Distinct roles for classical nuclear import receptors in the growth of multinucleated muscle cells.

Proper muscle function is dependent on spatial and temporal control of gene expression in myofibers. Myofibers are multinucleated cells that are formed, repaired and maintained by the process of myogenesis in which progenitor myoblasts proliferate, differentiate and fuse. Gene expression is dependent upon proteins that require facilitated nuclear import, however little is known about the regulation of nucleocytoplasmic transport during the formation of myofibers. We analyzed the role of karyopherin alpha (KPNA), a key classical nuclear import receptor, during myogenesis. We established that five karyopherin alpha paralogs are expressed by primary mouse myoblasts in vitro and that their steady-state levels increase in multinucleated myotubes, suggesting a global increase in demand for classical nuclear import during myogenesis. We used siRNA-mediated knockdown to identify paralog-specific roles for KPNA1 and KPNA2 during myogenesis. KPNA1 knockdown increased myoblast proliferation, whereas KPNA2 knockdown decreased proliferation. In contrast, no proliferation defect was observed with KPNA4 knockdown. Only knockdown of KPNA2 decreased myotube growth. These results identify distinct pathways involved in myoblast proliferation and myotube growth that rely on specific nuclear import receptors suggesting that regulation of classical nuclear import pathways likely plays a critical role in controlling gene expression in skeletal muscle.

Chemokine expression and control of muscle cell migration during myogenesis.

Adult regenerative myogenesis is vital for restoring normal tissue structure after muscle injury. Muscle regeneration is dependent on progenitor satellite cells, which proliferate in response to injury, and their progeny differentiate and undergo cell-cell fusion to form regenerating myofibers. Myogenic progenitor cells must be precisely regulated and positioned for proper cell fusion to occur. Chemokines are secreted proteins that share both leukocyte chemoattractant and cytokine-like behavior and affect the physiology of a number of cell types. We investigated the steady-state mRNA levels of 84 chemokines, chemokine receptors and signaling molecules, to obtain a comprehensive view of chemokine expression by muscle cells during myogenesis in vitro. A large number of chemokines and chemokine receptors were expressed by primary mouse muscle cells, especially during times of extensive cell-cell fusion. Furthermore, muscle cells exhibited different migratory behavior throughout myogenesis in vitro. One receptor-ligand pair, CXCR4-SDF-1alpha (CXCL12), regulated migration of both proliferating and terminally differentiated muscle cells, and was necessary for proper fusion of muscle cells. Given the large number of chemokines and chemokine receptors directly expressed by muscle cells, these proteins might have a greater role in myogenesis than previously appreciated.

The CHC22 clathrin-GLUT4 transport pathway contributes to skeletal muscle regeneration.

Mobilization of the GLUT4 glucose transporter from intracellular storage vesicles provides a mechanism for insulin-responsive glucose import into skeletal muscle. In humans, clathrin isoform CHC22 participates in formation of the GLUT4 storage compartment in skeletal muscle and fat. CHC22 function is limited to retrograde endosomal sorting and is restricted in its tissue expression and species distribution compared to the conserved CHC17 isoform that mediates endocytosis and several other membrane traffic pathways. Previously, we noted that CHC22 was expressed at elevated levels in regenerating rat muscle. Here we investigate whether the GLUT4 pathway in which CHC22 participates could play a role in muscle regeneration in humans and we test this possibility using CHC22-transgenic mice, which do not normally express CHC22. We observed that GLUT4 expression is elevated in parallel with that of CHC22 in regenerating skeletal muscle fibers from patients with inflammatory and other myopathies. Regenerating human myofibers displayed concurrent increases in expression of VAMP2, another regulator of GLUT4 transport. Regenerating fibers from wild-type mouse skeletal muscle injected with cardiotoxin also showed increased levels of GLUT4 and VAMP2. We previously demonstrated that transgenic mice expressing CHC22 in their muscle over-sequester GLUT4 and VAMP2 and have defective GLUT4 trafficking leading to diabetic symptoms. In this study, we find that muscle regeneration rates in CHC22 mice were delayed compared to wild-type mice, and myoblasts isolated from these mice did not proliferate in response to glucose. Additionally, CHC22-expressing mouse muscle displayed a fiber type switch from oxidative to glycolytic, similar to that observed in type 2 diabetic patients. These observations implicate the pathway for GLUT4 transport in regeneration of both human and mouse skeletal muscle, and demonstrate a role for this pathway in maintenance of muscle fiber type. Extrapolating these findings, CHC22 and GLUT4 can be considered markers of muscle regeneration in humans.

MOR23 promotes muscle regeneration and regulates cell adhesion and migration.

Odorant receptors (ORs) in the olfactory epithelium bind to volatile small molecules leading to the perception of smell. ORs are expressed in many tissues but their functions are largely unknown. We show multiple ORs display distinct mRNA expression patterns during myogenesis in vitro and muscle regeneration in vivo. Mouse OR23 (MOR23) expression is induced during muscle regeneration when muscle cells are extensively fusing and plays a key role in regulating migration and adhesion of muscle cells in vitro, two processes common during tissue repair. A soluble ligand for MOR23 is secreted by muscle cells in vitro and muscle tissue in vivo. MOR23 is necessary for proper skeletal muscle regeneration as loss of MOR23 leads to increased myofiber branching, commonly associated with muscular dystrophy. Together these data identify a functional role for an OR outside of the nose and suggest a larger role for ORs during tissue repair.