Myofibroblastic differentiation of rat valvular interstitial cells in culture

Background Familial genetic and genome wide studies associated FLNA, DCHS1, DZIP1 and TNS1 genes with mitral valve prolapse (MVP). These genes point to the cellular mechanical stress response as a unifying common pathway in MVP. The myofibroblastic differentiation of the valve interstitial cells (VICs) has been described as one of the main mechanisms associated with MVP. We recently developed a unique knock-in (KI) rat model expressing the Filamin A FlnA-P637Q mutation identified MVP patients. Objective To determine the primary cell culture conditions of the mitral VICs to limit myofibroblastic differentiation with the objective to analyze in vitro the impact of the FlnA-P637Q mutation on VICs responses to mechanical stress. Methods Mitral valves were dissected from 3 week-old WT and KI rats, then grown on collagen coated dishes in either 4.5 g/l or 1 g/l glucose DMEM supplemented with 10 or 2% Fetal Calf Serum (FCS). Growth rate and myofibroblastic differentiation of the cells were analyzed by immunofluorescence, western blot and qPCR experiments. Results Our preliminary results show that WT and KI cells grow with similar growth rates (t1/2= 24 h) in 4.5 g/l glucose/10% FCS DMEM. In addition, immunofluorescence and qPCR experiments show that both cell types exhibit significant smooth muscle actin (SMA-ACTA2) expression as soon as they are grown in culture (“Passage” P0). SMA expression further increases (× 1.5) when cells are trypsinized and amplified once (P1) or twice (P2). On the other hand, growing the cells in 1 g/l glucose/2% FCS resulted in a 45% decrease of SMA expression at P0 and P1. SMA expression finally reaches, at P2, the level of high glucose culture condition. Conclusion Our preliminary studies enabled us to establish culture conditions that limit myofibroblastic differentiation of WT and FlnA-P637Q mitral VICs at early “passages”. The response of these “quiescent” cells to mechanical stress and the impact of FlnA-P637Q mutation are now under study.