Relative Profibrotic Gene Expression in Cardiac Fibroblasts from Low Aerobic Capacity “Disease Prone” Rats Following Ischemic Reperfusion

Abstract

BackgroundIschemic heart disease is a major cause of morbidity. Regular active exercise is therapeutic after myocardial infarction, but up to 70% of individual exercise capacity is due to an intrinsic genetic component. Intrinsic capacity can be studied using high (HCR) and low (LCR) aerobic running capacity rat strains. The phenotypes differ by more than 5 fold in sedentary average running distance and time. The HCR rats have been characterized as “disease resistant”, while the LCRs are characterized as “disease prone”. In the heart, cardiac fibroblasts play a critical role in healing post myocardial infarction, but little is known about the effect of intrinsic aerobic capacity on early fibroblast gene expression following ischemia reperfusion.MethodsOn each study day, one HCR/LCR pair (n = 8 each) was anesthetized and hearts were rapidly excised. The hearts were immediately flushed with iced hyperkalemic, hyperosmotic, cardioplegia solution, and subjected to cold global ischemic arrest (80 min). Following arrest, the hearts underwent warm reperfusion (120 min) using a Langendorff style perfusion system. Following reperfusion, the heart was weighed and the LV was isolated. A mid ventricular ring was obtained to estimate infarction size (TTC), and part of the remaining tissue (~150 mg) was transferred to homogenation buffer on ice for subsequent isolation of fibroblasts. Cardiac fibroblasts from the LV of HCRs and LCR rats were isolated, cultured and characterized. Immunohistochemistry verified fibroblasts/myofibroblasts markers. RNA was isolated from the cultured fibroblasts, and RT‐PCR was used to determine baseline gene expression for Collagen I and III, MMP2, TIMP1, BMPR2, ATR1, SMAD5, and Bag3 in each phenotype.ResultsImmunocytochemistry successfully confirmed the presence of both fibroblasts and myofibroblasts markers. In these post IR fibroblast/myofibroblasts, LCRs showed significantly higher gene expression (49%) in the collagen I/collagen III ratio but lower expression of extracellular matrix regulatory genes including MMP2 (20%), TIMP1 (17%), and SMAD5 (15%) compared to HCRs counterparts. BMPR2 (50%), Agtr1a (25%), also were both decreased, and the anti‐apoptotic gene, Bag3, was significantly (67%) higher in LCRs.ConclusionThese results indicate that the LCR phenotype is predisposed to expressing a more fibrotic character in fibroblasts harvested immediately following IR, and may indicate poorer long term outcome/earlier onset heart failure following acute ischemic injury to the heart. While the phenotype produced no differences in the acute injury, there may be phenotypic differences that develop later during resolution of the injury.