ABSTRACT
BACKGROUND: Limb-girdle muscular dystrophy R1 calpain 3-related (LGMDR1) is an autosomal recessive muscular dystrophy due to mutations in the CAPN3 gene. While the pathophysiology of this disease has not been clearly established yet, Wnt and mTOR signaling pathways impairment in LGMDR1 muscles has been reported. RESULTS: A reduction in Akt phosphorylation ratio and upregulated expression of proteins implicated in glycolysis (HK-II) and in fructose and lactate transport (GLUT5 and MCT1) in LGMDR1 muscle was observed. In vitro analysis to establish mitochondrial and glycolytic functions of primary cultures were performed, however, no differences between control and patients were observed. Additionally, gene expression analysis showed a lack of correlation between primary myoblasts/myotubes and LGMDR1 muscle while skin fibroblasts and CD56- cells showed a slightly better correlation with muscle. FRZB gene was upregulated in all the analyzed cell types (except in myoblasts). CONCLUSIONS: Proteins implicated in metabolism are deregulated in LGMDR1 patients' muscle. Obtained results evidence the limited usefulness of primary myoblasts/myotubes for LGMDR1 gene expression and metabolic studies. However, since FRZB is the only gene that showed upregulation in all the analyzed cell types it is suggested its role as a key regulator of the pathophysiology of the LGMDR1 muscle fiber. The Wnt signaling pathway inactivation, secondary to FRZB upregulation, and GLUT5 overexpression may participate in the impaired adipogenesis in LGMD1R patients.
Subject(s)
Muscle Proteins , Muscular Dystrophies, Limb-Girdle , Humans , Muscle Proteins/genetics , Muscular Dystrophies, Limb-Girdle/genetics , Muscular Dystrophies, Limb-Girdle/metabolism , Muscle Fibers, Skeletal/metabolism , Wnt Signaling Pathway , Cell Culture Techniques , Muscle, Skeletal/metabolismABSTRACT
Limb-girdle muscular dystrophy R1 calpain 3-related (LGMDR1) is an autosomal recessive muscular dystrophy produced by mutations in the CAPN3 gene. It is a rare disease and there is no cure or treatment for the disease while the pathophysiological mechanism by which the absence of calpain 3 provokes the dystrophy in muscles is not clear. However, key proteins implicated in Wnt and mTOR signaling pathways, which regulate muscle homeostasis, showed a considerable reduction in their expression and in their phosphorylation in LGMDR1 patients' muscles. Finally, the administration of tideglusib and VP0.7, ATP non-competitive inhibitors of glycogen synthase kinase 3ß (GSK-3ß), restore the expression and phosphorylation of these proteins in LGMDR1 cells, opening the possibility of their use as therapeutic options.
Subject(s)
Glycogen Synthase Kinase 3 beta/antagonists & inhibitors , Muscular Dystrophies, Limb-Girdle/drug therapy , Nerve Tissue Proteins/antagonists & inhibitors , Signal Transduction/drug effects , Adenosine Triphosphate/metabolism , Allosteric Site/drug effects , CD56 Antigen/analysis , Calpain/deficiency , Calpain/genetics , Fibroblasts/drug effects , Fibroblasts/metabolism , Gene Expression Regulation/drug effects , Glycogen Synthase Kinase 3 beta/chemistry , Humans , Hydrazines/pharmacology , Hydrazines/therapeutic use , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Muscle Fibers, Skeletal/drug effects , Muscle Fibers, Skeletal/metabolism , Muscle Proteins/deficiency , Muscle Proteins/genetics , Muscular Dystrophies, Limb-Girdle/enzymology , Nerve Tissue Proteins/chemistry , Phosphorylation , Protein Processing, Post-Translational/drug effects , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , Proto-Oncogene Proteins c-akt/physiology , Quinolones/pharmacology , Quinolones/therapeutic use , TOR Serine-Threonine Kinases/antagonists & inhibitors , TOR Serine-Threonine Kinases/physiology , Thiadiazoles/pharmacology , Thiadiazoles/therapeutic use , Wnt Signaling Pathway/drug effectsABSTRACT
BACKGROUND: Limb-girdle muscular dystrophy recessive 1 calpain3-related (LGMDR1), previously known as LGMD2A, is a disease caused by mutations in the CAPN3 gene. It is characterized by progressive weakness and muscle degeneration. Frizzled related protein (FRZB), upregulated in LGMDR1, was identified as a key regulator of the crosstalk between Wnt and integrin signalling pathways. FRZB gene silencing showed a recovery in the expression of some of the costamere protein levels in myotubes. RESULTS: Here, we performed a comprehensive characterization of Frzb-/- mice muscles to study the absence of Frzb in skeletal muscle and eventual links with the molecular characteristics of LGMDR1 patient muscles. Frzb-/- mice showed reduced muscle size and strength. Gait analysis showed that Frzb-/- mice moved more slowly but no impaired regeneration capacity was observed after muscle injury. Additionally, Frzb-/- mice muscle showed an increased number of mesoangioblasts. Lack of Frzb gene in Frzb-/- mice and its increased expression in LGMDR1 patients, showed contrary regulation of Rora, Slc16a1, Tfrc and Capn3 genes. The reciprocal regulation of Frzb and Capn3 genes further supports this axis as a potential target for LGMDR1 patients. CONCLUSIONS: Our data confirm a role for Frzb in the regulation of Rora, Slc16a1, Tfrc, and Capn3 genes in muscle cells. In vivo, reduced muscle strength and gait in the Frzb-/- mice are intriguing features. The reciprocal relationship between FRZB and CAPN3 further supports a key role for this axis in patients with LGMDR1.