Abstract 1710: Knockdown of Nkx2.5 Results in Altered Sarcomere Organization and Cell Shape
Homeobox transcription factor, Nkx2.5 has been known to be a key factor in early cardiogenesis. Having previously demonstrated the persistent expression of Nkx2.5 in cardiac myocytes from the precardiac mesoderm through to the adult heart, we hypothesized that Nkx2.5 actively regulates a critical set of genes in postnatal cardiomyocytes to maintain proper cardiac function. In this study, we utilized cardiomyocytes with near complete depletion of Nkx2.5 to identify Nkx2.5-dependent gene regulation in neonatal cardiomyocytes by two molecular approaches: i) adenovius-RNAi targeting rat Nkx2.5 and ii) in vitro deletion of floxed-Nkx2.5 allele by adenoviral Cre-recombinase. Knockdown of Nkx2.5 in cardiomyocytes resulted in a significant change in cell shape and disorganized sarcomere structure. One potential molecular mechanism underlying sarcomere disorganization is decreased expression of Z-disc localizing protein, muscle LIM protein (MLP) and T-cap (titin-cap) that interacts with MLP. To understand Nkx2.5-dependent MLP regulation either directly or indirectly, we analyzed the -11.4 kb to +5 kb genomic region of the mouse MLP gene. Our results indicate that the region 500 bp proximal of the promoter is sufficient for robust induction of luciferase reporter activity (> 70 fold compared to the empty luciferase reporter construct) in cardiomyocytes, but not in noncardiomyocytes (? 4 fold). Cardiomyocyte-specific MLP gene expression depends partially on Nkx2.5 through a Nkx2.5 binding site located around -260 bp, suggesting direct effects of Nkx2.5 on MLP transcription. Understanding the process of sarcomere organization and maturation from embryonic to postnatal cardiomyocyte is presumably through characterization of regulatory factors in cardiomyocytes. Our results suggest Nkx2.5 is one of the key regulators in sarcomere formation or maturation in neonatal cardiomyocytes.