Failure of Prox1-conditional cardiomyocytes to grow and to maintain sarcomere organisation throughout development results in hypotrophic hearts that are insufficient to sustain life beyond birth. We observed disruption of the sarcomere at the level of both gene expression and protein localisation and have identified Prox1-bound enhancer elements for key sarcomere-associated genes, indicating that Prox1 regulates myofibrillar organisation both directly in terms of protein expression, and indirectly via intermediate factors which control sarcomere protein localisation and integration.
Prox1 deficiency impacts directly on sarcomeric components that facilitate Z-disc and thin filament interaction. Reduced expression of both Nrap
-conditional hearts, and the demonstration that these two genes are direct transcriptional targets of Prox1, is highly significant in terms of maintaining Z-disc stability. N-RAP has been proposed to act as a catalytic scaffold for the association of thin filament actin and Z-disc α-actinin during myofibrillogenesis (Dhume et al., 2006
), and Zyxin also interacts with α-actinin to facilitate actin assembly and organization (Crawford et al., 1992
; Frank et al., 2006
). During myofibrillogenesis, N-RAP and Zyxin are associated with cell-cell contacts that make up the developing intercalated discs, which ultimately mature during post-natal stages (Perriard et al., 2003
). Our confocal and transmission electron microscopy (TEM) observations that cell junctions (β-catenin-positive adherens-type junctions and desmosomes) are appropriately established and remain intact, between neighbouring cardiomyocytes in Prox1
-conditional mutants, suggests that the role of Prox1 is primarily to regulate Nrap
to facilitate cross-linking between actin and α-actinin in the Z-disc as one of the fundamental associations of the sarcomere (Fig. S4A
is also implicated in this study as a direct target of Prox1 and the modest, yet significant, reduction in the expression levels of Actn2
in a Prox1
mutant background, correlating with the Z-disc disruption, underlines the critical role of α-actinin in maintaining sarcomere integrity. Moreover, since Zyxin
-null mice are viable (Hoffman et al., 2003
), although the hearts have not been examined in detail for histological defects, the phenotype we describe represents a cumulative effect on the actin-α-actinin interaction: directly via α-actinin expression and localization and through interactions with co-factors, N-RAP and Zyxin.
Perturbation of the actin-α-actinin association, directly explains the Z-disc anomalies in Prox1
-null hearts. Whilst we cannot entirely exclude additional effects of loss of Prox1 function, normal levels of cardiomyocyte proliferation and apoptosis and the specificity of phenotype at the level of sarcomeric maintenance suggests that the latter is the primary defect in Prox1
-conditional mutants. Moreover, thin filament-Z-disc disruption will feedback directly onto the thin and thick filament arrangement of the sarcomere as observed at the level of TEM, resulting in a more global disorganization of myofibrils (Fig. S4B
). The latter may also explain the observed M-band defects, but equally these may relate more directly to mis-regulation of Nrap
in the Prox1
-mutant background, since N-RAP associates with the M-bands of maturing myofibrils where it acts as a catalytic scaffold (Lu et al., 2005).
Prox1 is not required for the initial stages of myofibrillogenesis since the phenotypic defects do not begin to manifest until E12.5; despite Prox1 expression in the myocardium of the heart at earlier stages (E10.5) of development and potential Cre-mediated knockdown of Prox1
occuring from E7.5 onwards. Sarcomere proteins are expressed immediately prior to the onset of beating and, once integrated into mature myofibrils, tend to have a relatively long half-life that varies between 3 and 10 days (Martin, 1981
). Moreover, between E8.25 and E10.5 the developing heart increases in mass primarily through addition of cells from the second cardiac lineage (Zaffran et al., 2004
). Therefore, there may be little requirement for newly synthesised structural proteins during these early stages of heart development. The initial activation of sarcomeric proteins is clearly carried out by alternate and as yet unidentified, transcriptional pathways with the role of Prox1 confined to regulating sarcomere maintenance and stability from E10.5 onwards, when all populations of cardiac cells have been acquired and the developing heart grows by a combination of both cardiomyocyte hyperplasia and hypertrophy. The fact that we observed significantly impaired hypertrophic growth following loss of Prox1
is secondary to the primary defect of disrupted assembly of sarcomere proteins. Developing cardiomyocytes elongate in a unidirectional manner by addition of sarcomeres to the existing myofibrils, the timing of which corresponds precisely with the onset of myocardial disruption and failure of the cells to elongate in Prox1
-conditional myocardium (refer to model in ).
In conclusion, Prox1 is essential for maintenance and maturation of the sarcomere in developing cardiomyocytes, which in turn is critical for hypertrophic growth and maturation of the embryonic myocardium. The identification of structural proteins, α-actinin, N-RAP and Zyxin, as direct targets of Prox1 suggests that mis-regulation of critical sarcomere components and their interacting protein partners is the primary cause of myofibril disruption in Prox1
-conditional myocardium. A number of other studies have described roles for transcription factors in initiating or maintaining cardiac muscle ultrastructure, during development and disease, most notably SRF (Balza, Jr. and Misra, 2006
; Nelson et al., 2005
) GATA4, Nkx2.5 and MEF2 (Akazawa and Komuro, 2003
) and Calcineurin/N-FAT (Bourajjaj et al., 2008
; Heineke and Molkentin, 2006
), however, to the best of our knowledge no study to-date has demonstrated direct transcriptional regulation of structural proteins in vivo.
Aberrant terminal differentiation and improper assembly of contractile protein filaments are associated with a number of cardiac myopathies (Engel, 1999
; Gregorio and Antin, 2000
; Seidman and Seidman, 2001
). Many of these disorders are caused by mutations in components of the myofibrillar apparatus itself including β-MHC, troponins T and I, titin and α-tropomyosin (Alcalai et al., 2008
; Chang and Potter, 2005
) or perturbations in the associated calcium-dependent signalling pathways (Frey et al., 2004
; Molkentin et al., 1998
). However, a large proportion of cardiomyopathy remains unexplained with no mutations found in sarcomere or sarcomere-related proteins. Our study not only provides novel insight into transcriptional regulation of cardiomyocyte ultrastructure and hypertrophy during development, but implicates Prox1 as a critical regulatory factor which may underlie the pathology of both inherited and acquired myopathic disease.