The influence of masticatory loading stimulus on mandibular development is not fully clear. In this paper, experimental alterations in the daily muscle use, caused by a changed diet consistency, were continuously monitored, while adaptations in bone and cartilage were examined. It is hypothesised that decreased muscular loading will result in a decrease in the growth factor expression and mandible growth. Fourteen 21-day-old Wistar strain male rats were randomly divided into two groups and fed on either a hard or soft diet for 14 weeks. An implanted radio-telemetric device recorded continuously muscle activity of the superficial masseter muscle. Chondroblast proliferation in the condylar cartilage was identified by insulin-like growth factor-1 receptor (IGF-1r) immunostaining. Furthermore, an X-ray was taken for cephalometric analysis. In the soft-diet group, the duty time of the superficial masseter muscle at higher activity levels was significantly lower than that in the hard-diet group. This decrease in muscular loading of the jaw system was accompanied by: a significant reduction in (i) articular cartilage thickness, (ii) expression of IGF-1r immunopositive cells and (iii) mandible ramus height. In conclusion, a decrease in masticatory demand during the growth period leads to insufficient mandibular development.

Summary Muscle atrophy is a complex process that occurs as a consequence of various stress events. Muscle atrophy-associated genes (atrogenes) such as atrogin-1/MAFbx and MuRF-1 are induced early in the atrophy process, and the increase in their expression precedes the loss of muscle weight. Although antioxidative nutrients suppress atrogene expression in skeletal muscle cells, the inhibitory effects of flavonoids on inflammation-induced atrogin-1/MAFbx expression have not been clarified. Here, we investigated the inhibitory effects of flavonoids on lipopolysaccharide (LPS)-induced atrogin-1/MAFbx expression. We examined whether nine flavonoids belonging to six flavonoid categories inhibited atrogin-1/ MAFbx expression in mouse C2C12 myotubes. Two major flavones, apigenin and luteolin, displayed potent inhibitory effects on atrogin-1/MAFbx expression. The pretreatment with apigenin and luteolin significantly prevented the decrease in C2C12 myotube diameter caused by LPS stimulation. Importantly, the pretreatment of LPS-stimulated myoblasts with these flavones significantly inhibited LPS-induced JNK phosphorylation in C2C12 myotubes, resulting in the significant suppression of atrogin-1/MAFbx promoter activity. These results suggest that apigenin and luteolin, prevent LPS-mediated atrogin-1/MAFbx expression through the inhibition of the JNK signaling pathway in C2C12 myotubes. Thus, these flavones, apigenin and luteolin, may be promising agents to prevent LPS-induced muscle atrophy.

BackgroundGrowing evidence suggests that small-interfering RNA (siRNA) can promote gene silencing in mammalian cells without induction of interferon synthesis or nonspecific gene suppression. Recently, a number of highly specific siRNAs targeted against disease-causing or disease-promoting genes have been developed. In this study, we evaluate the effectiveness of atelocollagen (ATCOL)-mediated application of siRNA targeting myostatin (Mst), a negative regulator of skeletal muscle growth, into skeletal muscles of muscular dystrophy model mice.Methods and FindingsWe injected a nanoparticle complex containing myostatin-siRNA and ATCOL (Mst-siRNA/ATCOL) into the masseter muscles of mutant caveolin-3 transgenic (mCAV-3Tg) mice, an animal model for muscular dystrophy. Scrambled (scr) -siRNA/ATCOL complex was injected into the contralateral muscles as a control. Two weeks after injection, the masseter muscles were dissected for histometric analyses. To investigate changes in masseter muscle activity by local administration of Mst-siRNA/ATCOL complex, mouse masseter electromyography (EMG) was measured throughout the experimental period via telemetry. After local application of the Mst-siRNA/ATCOL complex, masseter muscles were enlarged, while no significant change was observed on the contralateral side. Histological analysis showed that myofibrils of masseter muscles treated with the Mst-siRNA/ATCOL complex were significantly larger than those of the control side. Real-time PCR analysis revealed a significant downregulation of Mst expression in the treated masseters of mCAV-3Tg mice. In addition, expression of myogenic transcription factors was upregulated in the Mst-siRNA-treated masseter muscle, while expression of adipogenic transcription factors was significantly downregulated. EMG results indicate that masseter muscle activity in mCAV-3Tg mice was increased by local administration of the Mst-siRNA/ATCOL complex.ConclusionThese data suggest local administration of Mst-siRNA/ATCOL complex could lead to skeletal muscle hypertrophy and recovery of motor disability in mCAV-3Tg mice. Therefore, ATCOL-mediated application of siRNA is a potential tool for therapeutic use in muscular atrophy diseases.

