Epistatic rescue of Nkx2.5 adult cardiac conduction disease phenotypes by prospero-related homeobox protein 1 and HDAC3.
BACKGROUND
Nkx2.5 is one of the most widely studied cardiac-specific transcription factors, conserved from flies to man, with multiple essential roles in both the developing and adult heart. Specific dominant mutations in NKX2.5 have been identified in adult congenital heart disease patients presenting with conduction system anomalies and recent genome-wide association studies implicate the NKX2.5 locus, as causative for lethal arrhythmias ("sudden cardiac death") that occur at a frequency in the population of 1 in 1000 per annum worldwide. Haploinsufficiency for Nkx2.5 in the mouse phenocopies human conduction disease pathology yet the phenotypes, described in both mouse and man, are highly pleiotropic, implicit of unknown modifiers and/or factors acting in epistasis with Nkx2.5/NKX2.5.


OBJECTIVE
To identify bone fide upstream genetic modifier(s) of Nkx2.5/NKX2.5 function and to determine epistatic effects relevant to the manifestation of NKX2.5-dependent adult congenital heart disease.


RESULTS
A study of cardiac function in prospero-related homeobox protein 1 (Prox1) heterozygous mice, using pressure-volume loop and micromannometry, revealed rescue of hemodynamic parameters in Nkx2.5(Cre/+); Prox1(loxP/+) animals versus Nkx2.5(Cre/+) controls. Anatomic studies, on a Cx40(EGFP) background, revealed Cre-mediated knock-down of Prox1 restored the anatomy of the atrioventricular node and His-Purkinje network both of which were severely hypoplastic in Nkx2.5(Cre/+) littermates. Steady state surface electrocardiography recordings and high-speed multiphoton imaging, to assess Ca(2+) handling, revealed atrioventricular conduction and excitation-contraction were also normalized by Prox1 haploinsufficiency, as was expression of conduction genes thought to act downstream of Nkx2.5. Chromatin immunoprecipitation on adult hearts, in combination with both gain and loss-of-function reporter assays in vitro, revealed that Prox1 recruits the corepressor HDAC3 to directly repress Nkx2.5 via a proximal upstream enhancer as a mechanism for regulating Nkx2.5 function in adult cardiac conduction.


CONCLUSIONS
Here we identify Prox1 as a direct upstream modifier of Nkx2.5 in the maintenance of the adult conduction system and rescue of Nkx2.5 conduction disease phenotypes. This study is the first example of rescue of Nkx2.5 function and establishes a model for ensuring electrophysiological function within the adult heart alongside insight into a novel Prox1-HDAC3-Nkx2.5 signaling pathway for therapeutic targeting in conduction disease.
Institute of Child Health, University College London, UK.
Catherine A
CA
Risebro
Louisa K
LK
Petchey
Nicola
N
Smart
John
J
Gomes
James
J
Clark
Joaquim M
JM
Vieira
Joseph
J
Yanni
Halina
H
Dobrzynski
Sean
S
Davidson
Zia
Z
Zuberi
Andrew
A
Tinker
Bo
B
Shui
Yvonne I
YI
Tallini
Michael I
MI
Kotlikoff
Lucile
L
Miquerol
Robert J
RJ
Schwartz
Paul R
PR
Riley
D000818:Animals; D004843:Epistasis, Genetic / Q000235:genetics*; D006329:Heart Conduction System / Q000503:physiopathology*; D006331:Heart Diseases / Q000235:genetics* / Q000378:metabolism* / Q000503:physiopathology; D006655:Histone Deacetylases / Q000235:genetics* / Q000502:physiology; D000072576:Homeobox Protein Nkx-2.5; D018398:Homeodomain Proteins / Q000235:genetics* / Q000502:physiology; D051379:Mice; D008822:Mice, Transgenic; D041681:NIH 3T3 Cells; D010641:Phenotype; D014157:Transcription Factors / Q000235:genetics* / Q000502:physiology; D025521:Tumor Suppressor Proteins / Q000235:genetics* / Q000502:physiology; D000098551:Prospero-Related Homeobox 1 Protein; D000099186:Histone Deacetylase 3
D006655:Histone Deacetylases; D000072576:Homeobox Protein Nkx-2.5; D018398:Homeodomain Proteins; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; D000098551:Prospero-Related Homeobox 1 Protein; D000099186:Histone Deacetylase 3; C484469:Nkx2-5 protein, mouse