Effect of frizzled related protein (FRZB) on muscles: an inverse relationship between FRZB and calpain-3 with potential impact on muscle dystrophy and osteoarthritis

Purpose: To better understand the role of FRZB in muscles and identify novel mechanisms of disease that could be relevant to either osteoarthritis or muscular dystrophy. FRZB is an extracellular modulator of the Wnt signalling pathway and protects against the development of osteoarthritis in mice. The role of FRZB in cartilage and bone homeostasis has been documented. Frzb-/- mice also have reduced voluntary running performance compared to wildtype animals but a potential role for FRZB in muscle biology remains unknown. In the muscles of patients with limb girdle muscular dystrophy 2A (LGMD2A), a recessive disorder associated with loss of function mutations in the Calpain 3 gene, FRZB gene is strongly upregulated. Methods: Expression and protein levels of genes known to be involved in LGMD2A were analysed in dissected soleus, tibialis anterior and quadriceps muscles from wildtype and Frzb-/- mice. Quantification of gene expression and protein levels were carried out using BioRad SYBR 384 custom plate or quantitative PCR analysis (Taqman probes) and Western blot respectively. Cell culture: Satellite cells from these muscles were obtained by enzymatical digestion, quantified and characterized. Mesoangioblasts from wildtype and Frzb-/- mice were selected as alkaline phosphatase positive cells by FACS sorting, quantified and characterized, including their adipogenic differentiation potential by Oil Red O staining. In vivo muscle regeneration and gait analysis: Muscle injury was induced by cardiotoxin injection to evaluate the regenerative capacity of muscles isolated from wildtype and Frzb-/- mice. Gait analysis was performed using an automated Catwalk system. Chronic exercise protocol: wildtype and Frzb-/- mice were subjected to a 5-week exercise protocol on a treadmill, and muscles were analysed at morphological, RNA and protein levels by immunohistological stainings, qRT-PCR and Western blot. Results: Body size and muscle weight were slightly reduced in Frzb-/- mice compared to wildtypes. Gene analysis showed upregulation of Capn3, the mutant gene in LGDM2A, as well as of Rora, MyoD and Myog in Frzb-/- mice (downregulated in LGMD2A). Igf1, Tfrc, Slc16a1, Fn1 (upregulated in LGMD2A) and Ky genes were downregulated in Frzb-/- muscles compared to wildtype. Cell culture: Frzb-/- mice showed more AP+ cells but with lower expression of plateled derived growth factor receptor A (PDGFRA) and reduced adipogenic potential. On the other hand, satellite cells of Frzb-/- mice showed higher number of MyoD and Ki67 positive myonuclei as well as a higher proliferation rate. In vivo muscle regeneration and gait analysis: Muscle regeneration capacity after cardiotoxin injection was similar in WT and Frzb-/- mice. Gait analysis showed that Frzb-/- mice required more time for stand, swing and step cycle. The base of support distance between the hind paws was shorter in Frzb-/- mice and these mice used more different stepping patterns. Chronic exercise protocol: No differences were observed between WT and Frzb-/- mice regarding exercise. Conclusions: The inversed relationship between upregulation of Capn3 gene in Frzb-/- mice and the previously described upregulation of FRZB in LGMD2A patients, suggest that CAPN3 and FRZB genes control each other’s expression. The fact that muscle regeneration is not affected in Frzb-/- mice and the observation that muscle maturation in LGMD2A patients is impaired, suggest that inhibition of FRZB could be a therapeutic target in muscular dystrophies. This is further supported by the suggestion that in Frzb-/- mice, the transition from preadipocyte to differentiated adipocyte may be inhibited as less adipocytes are produced, contrary to LGMD2A patients, where adipocyte differentiation and fat are increased in muscle. The gene expression data and functional analysis including gait evaluation suggest that low FRZB levels may also affect muscle function and provide an additional mechanism by which FRZB can influence onset and severity of osteoarthritis.