Study Of Interactions And Mechanisms Between Cardiac Fibroblasts And Extracelluar Matrix During Hypoxia

Background: Hypoxia is one of major pathogenic factor of myocarial remodeling, repsented by ventricular hypertrophy, cardiac myocytes loss and interstitial fibrosis. From a clinical point of view, fibrosis is for the moment a major marker for cardiac failure and a crucial determinant of myocardial heterogeneity, increasing diastolic stiffness, and the propensity for reentry arrhythmias. The interstitial fibrosis of myocardium is resulted from the disproportionate increase in synthesis and/or inhibition of degradation of extracellular matrix (ECM) proteins. On the other hand, the ECM proteins exerts profound control over cells. Transmembrane receptors termed integrins provide a dynamic interaction of environmental cues and intracellular events, regulation of gene expression, cell growth, differentiation, migration, and death. Although cardiac fibroblasts(CFs) apppear to play a central role in the regulation of the ECM proteins of the heart and the effects of the ECM proteins mediated by integrins signaling are primarily in the regulation of cellular responses to pathophysiological signals, there are only few studies concerning the influence of hypoxia on the behaviors of CFs and its integrins signaling. Objective: The goal of this study was to explore the interactions and mechanisms between CFs and ECM during hypoxia.Methods:1. Ventricular tissue of adult Wistar rat was digested by a mixture of trypsin and collagenase, isolated cells were plated, and cardiac myocytes were separated from non-myocytes by differential plating. Studies were conducted on the 2～3 passage of CFs. CFs were cultured in vitro either in normoxic or hypoxic condition (2% O2).2. To explore the effects of hypoxia on the regulation of ECM by CFs:①The changes of collagen, fibronectin(FN), and osteopontin(OPN) mRNA expression was determined by RT-PCR;②The changes of collagen protein and total protein synthesis were determined by measuring the collagenase sensitive 3H-proline incorporation and the 3H-leucine incorporation respectively;③The protein level of OPN was examined by Western blotting;④The modification of CFs phenotype during hypoxia was measured by immunostaining.3. To analyze the changes of CFs integrins signaling during hypoxia:①The...