Nao Kinouchi

Atelocollagen-mediated local and systemic applications of myostatin-targeting siRNA increase skeletal muscle mass.

RNA interference (RNAi) offers a novel therapeutic strategy based on the highly specific and efficient silencing of a target gene. Since it relies on small interfering RNAs (siRNAs), a major issue is the delivery of therapeutically active siRNAs into the target tissue/target cells in vivo. For safety reasons, strategies based on vector delivery may be of only limited clinical use. The more desirable approach is to directly apply active siRNAs in vivo. Here, we report the effectiveness of in vivo siRNA delivery into skeletal muscles of normal or diseased mice through nanoparticle formation of chemically unmodified siRNAs with atelocollagen (ATCOL). ATCOL-mediated local application of siRNA targeting myostatin, a negative regulator of skeletal muscle growth, in mouse skeletal muscles or intravenously, caused a marked increase in the muscle mass within a few weeks after application. These results imply that ATCOL-mediated application of siRNAs is a powerful tool for future therapeutic use for diseases including muscular atrophy.

Adaptation of rat jaw muscle fibers in postnatal development with a different food consistency: an immunohistochemical and electromyographic study

The development of the craniofacial system occurs, among other reasons, as a response to functional needs. In particular, the deficiency of the proper masticatory stimulus affects the growth. The purpose of this study was to relate alterations of muscle activity during postnatal development to adaptational changes in the muscle fibers. Fourteen 21‐day‐old Wistar strain male rats were randomly divided into two groups and fed on either a solid (hard‐diet group) or a powder (soft‐diet group) diet for 63 days. A radio‐telemetric device was implanted to record muscle activity continuously from the superficial masseter, anterior belly of digastric and anterior temporalis muscles. The degree of daily muscle use was quantified by the total duration of muscle activity per day (duty time), the total burst number and their average length exceeding specified levels of the peak activity (5, 20 and 50%). The fiber type composition of the muscles was examined by the myosin heavy chain content of fibers by means of immunohistochemical staining and their cross‐sectional area was measured. All muscle fibers were identified as slow type I and fast type IIA, IIX or IIB (respectively, with increasing twitch contraction speed and fatigability). At lower activity levels (exceeding 5% of the peak activity), the duty time of the anterior belly of the digastric muscle was significantly higher in the soft‐diet group than in the hard‐diet group (P < 0.05). At higher activity levels (exceeding 20 and 50% of the peak activity), the duty time of the superficial masseter muscle in the soft‐diet group was significantly lower than that in the hard‐diet group (P < 0.05). There was no difference in the duty time of the anterior temporalis muscle at any muscle activity level. The percentage of type IIA fibers of the superficial masseter muscle in the soft‐diet group was significantly lower than that in the hard‐diet group (P < 0.01) and the opposite was true with regard to type IIB fibers (P < 0.05). The cross‐sectional area of type IIX and type IIB fibers of the superficial masseter muscle was significantly smaller in the soft‐diet group than in the hard‐diet group (P < 0.05). There was no difference in the muscle fiber composition and the cross‐sectional area of the anterior belly of the digastric and anterior temporalis muscles. In conclusion, for the jaw muscles of male rats reared on a soft diet, the slow‐to‐fast transition of muscle fiber was shown in only the superficial masseter muscle. Therefore, the reduction in the amount of powerful muscle contractions could be important for the slow‐to‐fast transition of the myosin heavy chain isoform in muscle fibers.

Atelocollagen-mediated systemic administration of myostatin-targeting siRNA improves muscular atrophy in caveolin-3-deficient mice

Small interfering RNA (siRNA)‐mediated silencing of gene expression is rapidly becoming a powerful tool for molecular therapy. However, the rapid degradation of siRNAs and their limited duration of activity require efficient delivery methods. Atelocollagen (ATCOL)‐mediated administration of siRNAs is a promising approach to disease treatment, including muscular atrophy. Herein, we report that ATCOL‐mediated systemic administration of a myostatin‐targeting siRNA into a caveolin‐3‐deficient mouse model of limb‐girdle muscular dystrophy 1C (LGMD1C) induced a marked increase in muscle mass and a significant recovery of contractile force. These results provide evidence that ATCOL‐mediated systemic administration of siRNAs may be a powerful therapeutic tool for disease treatment, including muscular atrophy.

