Selective Myostatin Inhibition Spares Sublesional Muscle Mass and Myopenia-Related Dysfunction after Severe Spinal Cord Contusion in Mice.

Clinically relevant myopenia accompanies spinal cord injury (SCI), and compromises function, metabolism, body composition, and health. Myostatin, a transforming growth factor (TGF)ß family member, is a key negative regulator of skeletal muscle mass. We investigated inhibition of myostatin signaling using systemic delivery of a highly selective monoclonal antibody - muSRK-015P (40 mg/kg) - that blocks release of active growth factor from the latent form of myostatin. Adult female mice (C57BL/6) were subjected to a severe SCI (65 kdyn) at T9 and were then immediately and 1 week later administered test articles: muSRK-015P (40 mg/kg) or control (vehicle or IgG). A sham control group (laminectomy only) was included. At euthanasia, (2 weeks post-SCI) muSRK-015P preserved whole body lean mass and sublesional gastrocnemius and soleus mass. muSRK-015P-treated mice with SCI also had significantly attenuated myofiber atrophy, lipid infiltration, and loss of slow-oxidative phenotype in soleus muscle. These outcomes were accompanied by significantly improved sublesional motor function and muscle force production at 1 and 2 weeks post-SCI. At 2 weeks post-SCI, lean mass was significantly decreased in SCI-IgG mice, but was not different in SCI-muSRK-015P mice than in sham controls. Total energy expenditure (kCal/day) at 2 weeks post-SCI was lower in SCI-immunoglobulin (Ig)G mice, but not different in SCI-muSRK-015P mice than in sham controls. We conclude that in a randomized, blinded, and controlled study in mice, myostatin inhibition using muSRK-015P had broad effects on physical, metabolic, and functional outcomes when compared with IgG control treated SCI animals. These findings may identify a useful, targeted therapeutic strategy for treating post-SCI myopenia and related sequelae in humans.

Specific inhibition of myostatin activation is beneficial in mouse models of SMA therapy.

Spinal muscular atrophy (SMA) is a neuromuscular disease characterized by loss of α-motor neurons, leading to profound skeletal muscle atrophy. Patients also suffer from decreased bone mineral density and increased fracture risk. The majority of treatments for SMA, approved or in clinic trials, focus on addressing the underlying cause of disease, insufficient production of full-length SMN protein. While restoration of SMN has resulted in improvements in functional measures, significant deficits remain in both mice and SMA patients following treatment. Motor function in SMA patients may be additionally improved by targeting skeletal muscle to reduce atrophy and improve muscle strength. Inhibition of myostatin, a negative regulator of muscle mass, offers a promising approach to increase muscle function in SMA patients. Here we demonstrate that muSRK-015P, a monoclonal antibody which specifically inhibits myostatin activation, effectively increases muscle mass and function in two variants of the pharmacological mouse model of SMA in which pharmacologic restoration of SMN has taken place either 1 or 24 days after birth to reflect early or later therapeutic intervention. Additionally, muSRK-015P treatment improves the cortical and trabecular bone phenotypes in these mice. These data indicate that preventing myostatin activation has therapeutic potential in addressing muscle and bone deficiencies in SMA patients. An optimized variant of SRK-015P, SRK-015, is currently in clinical development for treatment of SMA.

Intravital Imaging Reveals Ghost Fibers as Architectural Units Guiding Myogenic Progenitors during Regeneration.

How resident stem cells and their immediate progenitors rebuild tissues of pre-injury organization and size for proportional regeneration is not well understood. Using 3D, time-lapse intravital imaging for direct visualization of the muscle regeneration process in live mice, we report that extracellular matrix remnants from injured skeletal muscle fibers, "ghost fibers," govern muscle stem/progenitor cell behaviors during proportional regeneration. Stem cells were immobile and quiescent without injury whereas their activated progenitors migrated and divided after injury. Unexpectedly, divisions and migration were primarily bi-directionally oriented along the ghost fiber longitudinal axis, allowing for spreading of progenitors throughout ghost fibers. Re-orienting ghost fibers impacted myogenic progenitors' migratory paths and division planes, causing disorganization of regenerated muscle fibers. We conclude that ghost fibers are autonomous, architectural units necessary for proportional regeneration after tissue injury. This finding reinforces the need to fabricate bioengineered matrices that mimic living tissue matrices for tissue regeneration therapy.

Quantitative 3D Time Lapse Imaging of Muscle Progenitors in Skeletal Muscle of Live Mice.

