Garcinol Promotes the Formation of Slow-Twitch Muscle Fibers by Inhibiting p300-Dependent Acetylation of PGC-1α

Background The conversion of skeletal muscle fiber from fast twitch to slow-twitch is crucial for sustained contractile and stretchable events, energy homeostasis, and anti-fatigue ability. The purpose of our study was to explore the mechanism and effects of garcinol on the regulation of skeletal muscle fiber type transformation. Methods Forty 21-day-old male C57/BL6J mice (n = 10/diet) were fed a control diet or a control diet plus garcinol at 100 mg/kg (Low Gar), 300 mg/kg (Mid Gar), or 500 mg/kg (High Gar) for 12 weeks. The tibialis anterior (TA) and soleus muscles were collected for protein and immunoprecipitation analyses. Results Dietary garcinol significantly downregulated (P<0.05) fast MyHC expression and upregulated (P<0.05) slow MyHC expression in the TA and soleus muscles. Garcinol significantly increased (P<0.05) the activity of PGC-1α and markedly decreased (P<0.05) the acetylation of PGC-1α. In vitro and in vivo experiments showed that garcinol decreased (P<0.05) lactate dehydrogenase activity and increased (P<0.05) the activities of malate dehydrogenase and succinic dehydrogenase. In addition, the results of immunostaining C2C12 myotubes showed that garcinol treatment increased (P<0.05) the transformation of glycolytic muscle fiber to oxidative muscle fiber by 45.9%. Garcinol treatment and p300 interference reduced (P<0.05) the expression of fast MyHC but increased (P<0.05) the expression of slow MyHC in vitro. Moreover, the acetylation of PGC-1α was significantly decreased (P<0.05). Conclusion Garcinol promotes the transformation of skeletal muscle fibers from the fast-glycolytic type to the slow-oxidative type through the p300/PGC-1α signaling pathway in C2C12 myotubes.

The Effect of Mechanical Stretch on Myotube Growth Suppression by Colon-26 Tumor-Derived Factors

Preclinical models and in vitro experiments have provided valuable insight into the regulation of cancer-induced muscle wasting. Colon-26 (C26) tumor cells induce cachexia in mice, and conditioned media (CM) from these cells promotes myotube atrophy and catabolic signaling. While mechanical stimuli can prevent some effects of tumor-derived factors on myotubes, the impact of mechanical signaling on tumor-derived factor regulation of myosin heavy chain (MyHC) expression is not well understood. Therefore, we examined the effects of stretch-induced mechanical signaling on C2C12 myotube growth and MyHC expression after C26 CM exposure. C26 CM was administered to myotubes on day 5 of differentiation for 48 h. During the last 4 or 24 h of C26 CM exposure, 5% static uniaxial stretch was administered. C26 CM suppressed myotube growth and MyHC protein and mRNA expression. Stretch for 24 h increased myotube size and prevented the C26 CM suppression of MyHC-Fast protein expression. Stretch did not change suppressed MyHC mRNA expression. Stretch for 24 h reduced Atrogin-1/MAFbx, MuRF-1, and LC3B II/I ratio and increased integrin β1D protein expression and the myogenin-to-MyoD protein ratio. Stretch in the last 4 h of CM increased ERK1/2 phosphorylation but did not alter the CM induction of STAT3 or p38 phosphorylation. These results provide evidence that in myotubes pre-incubated with CM, the induction of mechanical signaling can still provide a growth stimulus and preserve MyHC-Fast protein expression independent of changes in mRNA expression.

The long noncoding RNA MyHC IIA/X-AS contributes to skeletal muscle myogenesis and maintains the fast fiber phenotype

