One of the earliest events during the progression of cardiac hypertrophy is thought to involve an inflammatory response where inflammatory cells and their proteases orchestrate myocardial repair. Although beneficial at early stages after myocardial injury, inflammatory cells release free radicals and proteolytic enzymes within the myocardium that may contribute to myocyte death and subsequent alterations in both the geometry and mechanical properties of the heart. We have found mast cell and neutrophil infiltration and protease activation (chymase, cathepsin G (CG)) associated with MMP activation and ECM degradation within 6-12 hrs after induction of aortocaval fistula (ACF), which persisted for 15 wks. Similar ventricular dilatation and decreases in cardiac contractility were also observed in rats injected with CG for 5 days suggesting that serine proteases may play a role in the early stages of volume overload-induced cardiac remodeling. We have further found in vitro that acute exposure of neonatal rat ventricular myocytes (NRVM) to CG promotes shedding of heparin-binding epidermal growth factor (HB-EGF) and stimulation of epidermal growth factor receptor (EGFR) activation. EGFR stimulation mediates most of the effect of CG that lead to focal adhesion dephosphorylation and caspases activation that culminate in NRVM detachment from matrix and death (also termed anoikis). This led to the hypothesis that inflammatory proteases are critical mediators of cardiac myocyte death that occur early during the progression to heart failure by regulating membrane shedding of pro-HB-EGF and subsequent disruption of focal adhesion signaling.
Aim 1 will establish the role of sheddases and HB-EGF in CG effect.
Aim 2 will identify downstream signaling pathways that link EGFR to focal adhesion disruption and subsequent activation of initiator caspases during CG-induced myocyte anoikis.
Aim 3 will determine the role of membrane shedding in volume overload-induced cardiac remodeling. Ectodomain products and sheddase activity will be measured by microdialysis combined with standard biochemical assays using pharmacological interventions and transgenic approaches. Collectively, this investigation will determine how membrane shedding and transmembrane proteins contribute to the inflammatory process during cardiac remodeling in areas of inflammation and whether serine proteases should be considered direct novel targets for therapeutic interventions. ? ?
