Loss of Prox1 in striated muscle causes slow to fast skeletal muscle fiber conversion and dilated cardiomyopathy.

Correct regulation of troponin and myosin contractile protein gene isoforms is a critical determinant of cardiac and skeletal striated muscle development and function, with misexpression frequently associated with impaired contractility or disease. Here we reveal a novel requirement for Prospero-related homeobox factor 1 (Prox1) during mouse heart development in the direct transcriptional repression of the fast-twitch skeletal muscle genes troponin T3, troponin I2, and myosin light chain 1. A proportion of cardiac-specific Prox1 knockout mice survive beyond birth with hearts characterized by marked overexpression of fast-twitch genes and postnatal development of a fatal dilated cardiomyopathy. Through conditional knockout of Prox1 from skeletal muscle, we demonstrate a conserved requirement for Prox1 in the repression of troponin T3, troponin I2, and myosin light chain 1 between cardiac and slow-twitch skeletal muscle and establish Prox1 ablation as sufficient to cause a switch from a slow- to fast-twitch muscle phenotype. Our study identifies conserved roles for Prox1 between cardiac and skeletal muscle, specifically implicated in slow-twitch fiber-type specification, function, and cardiomyopathic disease.

Epistatic rescue of Nkx2.5 adult cardiac conduction disease phenotypes by prospero-related homeobox protein 1 and HDAC3.

RATIONALE: 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. METHODS AND 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.