Damaged muscle fibers might masquerade as hybrid fibers – a cautionary note on immunophenotyping mouse muscle with mouse monoclonal antibodies

We report that, labeling mouse muscle tissue, with mouse monoclonal antibodies specific to slow or fast myosin heavy chain (sMyHC and fMyHC, respectively), can lead to artefactual labeling of damaged muscle fibers, as hybrid fibers (sMyHC+ and fMyHC+). We demonstrate that such erroneous immunophenotyping of muscle may be avoided, by performing colabeling or serial-section-labeling, to identify damaged fibers. The quadriceps femoris muscle group (QF) in 7-month-old, male, C57BL/6J mice had: 1.21 ± 0.21%, 98.34 ± 1.06%, 0.07 ± 0.01%, and 0.53 ± 0.85% fibers, that were, sMyHC+, fMyHC+, hybrid, and damaged, respectively. All fibers in the tibialis anterior muscle (TA) of 3-month-old, male, C57BL/6J mice were fMyHC+; and at 3 days after injurious eccentric contractions, there was no fiber-type shift, but ~ 18% fibers were damaged.

Perturbation of PTEN-PI3K/AKT Signalling Impaired Autophagy Modulation in Dystrophin-Deficient Myoblasts

Alteration of single protein regulation has given a massive implication in Muscular Dystrophy pathogenesis. Herein, we investigated the contribution of defected dystrophin that has impaired PI3K/Akt signalling and subsequently reduced autophagy in dystrophin-deficient myoblasts. In this study, dfd13 (dystrophin-deficient) and C2C12 (non-dystrophic) myoblasts were cultured in low mitogen condition for 10 days to induce differentiation. Analyses of protein expression has been done by using immunoblot technique, immunofluorescence and flow cytometry. In our myoblasts differentiation system, the dfd13 myoblasts did not achieved terminal differentiation as fewer myotube formation and fast-myosin heavy chain expression almost not detected. Immunoblot analysis showed that PTEN expression is profoundly increased in dfd13 myoblasts throughout the differentiation day. As a result, the PI3K activity is decreased and has caused serine/threonine kinase Akt inactivation. Both residues; Thr308 and Ser473, on Akt were found not phosphorylated. The mTOR activation by Ser2448 phosphorylation was decreased indicates an impairment for raptor and rictor binding. Unable to form complexes; mTORC1 target protein, p70S6K1 activation was found reduced at the same time explained un-phosphorylated-Akt at Ser473 by rictor-mTORC2. As one of Akt downstream protein, transcription factor FoxO3 regulation was found impaired as it was highly expressed and highly mainly localised in the nucleus in dfd13 towards the end of the differentiation day. This occurrence has caused higher activation of autophagy related genes; Beclin1, Atg5, Atg7, in dfd13 myoblasts. Autophagosome formation was increased as LC3B-I/II showed accumulation upon differentiation. However, ratio of LC3B lipidation and autophagic flux were shown decreased which exhibited dystrophic features. As a conclusion, destabilisation of plasma membrane owing to dystrophin mutation has caused the alteration of plasma membrane protein regulation particularly PTEN-PI3K, thus impaired autophagy modulation that critical for myoblasts development.

From Slow to Fast: Hypogravity-Induced Remodeling of Muscle Fiber Myosin Phenotype

Skeletal muscle consists of different fiber types arranged in a mosaic pattern. These fiber types are characterized by specific functional properties. Slow-type fibers demonstrate a high level of fatigue resistance and prolonged contraction duration, but decreased maximum contraction force and velocity. Fast-type fibers demonstrate a high contraction force and velocity, but profound fatigability. During the last decades, it has been discovered that all these properties are determined by the predominance of slow or fast myosin-heavy-chain (MyHC) isoforms. It was observed that gravitational unloading during space missions and simulated microgravity in ground-based experiments leads to the transformation of some slow-twitch muscle fibers into fast-twitch ones due to changes in the patterns of MyHC gene expression in the postural soleus muscle. The present review covers the facts and mechanistic speculations regarding myosin phenotype remodeling under conditions of gravitational unloading. The review considers the neuronal mechanisms of muscle fiber control and molecular mechanisms of regulation of myosin gene expression, such as inhibition of the calcineurin/NFATc1 signaling pathway, epigenomic changes, and the behavior of specific microRNAs. In the final portion of the review, we discuss the adaptive role of myosin phenotype transformations.