Figure 6. Nuclear up-regulation of calcium-dependent pro-remodelling transcription factors in pressure-overloaded hypertrophic and fibrotic K_ATP channel knockout hearts.

A and B, following transverse aortic constriction (TAC), nuclear translocation of the calcium-dependent pro-remodelling factor MEF2 was revealed using a MEF2C antibody in Kir6.2 knockout (KO), but not wild-type (WT) cardiomyocytes counterstained with the sarcomeric protein α-actinin antibody and the nuclear probe DAPI. C, electrophoretic mobility shift assay demonstrates K_ATP channel-dependent pressure overload-induced increase in specific binding of NF-AT to the B-type natriuretic peptide promoter element. Left ventricle nuclear extracts plus the radiolabelled DNA probe were loaded as follows: wild-type (WT) prior to (Pre, lane 1) and 48 h following TAC (lane 2); Kir6.2-KO (KO) Pre (lane 3) and 48 h following TAC (lane 4); KO 48 h following TAC in the presence of 50-fold excess of unlabelled DNA probe (lane 5) or unlabelled mutated DNA probe (lane 6). Note the up-regulated NF-AT complex formation with the promoter element (arrows) in TAC KO (lane 4) compared to Pre KO (lane 3) or WT counterparts (lane 1 and 2). The specificity of NF-AT binding to the DNA probe was validated by competition with an excess of cold probe used as control (C; lane 5). Point mutations (M) in the NF-AT binding region of the unlabelled probe prevented competition with the labelled consensus sequence (lane 6). The presented electrophoretic mobility shift assay is representative of a total of 6 wild-type and 6 Kir6.2-KO. D and E, the degree of fibrosis developing post-TAC is greater in KO (n = 5) than WT (n = 7) as indicated by blue staining on Masson trichrome exposed myocardial sections. †P < 0.05 TAC versus pre in E; *P < 0.05 KO TAC versus WT TAC in B and E.