Mice Deficient for the Stress-Induced Transcriptional Regulator p8 Have a Decreased Metalloprotease 9 Induction and Cardiac Fibrosis after Transverse Aortic Constriction

Abstract

Extracellular matrix (ECM) remodeling by cardiac fibroblasts is key to left ventricular (LV) remodeling and hence, heart failure progression. We have reported that the stress-inducible basic helix-loop-helix protein p8 is increased in vivo in failing human hearts. We have shown that p8 is required for TNF-induced production of matrix metalloprotease 9 (MMP) by rat cardiac fibroblasts; p8 associates with, and activates endogenous MMP9 promoter in a stimulus-dependent manner. In the present study we examine how p8 targeted deletion influences LV function and ECM remodeling after pressure overload induced by transverse aortic constriction (TAC). Age-matched p8 -/- and wild type (wt) mice (n=10+10) underwent TAC or Sham operation (n=5+5). Hearts were harvested for analysis 4 weeks following TAC. Hearts of wt and p8 -/- mice were comparable in size at baseline. Echocardiographic analysis at baseline indicated that all of the p8 -/- mice presented a small but significant LV dilation, wall thinning and a lower fractional shortening (n=15, p<0.01), resulting in a decreased heart functionality. In wt mice, LV p8 gene expression was barely detectable and was significantly increased after TAC (4.2 ± 1.3 fold). Hovewer, four weeks after TAC, both wt and p8 -/- mice developed a similar LV dilation and decrease in LV function. Gene expression of hypertrophic and fibrotic markers was analyzed in LV by rtPCR. p8 -/- mice exhibited an increased basal col1a2 and col3a1 gene expression (p<0.04 and p<0.01 respectively) and a lower TNF expression after TAC (p<0.01). Following pressure overload cardiac remodeling was accompanied by extensive fibrosis of the ventricular wall of wt mice, while there was little or no interstitial fibrosis in LV of p8 -/- mice, as determined by morphometric analysis of H&E and trichrome stained sections. Accordingly, analysis of newly synthesized soluble LV collagen using Sircoll colorimetric assay (p<0.05) showed that p8 -/- mice had a lower undenatured collagen content after TAC. Notably, LV from p8 -/- mice exhibited significantly less MMP9 protein expression after TAC (p<0.05). Our findings indicate for the first time that targeted deletion of p8 provokes a basal cardiac phenotype and attenuates LV MMP9 expression and collagen content induction following TAC-induced pressure overload, supporting a role for p8 during the onset of heart failure. Extracellular matrix (ECM) remodeling by cardiac fibroblasts is key to left ventricular (LV) remodeling and hence, heart failure progression. We have reported that the stress-inducible basic helix-loop-helix protein p8 is increased in vivo in failing human hearts. We have shown that p8 is required for TNF-induced production of matrix metalloprotease 9 (MMP) by rat cardiac fibroblasts; p8 associates with, and activates endogenous MMP9 promoter in a stimulus-dependent manner. In the present study we examine how p8 targeted deletion influences LV function and ECM remodeling after pressure overload induced by transverse aortic constriction (TAC). Age-matched p8 -/- and wild type (wt) mice (n=10+10) underwent TAC or Sham operation (n=5+5). Hearts were harvested for analysis 4 weeks following TAC. Hearts of wt and p8 -/- mice were comparable in size at baseline. Echocardiographic analysis at baseline indicated that all of the p8 -/- mice presented a small but significant LV dilation, wall thinning and a lower fractional shortening (n=15, p<0.01), resulting in a decreased heart functionality. In wt mice, LV p8 gene expression was barely detectable and was significantly increased after TAC (4.2 ± 1.3 fold). Hovewer, four weeks after TAC, both wt and p8 -/- mice developed a similar LV dilation and decrease in LV function. Gene expression of hypertrophic and fibrotic markers was analyzed in LV by rtPCR. p8 -/- mice exhibited an increased basal col1a2 and col3a1 gene expression (p<0.04 and p<0.01 respectively) and a lower TNF expression after TAC (p<0.01). Following pressure overload cardiac remodeling was accompanied by extensive fibrosis of the ventricular wall of wt mice, while there was little or no interstitial fibrosis in LV of p8 -/- mice, as determined by morphometric analysis of H&E and trichrome stained sections. Accordingly, analysis of newly synthesized soluble LV collagen using Sircoll colorimetric assay (p<0.05) showed that p8 -/- mice had a lower undenatured collagen content after TAC. Notably, LV from p8 -/- mice exhibited significantly less MMP9 protein expression after TAC (p<0.05). Our findings indicate for the first time that targeted deletion of p8 provokes a basal cardiac phenotype and attenuates LV MMP9 expression and collagen content induction following TAC-induced pressure overload, supporting a role for p8 during the onset of heart failure.