Local applications of myostatin-siRNA with atelocollagen increase skeletal muscle mass and recovery of muscle function.

Background Growing 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 Findings We 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. Conclusion These 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.

Effectiveness of cationic liposome‐mediated local delivery of myostatin‐targeting small interfering RNA in vivo

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‐γ (PPARγ) and CCAAT/enhancer binding protein‐α (CEBPα) 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 cost‐effective, they may represent a therapeutic tool for use in treating muscular diseases.

Skeletal anchorage for intrusion of bimaxillary molars in a patient with skeletal open bite and temporomandibular disorders

The treatment of severe skeletal anterior open bite is extremely difficult in adults, and orthognathic surgery is generally selected for its treatment. We report the case of an 18-year-old adult patient with skeletal anterior open bite and temporomandibular disorders who was successfully treated using temporary anchorage devices. She had an open bite of −2.0 mm and an increased facial height. Miniplates were implanted in both the maxilla and mandible, and molar intrusion resulted in counterclockwise rotation of the mandible over a period of 12 months. After active treatment, her upper and lower first molars were intruded by approximately 2 mm and her overbite became +2.5 mm. Her retrognathic profile improved with counterclockwise rotation of the mandible. Orthodontic treatment aided with skeletal anchorage is beneficial for intrusion of bimaxillary molars in patients with anterior open bite.

The increases in the skeletal muscle mass of the transgenic mice expressing the mutated myostatin affected craniofacial morphology

Abstract Myostatin, which is a member of the TGF-β superfamily, is a negative regulator of skeletal muscle formation. Myostatin null mice showed a doubling of their muscle mass compared to normal mice. In a previous study, we generated transgenic mice expressing the mutated myostatin containing an 11-nucleotide deletion that caused a frame-shift mutation. We identified that the transgenic mice exhibited dramatic increases in the skeletal muscle mass resulting from hyperplasia without hypertrophy. In this study, we examined the craniofacial morphology of the transgenic mice to assess the effects of the increased muscle mass on the skeletal growth and development. The craniums and mandibles of age-matched adult mutant mice and their normal littermates were compared. The byzigomatic width of the transgenic mice was larger than that of the control littermates. The mandible of the transgenic mice had increased in size. The coronoid process and angular process of the transgenic mice had developed particularly longer than those of the control littermates. The masseteric ridge of the transgenic mice, in which the deep masseter muscle was inserted, developed more than that of the control littermates. These results show that the increased muscle mass affected the regions of the cranium and mandible where the jaw muscles are specifically attached.

Effectiveness of presurgical nasoalveolar molding therapy on unilateral cleft lip nasal deformity

Objectives: To evaluate the effectiveness of pre-surgical nasoalveolar molding (PNAM) in patients with unilateral cleft lip nasal deformities. Methods: This was a retrospective study involving 29 patients with unilateral cleft lip and palate defects, of whom 13 were treated with palatal devices with nasal stents (PNAM group) and 16 were treated with palatal devices without nasal stents or surgical tapes (control group). Submental oblique photographs and orthodontic models were longitudinally obtained at the initial visit (T1) and immediately before (T2) and after cheiloplasty (T3). Asymmetry of the external nose, degree of columellar shifting, nasal tip/ala nose ratio, nasal base angle, interalveolar gap, and the sagittal difference in the alveolar gap were measured. The study was conducted in the Orthodontic Clinic at Tokushima University Hospital, Tokushima, Japan between 1997 and 2012. Results: At T1, there were no significant intergroup differences in the first 4 asymmetry parameters. At T2, the PNAM group showed a significant improvement in all values compared to the control group. At T3, the PNAM group showed significant improvement in nasal asymmetry and columellar shifting. Model analysis showed significantly greater changes in the inter-alveolar gap and the sagittal difference of the alveolar cleft gap from T1 to T2 in the PNAM group. Conclusion: The use of PNAM is indispensable for pre-surgical orthodontic treatment at the early postnatal age.

Co-Administration of Myostatin-Targeting siRNA and ActRIIB-Fc Fusion Protein Increases Masseter Muscle Mass and Fiber Size

Myostatin, a member of the TGF-β 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-β 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 ActRIIB-Fc 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.