MK5-MEDIATED FIBROBLAST FUNCTION AND EXTRACELLULAR MATRIX HOMEOSTASIS: THE DIFFERENCE BETWEEN QUIESCENT FIBROBLASTS AND ACTIVATED MYOFIBROBLASTS

Abstract

Cardiac fibroblast hyperplasia associated with enhanced matrix production and deposition is a major determinant of post-injury cardiac remodelling. MAP kinase-activated protein kinase-5 (MK5) is expressed in the heart but its physiological role in the heart is just beginning to be understood. Pressure overload-induced collagen expression is reduced in male MK5 haploinsufficient (MK5+/-) mice. Further, following myocardial infarction, both infarct size and scar size are reduced in male MK5+/- mice. Thus, MK5 may play a role in extracellular matrix (ECM) remodeling. This study sought to determine the role of MK5 in cardiac fibroblast function. Ventricular fibroblasts were isolated from male MK5+/+ or MK5-/- mice. When cultured on a rigid substrate, cardiac fibroblasts adopted an activated myofibroblast phenotype associated with increased αSMA expression. Further, MK5-deficient (MK5-/-; siRNA-mediated MK5 knockdown) myofibroblasts showed increased basal levels of collagen 1-α1 (COL1A1) mRNA and type 1 collagen secretion; however, addition of Ang-II failed to evoke a further increase in either. Confocal immunocytofluorescence revealed type 1 collagen immunoreactivity to be diffuse in MK5+/+ myofibroblasts but condensed in the perinuclear region of MK5-deficient myofibroblasts. When maintained on a compliant (8-kPa) substrate, fibroblasts remain quiescent with reduced αSMA expression. The Ang-II-induced increase in COL1A1 mRNA and type 1 collagen secretion was significantly decreased in MK5-deficient fibroblasts when compared with MK5+/+ fibroblasts. Further, MK5-deficiency impaired the ability of fibroblasts to contract collagen gels. Interestingly, pathway-targeted microarrays revealed that MK5-deficiency in myofibroblasts decreased the abundance of transcripts for ECM proteins such as integrin β3, latent TGF-β binding protein 1, thrombospondin 1, hepatocyte growth factor, and interleukin 13. In quiescent fibroblasts, reduced MK5 expression had little effect on the abundance of transcripts for ECM proteins. MK5 is involved in multiple aspects of fibroblast function and ECM dynamics and the role of MK5 is dependent on the activation status of these cells.