mitochondria markers along with Panx2 to determine its subcellular localization and imaged using confocal microscope. Cardiomyocytes were treated with thapsigargin (TG), an ER stress inducer and cell viability was measured by lactate dehydrogenase assay (LDH) and 3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Furthermore, the protein expression levels of UPR markers like PERK and IRE1α, and proptosis markers including cleaved caspase 1, NLRP3, and cleaved gasdermin were evaluated in our knockout cells and compared them to the control group. Cardiomyocytes were overexpressed with Panx2 and treated with TG to see whether it assists in cell survival or not. Results: Our confocal imaging results indicated that Panx2 is located intracellulary and is in close proximity to the SR. Our cell viability data shows that deficiency of Panx2 in cardiomyocytes significantly reduces its survival. We also observed that Panx2 knockout cells had more p-PERK and p-IRE1α expression indicating more ER stress compared to control cells. Deficiency of Panx2 resulted in inflammatory response that lead the cells to pyroptosis, which were indicated by elevated cleaved caspase1, NLRP3, and cleaved gasdermin protein expression. However, overexpressing the cells with Panx2 resulted in increased cell viability and reduced UPR and pyroptosis markers. Conclusion: Panx2 deficiency worsens thapsigargin induced injury in cardiomyocytes.