Mammalian skeletal muscles comprise different types of muscle fibers, and this muscle fiber heterogeneity is generally characterized by the expression of myosin heavy chain (MyHC) isoforms. A switch in MyHC expression leads to muscle fiber–type transition under various physiological and pathological conditions, but the underlying regulator coordinating the switch of MyHC expression remains largely unknown. Experiments reported in this study revealed the presence of a skeletal muscle–specific antisense transcript generated from the intergenic region between porcine MyHC IIa and IIx and is referred to here as MyHC IIA/X-AS. We found that MyHC IIA/X-AS is identified as a long noncoding RNA (lncRNA) that is strictly expressed in skeletal muscles and is predominantly distributed in the cytoplasm. Genetic analysis disclosed that MyHC IIA/X-AS stimulates cell cycle exit of skeletal satellite cells and their fusion into myotubes. Moreover, we observed that MyHC IIA/X-AS is more enriched in fast-twitch muscle and represses slow-type gene expression and thereby maintains the fast phenotype. Furthermore, we found that MyHC IIA/X-AS acts as a competing endogenous RNA that sponges microRNA-130b (miR-130b) and thereby maintains MyHC IIx expression and the fast fiber type. We also noted that miR-130b was proved to down-regulate MyHC IIx by directly targeting its 3′-UTR. Together, the results of our study uncovered a novel pathway, which revealed that lncRNA derived from the skeletal MyHC cluster could modulate local MyHC expression in trans, highlighting the role of lncRNAs in muscle fiber–type switching.

Altered expression of pectoral myosin heavy chain isoforms corresponds to migration status in the white-crowned sparrow ( Zonotrichia leucophrys gambelii )

Birds undergo numerous changes as they progress through life-history stages, yet relatively few studies have examined how birds adapt to both the dynamic energetic and mechanical demands associated with such transitions. Myosin heavy chain (MyHC) expression, often linked with muscle fibre type, is strongly correlated with a muscle's mechanical power-generating capability, thus we examined several morphological properties, including MyHC expression of the pectoralis, in a long-distance migrant, the white-crowned sparrow ( Zonotrichia leucophrys gambelii ) throughout the progression from winter, spring departure and arrival on breeding grounds. White-crowned sparrows demonstrated significant phenotypic flexibility throughout the seasonal transition, including changes in prealternate moult status, lipid fuelling, body condition and flight muscle morphology. Pectoral MyHC expression also varied significantly over the course of the study. Wintering birds expressed a single, newly classified adult fast 2 isoform. At spring departure, pectoral isoform expression included two MyHC isoforms: the adult fast 2 isoform along with a smaller proportion of a newly present adult fast 1 isoform. By spring arrival, both adult fast isoforms present at departure remained, yet expression had shifted to a greater relative proportion of the adult fast 1 isoform. Altering pectoral MyHC isoform expression in preparation for and during spring migration may represent an adaptation to modulate muscle mechanical output to support long-distance flight.

Myosin Heavy Chain Expression Can Vary over the Length of Jaw and Leg Muscles

Muscle fiber type classification can be determined by its myosin heavy chain (MyHC) composition based on a few consecutive sections. It is generally assumed that the MyHC expression of a muscle fiber is the same over its length since neural stimulation and systemic influences are supposed to be the same over its length. We analyzed this in detail in three muscle types: the temporalis (closer) and digastricus (opener; both first brachial arch), and the medial gastrocnemius (somite). Sections of the muscles were incubated with monoclonal antibodies against various MyHC isoforms, and the distribution of these isoforms within individual fibers was followed over a distance of approximately 1 mm. The staining intensity of a fiber was measured and compared with the other fibers in the section. In the temporalis, digastricus, and gastrocnemius, 46, 11, and 15%, respectively, of their MyHC-I fibers showed a variation in the staining intensity over the length of their fibers, as well as 47, 87, and 22%, respectively, of their MyHC-IIA fibers. Most variable fibers were found amongst those with an overall relative intermediate staining intensity, which are presumably hybrid fibers. We conclude that different parts of a muscle fiber can have different fiber type compositions and, thus, contractile properties. Some muscle parts might reach their maximum contraction peak sooner or later than a muscle part a few microns further away. Next to stimulation by the nerve and systemic influences, local influences might also have an impact on the MyHC expression of the fiber.

