Poster Session 1 - C3
1,2Haisam Shah, 1,3Shafkat Choudhury, 1Hangjun Zhang, 1Meghan McFadden, 1Yuqing Zhou, 1,2,4Anthony Gramolini, 1,2,4Scott Heximer
1 Translational Biology and Engineering Program, Ted Rogers Centre for Heart Research; 2 Department of Physiology, University of Toronto; 3 Department of Chemical Engineering & Applied Chemistry, University of Toronto, 4 Heart and Stroke/Richard Lewar Centre of Excellence in Cardiovascular Research
Myocardial infarction (MI) is the most common cause of heart failure (HF), a chronic condition characterized by pathological cardiac remodeling and fibrosis. Like pulmonary and renal fibrosis, cardiac fibrosis is thought to be mediated primarily by myofibroblasts, a population of ‘activated’ cardiac fibroblasts (CFs). By studying the acute proteomic changes that occur in CFs following an MI, we hope to identify novel biomarkers and potential therapeutic targets for fibrosis and heart failure. We started our studies by conducting M-mode, B-mode, and pulse-wave doppler echocardiography to demonstrate significant cardiac dysfunction in mice that underwent left anterior coronary descending artery (LAD) ligation, a standard model of MI. More precisely, compared to sham controls, mice that underwent LAD ligation exhibited a significant decrease in ejection fraction, fractional area change, stroke volume, and cardiac output seven days after surgery. Next, we developed and validated a protocol for adult murine CF isolation by showing that majority of the isolated cells were positive for CF markers, including collagen type 1, α1 and Vimentin. Fluorescent imaging successfully showed that approximately 80% of cells exhibited high Col1α1 promoter activity and 100% of the cells were positive for Vimentin expression. Furthermore, immunoblots were conducted to show the presence of Vimentin and lack of α-smooth muscle actin (αSMA), a characteristic marker of smooth muscle cells. To conduct deep proteomic analysis, we investigated the possibility of culturing adult CFs to increase cell and protein yields. However, brightfield imaging revealed that CFs undergo a change in morphology over the course of culture. Further examination with fluorescent imaging and immunoblotting demonstrated that αSMA expression increased over the course of culture, indicative of their differentiation into myofibroblasts. Therefore, we opted to use acutely dissociated CFs with overnight culture and/or acutely dissociated, FACS-sorted, CFs with no culture for subsequent MS studies. CFs were isolated from infarct and remote cardiac regions of seven-day post-MI and sham control mice. Liquid chromatography tandem MS (LC/MS-MS) revealed preferential clustering between samples from the same treatment group. Moreover, statistical analysis revealed region-specific differences in the protein expression of CFs isolated from MI and sham hearts. Follow-up studies will involve validating differentially expressed proteins using standard immunofluorescent and immunoblotting techniques. Studies will also investigate whether the manipulation of differentially expressed proteins and/or pathways can confer therapeutic benefits by inhibiting or reducing fibrosis, post-MI.