Control of Fibrotic Gene Expression in Cardiac Myocytes and Fibroblasts via the Rap1 GTPase

Abstract

Fibrosis describes the excessive deposition of extracellular matrix components by cardiac myofibroblasts (CFs), a process which frequently promotes the adverse effects of pathological conditions damaging cardiac muscle, such as myocardial infarction and arrhythmogenic cardiomyopathy. Roughly 1 million Americans suffer a myocardial infarction each year, after which necrotic cardiac myocytes (CMs) are replaced by CF‐derived extracellular matrix (ECM), in response to (a) biochemical signaling cues from necrotic CMs and (b) mechanical cues from the contracting myocardium and ventricle cavity pressure. Accumulation of ECM proteins (such as collagen) within the infarct scar is initially critical to prevent further tissue damage, but chronic and excessive collagen deposition is detrimental to regeneration of CMs and healing of the myocardium. While the physiological outcomes of cardiac fibrosis are well studied, the biochemical and mechano‐responsive signaling mechanisms which regulate fibrotic gene expression remain poorly investigated. The RhoA GTPase signaling pathway has been shown to play a central role in regulating fibrotic gene expression and is activated via many different pro‐fibrotic signaling factors and biomechanical stimuli (i.e., changes in ECM composition, organization and rigidity). The GTPase Rap1 is also very well‐known to mediate both inside‐out and outside‐in signaling via attachment of integrins to ECM proteins. In certain contexts, Rap1 has also been shown to antagonize RhoA signaling, but very little is known about its role in fibrotic gene expression. We therefore hypothesized that expression of Rap1 would inhibit fibrotic gene expression in cardiac myocytes and fibroblasts. Expression of either wildtype (WT) or constitutively active (63E) GFP‐tagged Rap1A constructs in cardiac fibroblasts resulted in a dramatic decrease in mRNA levels of ACTA2 (smooth muscle alpha‐actin), a commonly used marker of myofibroblast activation. Further, expression of WT or 63E Rap1A in either cardiac myocytes or fibroblasts resulted in a significant decrease in mRNA levels of ECM proteins (such as COL1A1, COL3A1, COL4A1 and FN1), as well as other common markers of fibrotic gene expression (CTGF and EGR1). Taken together, these data indicate that Rap1 signaling inhibits fibrotic gene expression and myofibroblast activation. Interestingly, expression of WT or 63E Rap1A in cardiac fibroblasts caused a dramatic increase in inflammatory markers (such as TNF, IL6, NFKB1A) and matrix remodeling enzymes (MMP3 and MMP9), genes which have been shown to play a role in increasing collagen turnover, reducing ECM rigidity/stiffness and repair of damaged myocardial tissue. Altogether, these data point to a significant role for Rap1 GTPase in reducing fibrotic gene expression and myofibroblast activation and suggest possible new therapeutic strategies to combat the damaging effects of cardiac fibrosis.