Injury-mediated stiffening persistently activates muscle stem cells through YAP and TAZ mechanotransduction.

The skeletal muscle microenvironment transiently remodels and stiffens after exercise and injury, as muscle ages, and in myopathic muscle; however, how these changes in stiffness affect resident muscle stem cells (MuSCs) remains understudied. Following muscle injury, muscle stiffness remained elevated after morphological regeneration was complete, accompanied by activated and proliferative MuSCs. To isolate the role of stiffness on MuSC behavior and determine the underlying mechanotransduction pathways, we cultured MuSCs on strain-promoted azide-alkyne cycloaddition hydrogels capable of in situ stiffening by secondary photocrosslinking of excess cyclooctynes. Using pre- to post-injury stiffness hydrogels, we found that elevated stiffness enhances migration and MuSC proliferation by localizing yes-associated protein 1 (YAP) and WW domain-containing transcription regulator 1 (WWTR1; TAZ) to the nucleus. Ablating YAP and TAZ in vivo promotes MuSC quiescence in postinjury muscle and prevents myofiber hypertrophy, demonstrating that persistent exposure to elevated stiffness activates mechanotransduction signaling maintaining activated and proliferating MuSCs.

Transplantation of Skeletal Muscle Stem Cells.

Transplanting adult stem cells provides a stringent test for self-renewal and the assessment of comparative engraftment in competitive transplant assays. Transplantation of satellite cells into mammalian skeletal muscle provided the first critical evidence that satellite cells function as adult muscle stem cells. Transplantation of a single satellite cell confirmed and extended this hypothesis, providing proof that the satellite cell is a bona fide adult skeletal muscle stem cell as reported by Sacco et al. (Nature 456(7221):502-506). Satellite cell transplantation has been further leveraged to identify culture conditions that maintain engraftment and to identify self-renewal deficits in satellite cells from aged mice. Conversion of iPSCs (induced pluripotent stem cells) to a satellite cell-like state, followed by transplantation, demonstrated that these cells possess adult muscle stem cell properties as reported by Darabi et al. (Stem Cell Rev Rep 7(4):948-957) and Mizuno et al. (FASEB J 24(7):2245-2253). Thus, transplantation strategies involving either satellite cells derived from adult muscles or derived from iPSCs may eventually be exploited as a therapy for treating patients with diseased or failing skeletal muscle. Here, we describe methods for isolating dispersed adult mouse satellite cells and satellite cells on intact myofibers for transplantation into recipient mice to study muscle stem cell function and behavior following engraftment .

Col4a1 mutations cause progressive retinal neovascular defects and retinopathy.

Mutations in collagen, type IV, alpha 1 (COL4A1), a major component of basement membranes, cause multisystem disorders in humans and mice. In the eye, these include anterior segment dysgenesis, optic nerve hypoplasia and retinal vascular tortuosity. Here we investigate the retinal pathology in mice carrying dominant-negative Col4a1 mutations. To this end, we examined retinas longitudinally in vivo using fluorescein angiography, funduscopy and optical coherence tomography. We assessed retinal function by electroretinography and studied the retinal ultrastructural pathology. Retinal examinations revealed serous chorioretinopathy, retinal hemorrhages, fibrosis or signs of pathogenic angiogenesis with chorioretinal anastomosis in up to approximately 90% of Col4a1 mutant eyes depending on age and the specific mutation. To identify the cell-type responsible for pathogenesis we generated a conditional Col4a1 mutation and determined that primary vascular defects underlie Col4a1-associated retinopathy. We also found focal activation of Müller cells and increased expression of pro-angiogenic factors in retinas from Col4a1(+/Δex41)mice. Together, our findings suggest that patients with COL4A1 and COL4A2 mutations may be at elevated risk of retinal hemorrhages and that retinal examinations may be useful for identifying patients with COL4A1 and COL4A2 mutations who are also at elevated risk of hemorrhagic strokes.

Formation of complex AChR aggregates in vitro requires alpha-dystrobrevin.

Efficient function at the neuromuscular junction requires high-density aggregates of acetylcholine receptors (AChRs) to be precisely aligned with the motor nerve terminal. A collaborative effort between the motor neuron and muscle intrinsic factors drives the formation and maintenance of these AChR aggregates. alpha-Dystrobrevin (alpha DB), a cytoplasmic protein found at the postsynaptic membrane, has been implicated in the regulation of AChR aggregate density and patterning. To investigate the contribution of alpha DB to the muscle intrinsic program regulating AChR aggregate development, we analyzed the formation of complex, pretzel-like AChR aggregates on primary muscle cell cultures derived from alpha DB knockout (alpha DB-KO) mice in the absence of nerve or agrin. In myotubes lacking alpha DB, complex AChR aggregates failed to form, whereas aggregates formed readily in wildtype myotubes. Five major isoforms of alpha DB are expressed in skeletal muscle: alpha DB1, alpha DB1(-), alpha DB2, alpha DB2(-), and alpha DB3. Expression of alpha DB1 or alpha DB1(-) in alpha DB-KO myotubes restored formation of complex AChR aggregates similar to those in wildtype myotubes. In contrast, individual expression of alpha DB2, alpha DB2(-), alpha DB3, or an alpha DB1 phosphorylation mutant resulted in the formation of few, if any, complex AChR aggregates. Collectively, these data suggest that alpha DB is a significant component of the muscle intrinsic program that mediates the formation of complex AChR aggregates and that alpha DB's tyrosine phosphorylation sites are of particular functional importance to this program. Although the muscle intrinsic program appears to influence synaptogenesis, the formation of complex mature AChR aggregates in alpha DB-KO mice (with the motor neuron present) suggests the motor neuron, not the muscle intrinsic program, is the major stimulus driving the maturation of AChRs from plaque to pretzel in vivo.