Katsuya Miyake

Efficient and Reproducible Myogenic Differentiation from Human iPS Cells: Prospects for Modeling Miyoshi Myopathy In Vitro

The establishment of human induced pluripotent stem cells (hiPSCs) has enabled the production of in vitro, patient-specific cell models of human disease. In vitro recreation of disease pathology from patient-derived hiPSCs depends on efficient differentiation protocols producing relevant adult cell types. However, myogenic differentiation of hiPSCs has faced obstacles, namely, low efficiency and/or poor reproducibility. Here, we report the rapid, efficient, and reproducible differentiation of hiPSCs into mature myocytes. We demonstrated that inducible expression of myogenic differentiation1 (MYOD1) in immature hiPSCs for at least 5 days drives cells along the myogenic lineage, with efficiencies reaching 70–90%. Myogenic differentiation driven by MYOD1 occurred even in immature, almost completely undifferentiated hiPSCs, without mesodermal transition. Myocytes induced in this manner reach maturity within 2 weeks of differentiation as assessed by marker gene expression and functional properties, including in vitro and in vivo cell fusion and twitching in response to electrical stimulation. Miyoshi Myopathy (MM) is a congenital distal myopathy caused by defective muscle membrane repair due to mutations in DYSFERLIN. Using our induced differentiation technique, we successfully recreated the pathological condition of MM in vitro, demonstrating defective membrane repair in hiPSC-derived myotubes from an MM patient and phenotypic rescue by expression of full-length DYSFERLIN (DYSF). These findings not only facilitate the pathological investigation of MM, but could potentially be applied in modeling of other human muscular diseases by using patient-derived hiPSCs.

Micromorphology of Resin-Dentin Interfaces Using One-Bottle Etch&Rinse and Self-Etching Adhesive Systems on Laser-Treated Dentin Surfaces: A Confocal Laser Scanning Microscope Analysis

This study evaluated the hybrid layer (HL) morphology created by three adhesive systems (AS) on dentin surfaces treated with Er:YAG laser using two irradiation parameters.

The C2A domain in dysferlin is important for association with MG53 (TRIM72)

In skeletal muscle, Mitsugumin 53 (MG53), also known as muscle-specific tripartite motif 72, reportedly interacts with dysferlin to regulate membrane repair. To better understand the interactions between dysferlin and MG53, we conducted immunoprecipitation (IP) and pull-down assays. Based on IP assays, the C2A domain in dysferlin associated with MG53. MG53 reportedly exists as a monomer, a homodimer, or an oligomer, depending on the redox state. Based on pull-down assays, wild-type dysferlin associated with MG53 dimers in a Ca2+-dependent manner, but MG53 oligomers associated with both wild-type and C2A-mutant dysferlin in a Ca2+-independent manner. In pull-down assays, a pathogenic missense mutation in the C2A domain (W52R-C2A) inhibited the association between dysferlin and MG53 dimers, but another missense mutation (V67D-C2A) altered the calcium sensitivity of the association between the C2A domain and MG53 dimers. In contrast to the multimers, the MG53 monomers did not interact with wild-type or C2A mutant dysferlin in pull-down assays. These results indicated that the C2A domain in dysferlin is important for the Ca2+-dependent association with MG53 dimers and that dysferlin may associate with MG53 dimers in response to the influx of Ca2+ that occurs during membrane injury. To examine the biological role of the association between dysferlin and MG53, we co-expressed EGFP-dysferlin with RFP-tagged wild-type MG53 or RFP-tagged mutant MG53 (RFP-C242A-MG53) in mouse skeletal muscle, and observed molecular behavior during sarcolemmal repair; it has been reported that the C242A-MG53 mutant forms dimers, but not oligomers. In response to membrane wounding, dysferlin accumulated at the injury site within 1 second; this dysferlin accumulation was followed by the accumulation of wild-type MG53. However, accumulation of RFP-C242A MG53 at the wounded site was impaired relative to that of RFP-wild-type MG53. Co-transfection of RFP-C242A MG53 inhibited the recruitment of dysferlin to the sarcolemmal injury site. We also examined the molecular behavior of GFP-wild-type MG53 during sarcolemmal repair in dysferlin-deficient mice which show progressive muscular dystrophy, and found that GFP-MG53 accumulated at the wound similar to wild-type mice. Our data indicate that the coordination between dysferlin and MG53 plays an important role in efficient sarcolemmal repair.

Development of an automated fluorescence microscopy system for photomanipulation of genetically encoded photoactivatable proteins (optogenetics) in live cells

Photomanipulation of genetically encoded light-sensitive protein activity, also known as optogenetics, is one of the most innovative recent microscopy techniques in the fields of cell biology and neurobiology. Although photomanipulation is usually performed by diverting the photobleaching mode of a confocal laser microscope, photobleaching by the laser scanning unit is not always suitable for photoactivation. We have developed a simple automated wide-field fluorescence microscopy system for the photomanipulation of genetically encoded photoactivatable proteins in live cells. An electrically automated fluorescence microscope can be controlled through MetaMorph imaging software, making it possible to acquire time-lapse, multiwavelength images of live cells. Using the journal (macro recording) function of MetaMorph, we wrote a macro program to change the excitation filter for photoactivation and illumination area during the intervals of image acquisition. When this program was run on the wide-field fluorescence microscope, cells expressing genetically encoded photoactivatable Rac1, which is activated under blue light, showed morphological changes such as lamellipodial extension and cell surface ruffling in the illuminated region. Using software-based development, we successfully constructed a fully automated photoactivation microscopy system for a mercury lamp-based fluorescence microscope.

