TGF-beta1 induced microRNA-21 (miR-21) up-regulation in human cardiac fibroblast precedes their transformation to myofibroblast
Rizvi F, Ross G, Holmuhamedov E, Mirza M, Jahangir A. TGF-beta1 induced microRNA-21 (miR-21) up-regulation in human cardiac fibroblast precedes their transformation to myofibroblast. J Patient-Centered Res Rev. 2014;1:58.
Presented at 2013 Aurora Scientific Day, Milwaukee, WI
Background/significance: The prevalence of atrial fibrillation (AF) increases with aging and aging-associated diseases. Although a common denominator underlying the substrate for aging-related AF is excessive extracellular matrix deposition, the molecular mechanisms regulating human atrial fibrosis are not fully understood. MicroRNAs (miRNA) are small noncoding regulatory RNAs that control gene expression by translational suppression and disruption of target mRNAs. Increased expression level of atrial microRNA-21 (miR-21) has been demonstrated in animal models and patients with AF with positive correlation with atrial collagen content. However, the time course of miR-21 elevation and fibroblast activation that results in collagen deposition in patients at risk for AF has not been characterized.
Purpose: The aim of this study was to delineate the temporal association of changes in the cytokine-induced expression of miR-21 to activation of fibroblast isolated from patients with no history or risk factors for AF, with risk factors, but no AF, and those with AF.
Methods: Passage 3 cultured human cardiac fibroblasts isolated from patients with no history of or risk factors (hypertension, heart failure, valve disease or age >45 years) for AF (Group 1), with risk factors but no history of AF (Group 2) and those with AF (Group 3) were used for this study. The effect of TGF-beta1 (5ng/ml), a central pathological mediator of fibrosis, on temporal expression of miR-21 and transformation of fibroblast to myofibroblast was determined using Real-time PCR with miR-21 primers and immunohistochemistry with antibodies against Vimentin as a marker for fibroblast and alpha-smooth muscle actin (alpha-SMA) for myofibroblasts. MiR-21 expression and number of alpha- SMA+ cells was compared between TGF-beta1 untreated and treated fibroblasts.
Results: TGF-beta1 exposure increased expression of miR-21 and the number of alpha-SMA+ cells in all groups. In fibroblasts from patient with no history of or risk factors for AF the level of expression of miR-21 reached a peak (350% of baseline) within 24 hours of TGF-beta1 treatment, followed by a time-dependent increase in the mRNA expression of alpha-SMA that reached a peak (400% of baseline) in 48 hours. The baseline protein expression level of alpha-SMA in unstimulated fibroblast isolated from patients with persistent AF was 1.5 fold higher than those with no history of AF and 3.0 fold higher than those with no risk factor or history of AF with relatively smaller increase in alpha- SMA expression following TGF-beta1, suggestive of endogenous activation of fibroblasts compared to those without AF.
Conclusion: The temporal relationship of miR-21expression following TGF-beta1 stimulation before fibroblast transformation to myofibroblast is suggestive of a regulatory role of miR-21 in fibroblast activation rather than a secondary effect of myofibroblast on miR-21 expression. Patients with persistent AF appear to have endogenously active fibroblast compared to those without risk factors or history of AF.