Facioscapulohumeral muscular dystrophy (FSHD) is the third most common myopathy, affecting 1 in 20,000 individuals. FSHD affects mainly the skeletal muscles with an asymmetric pattern of weakness involving facial, shoulder girdle and humeral muscles. Linkage studies mapped the FSHD locus at the subtelomeric region on the long arm of chromosome 4, 4q35. D4Z4 is the polymorphic region in 4q causally associated with disease and is composed by an array of 3.3 kb tandemly repeated elements. In healthy subjects the number of D4Z4 repeats varies between 11 and 150, while most patients carry an allele with 10 or fewer D4Z4 repeats. Despite FSHD was classified as autosomal dominant Mendelian disease its molecular basis are not fully characterized. Initially is has been hypothesized that the D4Z4 contraction determines a position effect mechanism that lead to the dysregulation in the expression of genes proximal to D4Z4 and these transcriptional abnormalities cause disease.
Among the multiple candidate genes causing FSHD, FRG1 was the first one identified in close proximity of the D4Z4 array. FRG1 mRNA is ubiquitously expressed in healthy tissues and was found overexpressed in muscle biopsies from FSHD patients. Consistently, transgenic mice overexpressing FRG1 develop muscular dystrophy similar to human disease. However the implication of FRG1 in FSHD is controversial.
The biological function and role in disease of FRG1 protein is yet to be determined. At present it is known that FRG1 is a RNA binding protein, which might be involved in RNA biogenesis and in RNA splicing.
FRG1 transgenics present a very early phenotype characterized by reduced muscle size, reduced number of myonuclei, reduced size, expression of the embryonic isoform of MyHC and fiber atrophy that precede muscle wasting.
It is thus possible that the alteration of FRG1 levels exerts its effects during embryonic development, playing its pathogenic role at this stage. FRG1 expression has been observed in different tissues of human healthy embryos. Furthermore, preliminary studies in other model organisms suggest a very early effect of FRG1 overexpression on muscle structures.
On this basis, we considered that the study of the FRG1 function during early muscles development might shed new lights on the role of FRG1 in FSHD. To this purpose, we generated a conditional knockout for FRG1.
With tamoxifen injection in pregnant mice, we determined the whole-embryo ablation of FRG1, and analyzed embryos at different ages to evaluate the effect of FRG1 absence.
To investigate possible effects of FRG1 during myogenesis we analyzed the expression of myogenic factors Pax3, Pax7, MyoD, Myf5, Pax3, Myogenin at different stages of embryonic life (from 9.5 to 12.5 and 14.5 day post coitum). This analysis revealed that Pax3 and MyoD are upregulated suggesting that Frg1 might have a role in the correct progression of myogenesis.
We also investigated whether FRG1 might have a role in the muscle differentiation program. Notably, in FRG1 transgenics muscle atrophy appears early, affects fast skeletal muscle fibers and correlates with downregulation of the glycolytic pathway. In addition FRG1 upregulation is associated with downregulation of Nur77, which controls the glycolytic pathway, lack of Akt phosphorylation and downregulation of the β2-adrenergic receptor.
To investigate whether FRG1 have a primary role on these alterations, we analyzed the expression of Nur77, Akt and B2AR in FRG1-knockout embryos and found the downregulation of Nur77 and the upregulation of pAkt (S473). These data confirm that the expression of Nur77 and Akt S473 phosphorylation correlate with the expression level of FRG1.
FRG1 might interfere with the signaling upstream or the effects downstream the Akt phosphorylation.