GSI Treatment Preserves Protein Synthesis in C2C12 Myotubes

It has been demonstrated that inhibiting Notch signaling through γ-secretase inhibitor (GSI) treatment increases myogenesis, AKT/mTOR signaling, and muscle protein synthesis (MPS) in C2C12 myotubes. The purpose of this study was to determine if GSI-mediated effects on myogenesis and MPS are dependent on AKT/mTOR signaling. C2C12 cells were assessed for indices of myotube formation, anabolic signaling, and MPS following GSI treatment in combination with rapamycin and API-1, inhibitors of mTOR and AKT, respectively. GSI treatment increased several indices of myotube fusion and MPS in C2C12 myotubes. GSI-mediated effects on myotube formation and fusion were completely negated by treatment with rapamycin and API-1. Meanwhile, GSI treatment was able to rescue MPS in C2C12 myotubes exposed to rapamycin or rapamycin combined with API-1. Examination of protein expression revealed that GSI treatment was able to rescue pGSK3β Ser9 despite AKT inhibition by API-1. These findings demonstrate that GSI treatment is able to rescue MPS independent of AKT/mTOR signaling, possibly via GSK3β modulation.

Valproic acid stimulates myogenesis in pluripotent stem cell-derived mesodermal progenitors in a NOTCH-dependent manner

Muscular dystrophies are debilitating neuromuscular disorders for which no cure exists. As this disorder affects both cardiac and skeletal muscle, patients would benefit from a cellular therapy that can simultaneously regenerate both tissues. The current protocol to derive bipotent mesodermal progenitors which can differentiate into cardiac and skeletal muscle relies on the spontaneous formation of embryoid bodies, thereby hampering further clinical translation. Additionally, as skeletal muscle is the largest organ in the human body, a high myogenic potential is necessary for successful regeneration. Here, we have optimized a protocol to generate chemically defined human induced pluripotent stem cell-derived mesodermal progenitors (cdMiPs). We demonstrate that these cells contribute to myotube formation and differentiate into cardiomyocytes, both in vitro and in vivo. Furthermore, the addition of valproic acid, a clinically approved small molecule, increases the potential of the cdMiPs to contribute to myotube formation that can be prevented by NOTCH signaling inhibitors. Moreover, valproic acid pre-treated cdMiPs injected in dystrophic muscles increase physical strength and ameliorate the functional performances of transplanted mice. Taken together, these results constitute a novel approach to generate mesodermal progenitors with enhanced myogenic potential using clinically approved reagents.

Inhibition of the Combinatorial Signaling of Transforming Growth Factor-Beta and NOTCH Promotes Myotube Formation of Human Pluripotent Stem Cell-Derived Skeletal Muscle Progenitor Cells

Understanding the signaling pathways that regulate the final differentiation of human myoblasts is essential for successful cell transplantation and drug screening for the treatment of muscular dystrophy. In an effort to improve myotube formation from hiPSC-derived myoblasts, we validated a collection of 13 small molecules in a newly established in vitro screening platform for the assessment of myotube formation. The analysis of myotube formation as measured by the fusion index showed that the combinational inhibition of the TGFβ signaling with NOTCH signaling enhances the ability of multi-nucleated myotube production. Combinational treatment of inhibitors for TGFβ and NOTCH signaling pathways improved myotube formation in a dose-dependent manner. This effect was achieved by inhibiting the combinatorial mechanism of signaling. The combination treatment of small molecules effective in inducing multinucleated myotubes was validated in healthy human primary myoblasts. In addition, it was also applied to DMD patient iPSC-derived myoblasts to enhance the generation of multinucleated myotubes.

Recycled algae-based carbon materials as electroconductive 3D printed skeletal muscle tissue engineering scaffolds

Skeletal muscle is an electrically and mechanically active tissue that contains highly oriented, densely packed myofibrils. The tissue has self-regeneration capacity upon injury, which is limited in the cases of volumetric muscle loss. Several regenerative therapies have been developed in order to enhance this capacity, as well as to structurally and mechanically support the defect site during regeneration. Among them, biomimetic approaches that recapitulate the native microenvironment of the tissue in terms of parallel-aligned structure and biophysical signals were shown to be effective. In this study, we have developed 3D printed aligned and electrically active scaffolds in which the electrical conductivity was provided by carbonaceous material (CM) derived from algae-based biomass. The synthesis of this conductive and functional CM consisted of eco-friendly synthesis procedure such as pre-carbonization and multi-walled carbon nanotube (MWCNT) catalysis. CM obtained from biomass via hydrothermal carbonization (CM-03) and its ash form (CM-03K) were doped within poly(ɛ-caprolactone) (PCL) matrix and 3D printed to form scaffolds with aligned fibers for structural biomimicry. Scaffolds were seeded with C2C12 mouse myoblasts and subjected to electrical stimulation during the in vitro culture. Enhanced myotube formation was observed in electroactive groups compared to their non-conductive counterparts and it was observed that myotube formation and myotube maturity were significantly increased for CM-03 group after electrical stimulation. The results have therefore showed that the CM obtained from macroalgae biomass is a promising novel source for the production of the electrically conductive scaffolds for skeletal muscle tissue engineering.