Human Cardiac Fibroblasts-adult ventrical (HCF-av)

Long-Term Culture of Human Cardiac Fibroblasts Leads to Myofibroblast Transition

Activation of cardiac fibroblasts into myofibroblasts underlies pathological cardiac fibrosis, which can cause arrhythmias and heart failure. Hall et al. sought to determine if the myofibroblast phenotype is reversible in human cardiac fibroblasts cultured on soft substrates or with inhibition of TGF-β receptors.

Human cardiac fibroblasts (HCFs) were cultured for up to 10 passages on stiff polystyrene plates (E = 3 GPa) to activate the myofibroblast phenotype. Cells were then treated with 10 ng/mL TGF-β for 2-4 days to induce further activation. Fibroblast activation was indicated by expression of α-SMA and collagen 1. HCFs cultured on stiff substrates for long term had baseline activation; α-SMA expression was confirmed by western blot and immunofluorescence (Fig. 1, panels b, d, e). However, when treated with TGF-β for 4 days, there was no significant increase in expression of α-SMA or collagen 1 at the mRNA (Fig. 1a) or protein level (Fig. 1b) compared to controls. TGF-β had no effect on proliferation (Fig. 1c) or cell size. Immunofluorescence also revealed that ~70% of HCFs were α-SMA positive. There was no significant difference in the percent of α-SMA positive cells with TGF-β treatment (Figs. 1d, f), and secondary antibody controls were used to rule out non-specific binding of the α-SMA antibody. Lack of response to TGF-β was confirmed in HCFs cultured for a range of passages p6-p9 (Fig. 1f). Both the total expression of α-SMA and the percent of α-SMA positive cells did not significantly change throughout long term culture (Fig. 1f). There was a non-significant decrease in α-SMA positive cells with time, which may indicate de-differentiation. This data shows that culturing human cardiac fibroblasts on stiff plastic substrates is sufficient to irreversibly transition into myofibroblasts and TGF-β stimulation is unable to upregulate fibroblast activation.