Development of the masticatory system is influenced by functional needs. Furthermore, masticatory exercise can improve masticatory function. The aim of this study was to evaluate the potential effect of the gum chewing exercise on the maximum bite force (MBF) in adult subjects with different facial morphologies. MBF was measured by a portable occlusal force gauge and lateral cephalogram was used for evaluation of craniofacial morphology in 19 individuals (7 males and 12 females) with a mean age of 25.4 years (SD ± 4.3). The volunteers underwent gum chewing exercise for 5 min twice a day for 4 weeks. MBF was measured before (T1) and after the 4‐week exercise (T2). The facial morphology of the subjects was classified into the brachy (n = 7), mesio (n = 7), and dolicho (n = 5) facial types. In all three groups, exercise was associated with a significant increase in MBF, though the percent increase was highest in the dolicho facial type. We conclude that gum chewing exercise can improve masticatory performance, especially in individuals with dolicho facial morphology.

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Skeletal muscle atrophy occurs when the rate of protein degradation exceeds that of protein synthesis in various catabolic conditions, such as fasting, disuse, aging, and chronic diseases. Insulin-like growth factor-1 (IGF-1) signaling stimulates muscle growth and suppresses muscle protein breakdown. In atrophied muscles, ubiquitin ligase, Cbl-b, increases and stimulates the ubiquitination and degradation of IRS-1, an intermediate in IGF-1 signaling pathway, resulting in IGF-1 resistance. In this study, we evaluated the efficacy of atelocollagen (ATCOL)-transported anti-ubiquitination oligopeptide (Cblin: Cbl-b inhibitor) (consisting of tyrosine phosphorylation domain of IRS-1) in starved C2C12 myotubes. The amount of IRS-1 protein was lower in starved versus unstarved myotubes. The Cblin-ATCOL complex inhibited IRS-1 degradation in a concentration-dependent manner. Myotubes incubated with Cblin-ATCOL complex showed significant resistance to starvation-induced atrophy (p<0.01). Furthermore, the Cblin-ATCOL complex significantly inhibited any decrease in Akt phosphorylation (p<0.01) and localization of FOXO3a to the nucleus in starved myotubes. These results suggest that Cblin prevented starvation-induced C2C12 myotube atrophy by maintaining the IGF-1/Akt/FOXO signaling. Therefore, attachment of anti-ubiquitination oligopeptide, Cblin, to ATCOL enhances its delivery to myotubes and could be a potentially effective strategy in the treatment of atrophic myopathies.

Summary Myostatin, a member of the TGF-␤ superfamily, is a negative regulator of skeletal muscle cell growth and differentiation, and binds with high affinity to the activin type IIB receptor (ActRIIB). The soluble ligand-binding domain of ActRIIB fused to the Fc domain of IgG (ActRIIB-Fc) potently binds and inhibits TGF-␤ family members in muscle, leading to rapid and marked muscle growth. The present study was designed to assess the effectiveness of the co-delivery of myostatin-targeting siRNA (Mstn-siRNA) and ActRIIBFc into skeletal muscle as a potential treatment of atrophic myopathies. Eleven-week-old, male C57BL/6 mice were injected with atelocollagen (ATCOL)-mediated Mstn-siRNA with/ without ActRIIB-Fc locally into the masseter muscle twice a week. Inhibition of myostatin function by the combination of Mstn-siRNA and ActRIIB-Fc increased muscle weight and myofibril size in murine masseter muscle. Real-time RT-PCR analysis revealed significant downregulation of myostatin mRNA expression in both the Mstn-siRNA-treated and the combination treatment group. Furthermore, myogenin mRNA expression was upregulated in the combination treatment group, while MuRF-1 and Atrogin-1 mRNA expression was downregulated compared to administration of each compound alone. These findings suggest that double inhibition of myostatin is a potentially useful treatment strategy to increase muscle mass and fiber size and could be a useful treatment of patients with various muscle atrophies, including muscular dystrophy.

This study evaluated the effectiveness of local administration of cationic liposome-delivered myostatin-targeting siRNA. Myostatin (Mst)-siRNA and scrambled (scr)-siRNA-lipoplexes were injected into the masseter muscles of wild type and dystrophin-deficient mdx mice, which model Duchenne muscular dystrophy. One week after injection, the masseter muscles were dissected for histometric analyses. To evaluate changes in masseter muscle activity, masseter electromyographic (EMG) measurements were performed. One week after local administration of Mst-siRNA-lipoplexes, masseter muscles and myofibrils were significantly larger compared to control masseter muscles treated with scr-siRNA-lipoplexes. Real-time polymerase chain reaction (PCR) analyses revealed significant upregulation of the myogenic regulatory factors MyoD and myogenin and significant downregulation of the adipogenic transcription factors peroxisome proliferator-activated receptor-c (PPARc) and CCAAT/enhancer binding protein-a (CEBPa) in masseter muscles treated with Mst-siRNA-lipoplexes. The duty times of masseter muscle activity exceeding 5% showed a slight tendency to increase in both wild type and mdx mice. Therefore, cationic liposome-mediated local administration of Mst-siRNA could increase muscular size and improve muscle activity. Since cationic liposomes delivered siRNA to muscles effectively and are safe and costeffective, they may represent a therapeutic tool for use in treating muscular diseases.