Abstract
Nkx2.5 is a cardiac specific homeobox gene critical for normal heart development. We previously identified Nkx2.5 as a target of sumoylation, a posttranslational modification implicated in a variety of cellular activities. Sumoylation enhanced Nkx2.5 activity via covalent attachment to the lysine residue 51, the primary SUMO acceptor site. However, how sumoylation regulates the activity of Nkx2.5 in vivo remains unknown. We generated transgenic mice overexpressing sumoylation deficient mutant K51R (conversion of lysine 51 to arginine) specifically in mouse hearts under the control of cardiac α-myosin heavy chain (α-MHC) promoter (K51R-Tg). Expression of the Nkx2.5 mutant transgene in the wild type murine hearts did not result in any overt cardiac phenotype. However, in the presence of Nkx2.5 haploinsufficiency, cardiomyocyte-specific expression of the Nkx2.5 K51R mutant led to congenital heart diseases (CHDs), accompanied with decreased cardiomyocyte proliferation. Also, a number of human CHDs-associated Nkx2.5 mutants exhibited aberrant sumoylation. Our work demonstrates that altered sumoylation status may underlie the development of human CHDs associated with Nkx2.5 mutants.
Highlights
Nkx2.5, a cardiac specific homeobox gene, is one of the earliest markers for cardiac progenitor cells and is central for normal fetal cardiac structural morphogenesis
As an initial step to investigate whether altered sumoylation status underpinned those Nkx2.5 mutants that were associated with congenital heart diseases (CHDs), sumoylation assay using Ni2+-NTA chromatography was performed on Hela cell lysates expressing His6-tagged SUMO-1 and Nkx2.5 wild type or one of seven point mutants created on murine Nkx2.5 protein, R25C, T177M, N187K, R188G, Y190C, C265Y and V309L (Fig. 1A)
Based on our previous findings that Nkx2.5 was a sumoylation substrate [18], in the present study we investigated the effects of CHDs-associated Nkx2.5 missense mutations on Nkx2.5 sumoylation and the impact of accumulated unsumoylatable Nkx2.5 mutant K51R on murine heart development and function
Summary
Nkx2.5, a cardiac specific homeobox gene, is one of the earliest markers for cardiac progenitor cells and is central for normal fetal cardiac structural morphogenesis. The critical role of Nkx2.5 in the conduction system was revealed by observations in both human patients with heterozygous Nkx2.5 mutants and in animal models [5,6]. Overexpressing wild type Nkx2.5 or one of its homeodomain (HD) mutants such as I183P in murine cardiomyocytes under the control of b-myosin heavy chain promoter resulted in embryonic lethality and/or heart failure, which was a consequence of dilated cardiomyopathy and/or a defective conduction system [7]. Transgene expression of the I183P mutant, driven by a-myosin heavy chain promoter (a-MHC), led to cardiac dysfunction, but with later onset [8] and with phenotypic discrepancy [8,9]. Postnatal cardiac specific ablation of Nkx2.5 by tamoxifeninducible Cre expression starting at 2 weeks of age led to a conduction defect and dilated cardiomyopathy [10]. Nkx2.5 has an essential role in the formation of cardiac structure and in the maintenance of cardiac function
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