Abstract 14359: Abnormal Extracellular Matrix Remodeling in an Nkx2-5+/- Model of Dilated Cardiomyopathy

Mice heterozygous for the cardiac transcription factor Nkx2-5 are viable but have an abnormal conduction system. To identify conduction cells we employed a double transgenic strategy by crossing Nkx2-5 +/- mice with a knock-out/knock-in Connexin 40 (Cx40) allele replaced with enhanced green fluorescent protein (EGFP)(Cx40 EGFP/+ ). We subjected both the Nkx2-5 +/- and Nkx2-5 +/- /Cx40 EGFP/+ mice to 4 weeks of pressure-overload (PO) via transaortic constriction and found the left ventricle (LV) responded with a reproducibly dilated cardiomyopathy phenotype in contrast to the LV hypertrophy and preserved function in Cx40 EGFP/+ and wild-type controls (EF declined to 40-42% with associated dysynchrony). To elucidate mechanisms underlying the dilated phenotypes found in Nkx2-5 cohorts, mRNAs were analyzed by specialized gene chip arrays. We found no significant differences in markers of apoptotic, growth and transcription factor genes unrelated to Nkx2-5. However, there were marked and significant abnormalities of genes known to regulate the extra-cellular matrix (ECM) including increased metalloproteinase (MMP) -2(+3.1 fold) and MMP-14 (+12.5 fold) and decreases in tissue inhibitor of metalloproteinase (TIMP) -3 (-76.6 fold) and TIMP-4 (-3.7 fold). Confirmatory MMP-2 zymography showed the active form increased 5.8 fold. There were also changes identified within the ECM with increases in cardiac fibroblast specific collagen type 1a (+57.6 fold) and 5a (+7.5 fold). Immunofluorescence imaging for collagen 1a after PO in the WT mice showed high levels typical for myocardial fibrosis but the dilated Nkx2-5 +/- /Cx40 EGFP/+ transgenic failed to mount a fibrotic collagen response and was confirmed with picro sirius red staining for total collagen. Finally, we found gene expression was decreased for Smad1 (-16-fold), -5 (-5.9-fold) and -7 (-24-fold) with corresponding increases in latent TGF-β binding protein-1 (+3.3-fold) and -2 (+56-fold), compared to controls, suggesting that perturbed Smad/TGF-β signaling may underlie the dilated Nkx2-5 +/- /Cx40 EGFP/+ phenotype. ECM remodeling is a component of heart failure syndromes. Our data suggest cardiomyocytes lacking Nkx2-5 are unable to correctly signal to the ECM after PO.