Contribution of Dysferlin Deficiency to Skeletal Muscle Pathology in Asymptomatic and Severe Dystroglycanopathy Models: Generation of a New Model for Fukuyama Congenital Muscular Dystrophy

Defects in dystroglycan glycosylation are associated with a group of muscular dystrophies, termed dystroglycanopathies, that include Fukuyama congenital muscular dystrophy (FCMD). It is widely believed that abnormal glycosylation of dystroglycan leads to disease-causing membrane fragility. We previously generated knock-in mice carrying a founder retrotransposal insertion in fukutin, the gene responsible for FCMD, but these mice did not develop muscular dystrophy, which hindered exploring therapeutic strategies. We hypothesized that dysferlin functions may contribute to muscle cell viability in the knock-in mice; however, pathological interactions between glycosylation abnormalities and dysferlin defects remain unexplored. To investigate contributions of dysferlin deficiency to the pathology of dystroglycanopathy, we have crossed dysferlin-deficient dysferlin sjl/sjl mice to the fukutin-knock-in fukutin Hp/− and Large-deficient Large myd/myd mice, which are phenotypically distinct models of dystroglycanopathy. The fukutin Hp/− mice do not show a dystrophic phenotype; however, (dysferlin sjl/sjl: fukutin Hp/−) mice showed a deteriorated phenotype compared with (dysferlin sjl/sjl: fukutin Hp/+) mice. These data indicate that the absence of functional dysferlin in the asymptomatic fukutin Hp/− mice triggers disease manifestation and aggravates the dystrophic phenotype. A series of pathological analyses using double mutant mice for Large and dysferlin indicate that the protective effects of dysferlin appear diminished when the dystrophic pathology is severe and also may depend on the amount of dysferlin proteins. Together, our results show that dysferlin exerts protective effects on the fukutin Hp/− FCMD mouse model, and the (dysferlin sjl/sjl: fukutin Hp/−) mice will be useful as a novel model for a recently proposed antisense oligonucleotide therapy for FCMD.

P.5.1 Sarcolemmal repair and reorganization of microtubule

Dysferlin is a sarcolemmal protein that is defective in Miyoshi myopathy and LGMD2B, and is involved in sarcolemmal repair. Proteomics analyses have identified multiple dysferlin-binding partners including cytoskeletal proteins. Affixin, a binding partner of dysferlin, regulates cytoskeletal actin. We reported that affixin, β- and γ-actin accumulated at the sarcolemmal injury site of wild-type mice in response to membrane rapture. α-tubulin and microtubule-associated proteins have been reported as dysferlin binding partners, however, their involvement in sarcolemmal repair remains unclear. The purpose of this study is to examine involvement of microtubule in sarcolemmal repair. To clarify molecular behavior of α-tubulin in sarcolemmal repair, GFP2-tagged human α-tubulin was expressed in mouse FDB muscle (C57BL/6J and dysferlin-deficient SJL) by electroporation. Membrane wound-repair assay of single myofiber was performed using confocal microscope equipped two-photon laser. GFP2-α-tubulin expressed in wild-type mice was localized at sarcolemma and T-tubule, and observed as a striated pattern. This striation pattern of GFP2-α-tubulin was slightly disorganized in SJL mice. After sarcolemmal injury, GFP2-α-tubulin in wild-type mice was disassembled and accumulates slower compared to GFP-β- and γ -actin. In SJL mice, GFP2-α-tubulin around the injury site was depolymerized, however, accumulation of GFP2-α-tubulin was not observed. α-Tubulin may also have a role in sarcolemmal repair.

G.P.48 Affixin is involved in sarcolemmal repair

Abstract Dysferlin is a sarcolemmal protein that is defective in Miyoshi myopathy and LGMD2B. In the presence of Ca2+, dysferlin accumulates around the damaged membrane site and is suggested to mediate sarcolemmal repair. We previously reported that affixin is a dysferlin-binding protein and co-localizes with dysferlin at the sarcolemma of normal human skeletal muscle. The association of dysferlin with affixin was confirmed by immunoprecipitation study using normal human skeletal muscles and COS-7 transfectants. The immunoreactivity of affixin was reduced in sarcolemma of dysferlinopathy muscles. We also reported that affixin activates Rac1 via GDP/GTP nucleotide exchange factor (GEF) and regulates the reorganization of cytoskeletal actin. The purpose of this study is to examine the effect of calcium on the association of dysferlin with affixin and to test the possibility that affixin is involved in sarcolemmal repair. The calcium-dependency of dysferlin–affixin association was examined by pull-down assay using lysates from COS-7 transfectants expressing human dysferlin and bacterially expressed affixin CH1 domain. To clarify molecular behavior of affixin in sarcolemmal repair, mCherry-tagged human affixin was expressed in FDB of mice (C57BL/6J and dysferlin-deficient A/J) by electroporation. Membrane wound-repair assay of single myofiber was performed using confocal microscope equipped two-photon laser. Pull-down assay revealed that dysferlin associates with affixin in a calcium-dependent manner. However, the association of dysferlin and caveolin-3 was not affected in different calcium concentrations. mCherry–affixin accumulates at the wounded site in C57BL/6J mice, however accumulation of affixin was not observed in A/J mice. These results suggest affixin involvement in sarcolemmal repair. We are analyzing movement of mCherry–affixin and dysferlin–GFP during sarcolemmal repair.