Inhibiting Fibronectin Attenuates Fibrosis and Improves Cardiac Function in a Model of Heart Failure.

BACKGROUND: Fibronectin (FN) polymerization is necessary for collagen matrix deposition and is a key contributor to increased abundance of cardiac myofibroblasts (MFs) after cardiac injury. We hypothesized that interfering with FN polymerization or its genetic ablation in fibroblasts would attenuate MF and fibrosis and improve cardiac function after ischemia/reperfusion (I/R) injury. METHODS: Mouse and human MFs were used to assess the impact of the FN polymerization inhibitor (pUR4) in attenuating pathological cellular features such as proliferation, migration, extracellular matrix deposition, and associated mechanisms. To evaluate the therapeutic potential of inhibiting FN polymerization in vivo, wild-type mice received daily intraperitoneal injections of either pUR4 or control peptide (III-11C) immediately after cardiac surgery for 7 consecutive days. Mice were analyzed 7 days after I/R to assess MF markers and inflammatory cell infiltration or 4 weeks after I/R to evaluate long-term effects of FN inhibition on cardiac function and fibrosis. Furthermore, inducible, fibroblast-restricted, FN gene-ablated (Tcf21MerCreMer; Fnflox) mice were used to evaluate cell specificity of FN expression and polymerization in the heart. RESULTS: pUR4 administration on activated MFs reduced FN and collagen deposition into the extracellular matrix and attenuated cell proliferation, likely mediated through decreased c-myc signaling. pUR4 also ameliorated fibroblast migration accompanied by increased ß1 integrin internalization and reduced levels of phosphorylated focal adhesion kinase protein. In vivo, daily administration of pUR4 for 7 days after I/R significantly reduced MF markers and neutrophil infiltration. This treatment regimen also significantly attenuated myocardial dysfunction, pathological cardiac remodeling, and fibrosis up to 4 weeks after I/R. Last, inducible ablation of FN in fibroblasts after I/R resulted in significant functional cardioprotection with reduced hypertrophy and fibrosis. The addition of pUR4 to the FN-ablated mice did not confer further cardioprotection, suggesting that the salutary effects of inhibiting FN polymerization may be mediated largely through effects on FN secreted from the cardiac fibroblast lineage. CONCLUSIONS: Inhibiting FN polymerization or cardiac fibroblast gene expression attenuates pathological properties of MFs in vitro and ameliorates adverse cardiac remodeling and fibrosis in an in vivo model of heart failure. Interfering with FN polymerization may be a new therapeutic strategy for treating cardiac fibrosis and heart failure.

G Protein Coupled Receptor-mediated Transactivation of Extracellular Proteases.

G protein-coupled receptors (GPCRs) comprise the largest family of receptors in humans. Traditional activation of GPCRs involves binding of a ligand to the receptor, activation of heterotrimeric G proteins and induction of subsequent signaling molecules. It is now known that GPCR signaling occurs through G protein-independent pathways including signaling through ß-arrestin and transactivation of other receptor types. Generally, transactivation occurs when activation of one receptor leads to the activation of another receptor(s). GPCR-mediated transactivation is an essential component of GPCR signaling, as activation of other receptor types, such as receptor tyrosine kinases, allows GPCRs to expand their signal transduction and affect various cellular responses. Several mechanisms have been identified for receptor transactivation downstream of GPCRs, one of which involves activation of extracellular proteases, such as a disintegrin and metalloprotease, and matrix metalloproteases . These proteases cleave and release ligands that are then able to activate their respective receptors. A disintegrin and metalloprotease, and matrix metalloproteases can be activated via various mechanisms downstream of GPCR activation, including activation via second messenger, direct phosphorylation, or direct G protein interaction. Additional understanding of the mechanisms involved in GPCR-mediated protease activation and subsequent receptor transactivation could lead to identification of new therapeutic targets.

G Protein-Coupled Receptor-G-Protein ßγ-Subunit Signaling Mediates Renal Dysfunction and Fibrosis in Heart Failure.

Development of CKD secondary to chronic heart failure (CHF), known as cardiorenal syndrome type 2 (CRS2), clinically associates with organ failure and reduced survival. Heart and kidney damage in CRS2 results predominantly from chronic stimulation of G protein-coupled receptors (GPCRs), including adrenergic and endothelin (ET) receptors, after elevated neurohormonal signaling of the sympathetic nervous system and the downstream ET system, respectively. Although we and others have shown that chronic GPCR stimulation and the consequent upregulated interaction between the G-protein ßγ-subunit (Gßγ), GPCR-kinase 2, and ß-arrestin are central to various cardiovascular diseases, the role of such alterations in kidney diseases remains largely unknown. We investigated the possible salutary effect of renal GPCR-Gßγ inhibition in CKD developed in a clinically relevant murine model of nonischemic hypertrophic CHF, transverse aortic constriction (TAC). By 12 weeks after TAC, mice developed CKD secondary to CHF associated with elevated renal GPCR-Gßγ signaling and ET system expression. Notably, systemic pharmacologic Gßγ inhibition by gallein, which we previously showed alleviates CHF in this model, attenuated these pathologic renal changes. To investigate a direct effect of gallein on the kidney, we used a bilateral ischemia-reperfusion AKI mouse model, in which gallein attenuated renal dysfunction, tissue damage, fibrosis, inflammation, and ET system activation. Furthermore, in vitro studies showed a key role for ET receptor-Gßγ signaling in pathologic fibroblast activation. Overall, our data support a direct role for GPCR-Gßγ in AKI and suggest GPCR-Gßγ inhibition as a novel therapeutic approach for treating CRS2 and AKI.

Extracellular matrix-mediated cellular communication in the heart.

The extracellular matrix (ECM) is a complex and dynamic scaffold that maintains tissue structure and dynamics. However, the view of the ECM as an inert architectural support has been increasingly challenged. The ECM is a vibrant meshwork, a crucial organizer of cellular microenvironments. It plays a direct role in cellular interactions regulating cell growth, survival, spreading, proliferation, differentiation and migration through the intricate relationship among cellular and acellular tissue components. This complex interrelationship preserves cardiac function during homeostasis; however it is also responsible for pathologic remodeling following myocardial injury. Therefore, enhancing our understanding of this cross-talk may provide mechanistic insights into the pathogenesis of heart failure and suggest new approaches to novel, targeted pharmacologic therapies. This review explores the implications of ECM-cell interactions in myocardial cell behavior and cardiac function at baseline and following myocardial injury.