Silencing of Mustn1 inhibits myogenic fusion and differentiation

Mustn1 (Mustang, musculoskeletal temporally activated novel gene) was originally identified in fracture callus tissue, but its greatest expression is detected in skeletal muscle. Thus, we conducted experiments to investigate the expression and function of Mustn1 during myogenesis. Temporally, quantitative real-time PCR analysis of muscle samples from embryonic day 17 to 12 mo of age reveals that Mustn1 mRNA expression is greatest at 3 mo of age and beyond, consistent with the expression pattern of Myod. In situ hybridization shows abundant Mustn1 expression in somites and developing skeletal muscles, while in adult muscle, Mustn1 is localized to some peripherally located nuclei. Using RNA interference (RNAi), we investigated the function of Mustn1 in C2C12 myoblasts. Though silencing Mustn1 mRNA had no effect on myoblast proliferation, it did significantly impair myoblast differentiation, preventing myofusion. Specifically, when placed in low-serum medium for up to 6 days, Mustn1-silenced myoblasts elongated poorly and were mononucleated. In contrast, control RNAi-treated and parental myoblasts presented as large, multinucleated myotubes. Further supporting the morphological observations, immunocytochemistry of Mustn1-silenced cells demonstrated significant reductions in myogenin (Myog) and myosin heavy chain (Myhc) expression at 4 and 6 days of differentiation as compared with control and parental cells. The decreases in Myog and Myhc protein expression in Mustn1-silenced cells were associated with robust (∼3-fold or greater) decreases in the expression of Myod and desmin ( Des), as well as the myofusion markers calpain 1 ( Capn1), caveolin 3 ( Cav3), and cadherin 15 (M-cadherin; Cadh15). Overall, we demonstrate that Mustn1 is an essential regulator of myogenic differentiation and myofusion, and our findings implicate Myod and Myog as its downstream targets.

R429 – Irradiation Changes MyHC Expression in Rat Larynx

Problem Laryngeal cancer treatment involves radiotherapy, laser treatment, and/or surgery. Radiotherapy does not impair voice quality as much as laser treatment or surgery but can induce muscle wasting, fibrosis, and dry mouth symptoms. To elucidate the molecular mechanisms involved in such changes, we investigated the effect of irradiation on myosin heavy chain (MyHC) expression in laryngeal muscles. Methods Rats were irradiated with one dose of 10, 15, 20, 25, 30, or 35 Gy and sacrificed 2 weeks later. Other rats were irradiated with 20 Gy and sacrificed 3 days or 2, 4, or 12 weeks later. The thyroarytenoid (TA), posterior cricoarytenoid (PCA), cricothyroid (CT), and digastric (DG) muscles were subjected to SYBR Green real time-reverse transcriptase-PCR. Results Radiation exceeding 20 Gy killed all rats within 7-12 days. Two weeks after irradiation with 10, 15, or 20 Gy, MyHC type IIa expression had decreased in a dose-dependent manner in the PCA and CT muscles and in a dose-independent manner in the TA muscle. The expression of MyHC IIx and IIL also decreased but in a dose-dependent manner in the TA muscle and a dose-independent manner in the PCA and CT muscles. In the 20 Gy-irradiated rats, MyHC IIx, IIL, and especially IIa expression declined over 12 weeks. Conclusion The laryngeal muscles responded differently to radiation but showed a time-dependent and long-lasting decrease in the expression of all MyHC isoforms. MyHC IIa expression in the PCA and CT muscles may be more sensitive to irradiation than the other MyHC isoforms. Significance All of these observations together support the notion that if the radiation-induced changes in MyHC expression in the larynx can be ameliorated by an as-yet undefined method, it may be possible to restore to normal the laryngeal muscles of larynx cancer patients who have been trated with radiation therapy, therepy presevering their voice quality. Support This work was supported by the Korea Research Foundation Grant funded by the Korea Govement (MOE-HRD)” (KRF-2006-331-E00235).

Immunohistochemical Analysis of Myosin Heavy Chain Expression in Laryngeal Muscles of the Rabbit, Cat, and Baboon

We studied myosin heavy chain (MyHC) expression and fiber type distribution in laryngeal muscles in the rabbit, cat, and baboon using immunohistochemistry with highly MyHC-specific antibodies. Two types of variation in MyHC expression were found: between muscles of different function within species and within specific muscles between species. Within species, thyroarytenoid (Ta), an adductor, had faster MyHCs and fiber type profiles than the abductor, posterior cricoarytenoid (PCA), which expressed faster MyHCs than the vocal fold tensor, cricothyroid (CT). Between species, laryngeal muscles generally expressed faster MyHCs in small animals than in larger ones: extraocular (EO) MyHC was expressed in the Ta and PCA of the rabbit but not in the cat and baboon, whereas 2B MyHC was expressed in these muscles of the cat but not of the baboon. The CT expressed only MyHC isoforms and fiber types found in the limb muscles of the same species. These results are discussed in light of the hypothesis that the between-species variations in laryngeal muscle fiber types are evolutionary adaptations in response to changes in body mass and respiratory frequency. Within-species variations in fiber types ensure that protective closure of the glottis is always faster than movements regulating airflow during respiration.