The Optimized Quantum Dots Mediated Thermometry Reveals the Efficiency of Myosin Extracted from Muscle Mini Bundles

Background: The quantum dots (QD) has been investigated as thermometrical sensor in biological microenvironment and applied to measure the muscle efficiency with underlying mechanisms, i.e., a reduction in fluorescent intensity of QD reflects an increase in temperature caused by heat release during ATP hydrolysis, denoting the efficiency of the motor protein myosin. The aim of this study is to optimize the QD mediated thermometry for measuring the efficiency of freshly extracted myosin from muscle mini bundles rather than pre-purified myosin and test this approach in preparations with different myosin isoform.Methods: The protocol of myosin extraction used in the single muscle fiber in vitro motility assay was modified slightly for extracting myosin from the muscle mini bundles. Moreover, the quantitation of extracted myosin was calculated from the total extracted protein since the ratio of myosin to total protein was constant, performing through spectrophotometric measurement of UV absorbance at 280 nm. The change in fluorescence intensity of QD thermometry measurement of myosin ATPase enzymatic reaction was plotted over time, and the slope of the linear negative regression between time course and relatively decreased fluorescence intensity was used to reflect the efficiency of extracted myosin. Results: The optimized QD mediated thermometry is established for evaluating the efficiency of myosin extracted from muscle mini bundles. Moreover, myosin isoform specific differences in the myosin efficiency were observed in comparison between slow myosin and fast myosin, i.e., the low myosin has lower efficiency than fast myosin, evidenced by a higher heat release.Conclusions: The optimized QD mediated thermometric measure of myosin efficiency in muscle mini bundles provides a nanoscale approach to evaluate myosin function based on a minimal amount of muscle, which is essentially required in the muscle research.

Single-molecule analysis reveals that regulatory light chains fine-tune skeletal myosin II function

Myosin II is the main force-generating motor during muscle contraction. Myosin II exists as different isoforms that are involved in diverse physiological functions. One outstanding question is whether the myosin heavy chain (MHC) isoforms alone account for these distinct physiological properties. Unique sets of essential and regulatory light chains (RLCs) are known to assemble with specific MHCs, raising the intriguing possibility that light chains contribute to specialized myosin functions. Here, we asked whether different RLCs contribute to this functional diversification. To this end, we generated chimeric motors by reconstituting the MHC fast isoform (MyHC-IId) and slow isoform (MHC-I) with different light-chain variants. As a result of the RLC swapping, actin filament sliding velocity increased by ∼10-fold for the slow myosin and decreased by >3-fold for the fast myosin. Results from ensemble molecule solution kinetics and single-molecule optical trapping measurements provided in-depth insights into altered chemo-mechanical properties of the myosin motors that affect the sliding speed. Notably, we found that the mechanical output of both slow and fast myosins is sensitive to the RLC isoform. We therefore propose that RLCs are crucial for fine-tuning the myosin function.

Single molecule analysis reveals the role of regulatory light chains in fine-tuning skeletal myosin-II function

Myosin II is the main force generating motor during muscle contraction. Myosin II exists as different isoforms, involved in diverse physiological functions. The outstanding question is whether the myosin heavy chain (MHC) isoforms alone account for the distinct physiological properties. Unique sets of essential and regulatory light chains (RLCs) assembled with specific MHCs raises an interesting possibility of specialization of myosin functions via light chains (LCs). Here, we ask whether different RLCs contribute to the functional diversification. To investigate this, we generated chimeric motors by reconstituting MHC fast isoform (MyHC-IId) and slow isoform (MHC-I) with different light chain variants. As a result of RLCs swapping, actin filament sliding velocity increased by ∼ 10 fold for the slow myosin and decreased by >3 fold for the fast myosin. Ensemble molecule solution kinetics and single molecule optical trapping measurements provided in-depth insights into altered chemo mechanical properties of the myosin motors, thereby affecting the sliding speed. We find that both slow and fast myosins mechanical output is sensitive to the RLC isoform and propose that RLCs are crucial in fine-tuning of the myosin function.

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.

Beef tenderness and intramuscular fat proteomic biomarkers: muscle type effect

Tenderness and intramuscular fat content are key attributes for beef sensory qualities. Recently some proteomic analysis revealed several proteins which are considered as good biomarkers of these quality traits. This study focuses on the analysis of 20 of these proteins representative of several biological functions: muscle structure and ultrastructure, muscle energetic metabolism, cellular stress and apoptosis. The relative abundance of the proteins was measured by Reverse Phase Protein Array (RPPA) in five muscles known to have different tenderness and intramuscular lipid contents: Longissimus thoracis (LT), Semimembranosus (SM), Rectus abdominis (RA), Triceps brachii (TB) and Semitendinosus (ST). The main results showed a muscle type effect on 16 among the 20 analyzed proteins. They revealed differences in protein abundance depending on the contractile and metabolic properties of the muscles. The RA muscle was the most different by 11 proteins differentially abundant comparatively to the four other muscles. Among these 11 proteins, six were less abundant namely enolase 3 (ENO3), phosphoglucomutase 1 (PGK1), aldolase (ALDOA), myosin heavy chain IIX (MyHC-IIX), fast myosin light chain 1 (MLC1F), triosephosphate isomerase 1 (TPI1) and five more abundant: Heat shock protein (HSP27, HSP70-1A1, αB-crystallin (CRYAB), troponin T slow (TNNT1), and aldolase dehydrogenase 1 (ALDH1A1). Four proteins: HSP40, four and a half LIM domains protein 1 (FHL1), glycogen phosphorylase B (PYGB) and malate dehydrogenase (MDH1) showed the same abundance whatever the muscle. The correlations observed between the 20 proteins in all the five muscles were used to construct a correlation network. The proteins the most connected with the others were in the following order MyHC-IIX, CRYAB, TPI1, PGK1, ALDH1A1, HSP27 and TNNT1. This knowledge is important for understanding the biological functions related to beef tenderness and intramuscular fat content.

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.