Abstract
BackgroundThe absence of dystrophin has gave a massive impact on myotube development in Muscular Dystrophy pathogenesis. One of the conserved signaling pathways involved in skeletal muscle differentiation is the PI3K/Akt/mTOR pathway that plays a vital role in autophagy regulation. To further understand and establish targeted therapy in dystrophin-deficient myoblasts, protein expression profiling has been determined which provides information on perturbed autophagy modulation and activation.MethodsIn this study, a dystrophin-deficient myoblast cell line established from the skeletal muscle of a dystrophic (mdx) mouse was used as a model. The dfd13 (dystrophin-deficient) and C2C12 (non-dystrophic) myoblasts were cultured in low mitogen conditions for 10 days to induce differentiation. The cells were subjected to total protein extraction prior to Western blotting assay technique. Protein sub-fractionation has been conducted to determine protein localization. The live-cell analysis of autophagy assay was done using a flow cytometer.ResultsIn our culture system, the dfd13 myoblasts did not achieve terminal differentiation. PTEN expression was profoundly increased in dfd13 myoblasts throughout the differentiation day subsequently indicates perturbation of PI3K/Akt/mTOR regulation. In addition, rictor-mTORC2 was also found inactivated in this event. This occurrence has caused FoxO3 misregulation leads to higher activation of autophagy-related genes in dfd13 myoblasts. Autophagosome formation was increased as LC3B-I/II showed accumulation upon differentiation. However, the ratio of LC3B lipidation and autophagic flux were shown decreased which exhibited dystrophic features.ConclusionPerturbation of the PTEN-PI3K/Akt pathway triggers excessive autophagosome formation and subsequently reduced autophagic flux within dystrophin-deficient myoblasts where these findings are of importance to understand Duchenne Muscular Dystrophy (DMD) patients. We believe that some manipulation within its regulatory signaling reported in this study could help restore muscle homeostasis and attenuate disease progression.1FSGSuke3ozHkrGhS-2dsfVideo
Highlights
The absence of dystrophin has gave a massive impact on myotube development in Muscular Dystro‐ phy pathogenesis
Dystrophin‐deficient myoblasts do not achieve terminal differentiation In vitro differentiation of myoblasts can be induced in culture through the use of a low mitogen medium for a few days
Both C2C12 and dfd13 myoblasts were cultured in low mitogen medium for 10 days before determining their terminal differentiation capacity
Summary
The absence of dystrophin has gave a massive impact on myotube development in Muscular Dystro‐ phy pathogenesis. One of the conserved signaling pathways involved in skeletal muscle differentiation is the PI3K/ Akt/mTOR pathway that plays a vital role in autophagy regulation. Yazid and Hung‐Chih Cell Commun Signal (2021) 19:105 myoblasts from an mdx mouse (DMD model) affects transmembrane protein stability, as well as the proteinanchored cytoplasmic layer of the cell membrane changing signaling [3, 4]. One of the conserved signaling pathways for skeletal muscle differentiation is the PI3K/ Akt/mTOR pathway. The PTEN-PI3K/Akt/mTOR signaling pathway regulates cell proliferation, differentiation, autophagy, and apoptosis [5,6,7]. The elevation of PTEN expression and its activity has been shown to contribute to profound PI3K/Akt signaling pathway deregulation in dystrophin-deficient dog muscle [10]
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