Fig. 5.

KLF15 inhibits GATA4 and MEF2 function. (A) Hearts from KLF15−/− mice exhibit enhanced hypertrophic gene expression. Northern blot analysis was performed for ANF/BNP in KLF15 +/+ and −/− mice after sham-operation and AAC. (B) KLF15 deficiency does not affect GATA4 and MEF2 protein expression. Total protein isolated from ventricular tissue of KLF15 +/+ and −/− mice was used in Western blot analysis. (C) KLF15 inhibits basal and MEF2-inducible activity on the ANF promoter (Upper) and also inhibits basal and GATA4-inducible activity of the BNP promoter (Lower). H9c2 cells were transfected with -638-ANF-luc or -112-BNP-luc reporter constructs, and with indicated combinations of expression plasmids for KLF15, MEF2A, GATA4, and pCDNA3.1 (n = 9 per group; P values < 0.05 were considered significant and are indicated in the figure). (D) KLF15 attenuates the ability of MEF2 and GATA4 to bind target DNA in NRVM. Nuclear extracts from NRVM overexpressing EV or KLF15 were used in binding with ³²P-labeled DNA probes derived from the Atr promoter (top EMSA for MEF2) or BNP promoter (bottom EMSA for GATA4). Cold competition was performed by using 100× molar excess of unlabeled probe. Supershift studies were performed by preincubating reactions with α-MEF2 antibody (upper gel shift) or α-GATA4 antibody (lower gel shift). (E) KLF15 −/− hearts exhibit enhanced MEF2 and GATA4 DNA binding. Nuclear extracts were harvested from KLF15 +/+ and −/− mice after sham surgery vs. AAC. EMSA for MEF2 and GATA4 were performed by using the same probes as used in D.