669 Application of In-cell Western Blot to Study Burn-induced Cardiac Dysfunction

Abstract

Abstract Introduction Traditional Western blotting (WB) may not reflect true target protein amount due to interference from enzymic activity during sample preparation. In-Cell Western (ICW) blotting has proved to have advantages over the traditional WB. However, ICW has not been used to study burn-induced cardiac dysfunction. This study will explore the efficacy of ICW in the study of burn-induced cardiac dysfunction. Methods Human cardiomyocytes (Ac16) were cultured in serum from rats with/without 60% of total body surface area (TBSA) burn injury. Cultured cells were permeabilized by Tritons-100 X, 1st anti-targeted protein antibodies were added and incubated, and, finally, two fluorescence-conjugated 2nd antibodies (one red and one blue) were added and incubated. LI-COR Odyssey CLx scanner was employed to obtain images and LI-COR Image Studio 4.0 software was used to analyze image and GraphPad Prism 8.0 software was used to determine image density. Results ICW proved to be a good tool to study burn injury. Our ICW data showed that burn serum resulted in 1) increased cardiomyocyte PARP1 (leading to increased protein acetylation); 2) increased damage of cardiomyocyte mitochondrial DNA replication (via increase in mitochondrial Fis1and BCBN proteins and decrease in parkin and POLG proteins); and 3) a decrease of cardiomyocyte mitochondrial biogenesis (with increase in inflammation-related proteins, apoptosis-related proteins, and decrease Brf2-ARE-related proteins). Conclusions This study provides preliminary evidence that ICW may be a novel tool to study burn injury and confirms that burn-injury induced cardiomyocyte mitochondrial dysfunction occurs via damage of mitDNA replication, involving inflammation, apoptosis, and mitochondrial biogenesis pathways.