Abstract
Many etiologies of heart disease are characterized by expansion and remodeling of the cardiac extracellular matrix (ECM or matrix) which results in cardiac fibrosis. Cardiac fibrosis is mediated in cardiac fibroblasts by TGF‐β 1/R‐Smad2/3 signaling. Matrix component proteins are synthesized by activated resident cardiac fibroblasts known as myofibroblasts (MFB). These events are causal to heart failure with diastolic dysfunction and reduced cardiac filling. We have shown that exogenous Ski, a TGF‐β 1/Smad repressor, localizes in the cellular nucleus and deactivates cardiac myofibroblasts. This deactivation is associated with reduction of myofibroblast marker protein expression in vitro, including alpha smooth muscle actin (α‐SMA) and extracellular domain‐A (ED‐A) fibronectin. We hypothesize that Ski also acutely regulates MMP expression in cardiac MFB. While acute Ski overexpression in cardiac MFB in vitro was not associated with any change in intracellular MMP‐9 protein expression versus LacZ‐treated controls,exogenous Ski caused elevated MMP‐9 mRNA expression and increased MMP‐9 protein secretion versus controls. Zymographic analysis revealed increased MMP‐9‐specific gelatinase activity in myofibroblasts overexpressing Ski versus controls. Moreover, Ski expression was attended by reduced paxillin and focal adhesion kinase phosphorylation (FAK ‐ Tyr 397) versus controls. As myofibroblasts are hypersecretory and less motile relative to fibroblasts, Ski's reduction of paxillin and FAK expression may reflect the relative deactivation of myofibroblasts. Thus, in addition to its known antifibrotic effects, Ski overexpression elevates expression and extracellular secretion/release of MMP‐9 and thus may facilitate internal cytoskeletal remodeling as well as extracellular ECM components. Further, as acute TGF‐β 1 treatment of primary cardiac MFB is known to cause rapid translocation of Ski to the nucleus, our data support an autoregulatory role for Ski in mediating cardiac ECM accumulation.
Highlights
The cardiac extracellular matrix (ECM) is a load-bearing connective tissue that is dysregulated in most etiologies of heart disease
Effects of exogenous Ski expression on matrix metalloproteinases (MMPs) expression and activity in cardiac MFB To investigate the effects of adenoviral Ski over-expression on intracellular cardiac P1 MFB MMP-2 and MMP-9 expression, respectively, we infected cells with Skiexpressing adenovirus or LacZ-expressing control adenovirus at 100 multiplicities of infection (MOI) for 48 h (Fig. 1A)
Comparison of different treatment groups revealed that protein expression of intracellular MMP-9 was not significantly different in MFB that were overexpressing Ski versus controls (Fig. 1A and C); this finding notwithstanding, we observed a significant increase in the level of secreted MMP-9 protein when first-passage (P1) MFB were infected with AdSki for 48 h at both 50 and 150 MOI, as compared to AdLacZ controls (Fig. 3A and B)
Summary
The cardiac extracellular matrix (ECM) is a load-bearing connective tissue that is dysregulated in most etiologies of heart disease. The excessive deposition of ECM component proteins, such as fibrillar collagen, is manifested as cardiac fibrosis and contributes to heart failure (Martin and Blaxall 2012; Garza et al 2015; Travers et al 2016). In many cardiac disease etiologies, the phenoconversion of CF to hyper-synthetic cardiac myofibroblasts (MFB) precedes the inappropriate accumulation of fibrotic material and this increases global cardiac stiffness which may lead to decreased cardiac output and eventual heart failure (Weber et al 2013)
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