This proof-of-concept study using an in vitro experimental cell culture model identifies the protective role of simvastatin against TGF-β1-induced hVF transformation into activated myofibroblasts through activation of protein phosphatase, a novel target that can be therapeutically modulated to curb excessive cardiac fibrosis associated with maladaptive cardiac remodeling.

Fibroblasts, the most abundant cells in the heart, contribute to cardiac fibrosis, the substrate for the development of arrythmogenesis, and therefore are potential targets for preventing arrhythmic cardiac remodeling. A chamber-specific difference in the responsiveness of fibroblasts from the atria and ventricles toward cytokine and growth factors has been described in animal models, but it is unclear whether similar differences exist in human cardiac fibroblasts (HCFs) and whether drugs affect their proliferation differentially. Using cardiac fibroblasts from humans, differences between atrial and ventricular fibroblasts in serum-induced proliferation, DNA synthesis, cell cycle progression, cyclin gene expression, and their inhibition by simvastatin were determined. The serum-induced proliferation rate of human atrial fibroblasts was more than threefold greater than ventricular fibroblasts with faster DNA synthesis and higher mRNA levels of cyclin genes. Simvastatin predominantly decreased the rate of proliferation of atrial fibroblasts, with inhibition of cell cycle progression and an increase in the G0/G1 phase in atrial fibroblasts with a higher sensitivity toward inhibition compared with ventricular fibroblasts. The DNA synthesis and mRNA levels of cyclin A, D, and E were significantly reduced by simvastatin in atrial but not in ventricular fibroblasts. The inhibitory effect of simvastatin on atrial fibroblasts was abrogated by mevalonic acid (500 μM) that bypasses 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibition. Chamber-specific differences exist in the human heart because atrial fibroblasts have a higher proliferative capacity and are more sensitive to simvastatin-mediated inhibition through HMG-CoA reductase pathway. This mechanism may be useful in selectively preventing excessive atrial fibrosis without inhibiting adaptive ventricular remodeling during cardiac injury.

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Background: HMG-CoA reductase inhibitors (statins) have been shown to reduce the incidence of atrial fibrillation (AF) and its progression but the underlying mechanisms are not fully elucidated. Since atrial fibrosis plays a major role in the development of the substrate for AF progression, we hypothesized that statins antagonize the effect of profibrotic cytokines, reducing their stimulatory effect on fibroblast proliferation, differentiation and activation. Methods: The effect of TGF-β1, a major profibrotic cytokine, on fibroblast proliferation and activation of myofibroblasts was assessed by expression of alpha smooth muscle actin (α-SMA) message (qPCR) and proteins (western and immunofluorocytochemistry) in the presence and absence of simvastatin (1-10μM). The inhibitory effect of simvastatin on SMAD 2/3 phosphorylation (western) and its nuclear translocation by TGF-β1 (5ng) was determined by immunofluorescence antibodies using fluorescent microscopy. Results: TGF-β1 treatment increased fibroblast proliferation (cell count) by 63% compared to control (p<0.001) at 96 hours, which was inhibited by simvastatin by 61% (p<0.001) (Fig a). Simvastatin also reduced TGF-β1-mediated myofibroblast differentiation (α-SMA positive) by 75% (p=0.02); (Fig b and c). TGF-β1 increased SMAD2/3 phosphorylation with increase in nuclear localization was inhibited by simvastatin. Conclusion: Simvastatin inhibits TGFβ-1-mediated cardiac fibroblast proliferation and myofibroblast differentiation by antagonizing SMAD phosphorylation and its translocation into the nucleus.

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