For non-optically clear mammalian tissues, it is now possible to use multi-photon microscopy to penetrate deep into the tissue and obtain detailed single cell images in a live animal, i.e., intravital imaging. This technique is in principle applicable to any fluorescently marked cell, and we have employed it to observe stem cells during the regenerative process. Stem cell-mediated skeletal muscle regeneration in the mouse model has been classically studied at specific time points by sacrificing the animal and harvesting the muscle tissue for downstream analyses. A method for direct visualization of muscle stem cells to gain real-time information over a long period in a live mammal has been lacking. Here we describe a step-by-step protocol adapted from Webster et al. (2016) to quantitatively measure the behaviors of fluorescently labeled (GFP, EYFP) muscle stem and progenitor cells during homeostasis as well as following muscle injury.

Acute tissue injury activates satellite cells and promotes sarcoma formation via the HGF/c-MET signaling pathway.

Some patients with soft-tissue sarcoma (STS) report a history of injury at the site of their tumor. Although this phenomenon is widely reported, there are relatively few experimental systems that have directly assessed the role of injury in sarcoma formation. We recently described a mouse model of STS whereby p53 is deleted and oncogenic Kras is activated in muscle satellite cells via a Pax7(CreER) driver following intraperitoneal injection with tamoxifen. Here, we report that after systemic injection of tamoxifen, the vast majority of Pax7-expressing cells remain quiescent despite mutation of p53 and Kras. The fate of these muscle progenitors is dramatically altered by tissue injury, which leads to faster kinetics of sarcoma formation. In adult muscle, quiescent satellite cells will transition into an active state in response to hepatocyte growth factor (HGF). We show that modulating satellite cell quiescence via intramuscular injection of HGF increases the penetrance of sarcoma formation at the site of injection, which is dependent on its cognate receptor c-MET. Unexpectedly, the tumor-promoting effect of tissue injury also requires c-Met. These results reveal a mechanism by which HGF/c-MET signaling promotes tumor formation after tissue injury in a mouse model of primary STS, and they may explain why some patients develop a STS at the site of injury.

c-MET regulates myoblast motility and myocyte fusion during adult skeletal muscle regeneration.

Adult muscle stem cells, satellite cells (SCs), endow skeletal muscle with tremendous regenerative capacity. Upon injury, SCs activate, proliferate, and migrate as myoblasts to the injury site where they become myocytes that fuse to form new muscle. How migration is regulated, though, remains largely unknown. Additionally, how migration and fusion, which both require dynamic rearrangement of the cytoskeleton, might be related is not well understood. c-MET, a receptor tyrosine kinase, is required for myogenic precursor cell migration into the limb for muscle development during embryogenesis. Using a genetic system to eliminate c-MET function specifically in adult mouse SCs, we found that c-MET was required for muscle regeneration in response to acute muscle injury. c-MET mutant myoblasts were defective in lamellipodia formation, had shorter ranges of migration, and migrated slower compared to control myoblasts. Surprisingly, c-MET was also required for efficient myocyte fusion, implicating c-MET in dual functions of regulating myoblast migration and myocyte fusion.

The budding yeast nuclear envelope adjacent to the nucleolus serves as a membrane sink during mitotic delay.

The mechanisms that dictate nuclear shape are largely unknown. Here we screened the budding yeast deletion collection for mutants with abnormal nuclear shape. A common phenotype was the appearance of a nuclear extension, particularly in mutants in DNA repair and chromosome segregation genes. Our data suggest that these mutations led to the abnormal nuclear morphology indirectly, by causing a checkpoint-induced cell-cycle delay. Indeed, delaying cells in mitosis by other means also led to the appearance of nuclear extensions, whereas inactivating the DNA damage checkpoint pathway in a DNA repair mutant reduced the fraction of cells with nuclear extensions. Formation of a nuclear extension was specific to a mitotic delay, because cells arrested in S or G2 had round nuclei. Moreover, the nuclear extension always coincided with the nucleolus, while the morphology of the DNA mass remained largely unchanged. Finally, we found that phospholipid synthesis continued unperturbed when cells delayed in mitosis, and inhibiting phospholipid synthesis abolished the formation of nuclear extensions. Our data suggest a mechanism that promotes nuclear envelope expansion during mitosis. When mitotic progression is delayed, cells sequester the added membrane to the nuclear envelope associated with the nucleolus, possibly to avoid disruption of intranuclear organization.

Vesicle trafficking maintains nuclear shape in Saccharomyces cerevisiae during membrane proliferation.

The parameters that control nuclear size and shape are poorly understood. In yeast, unregulated membrane proliferation, caused by deletion of the phospholipid biosynthesis inhibitor SPO7, leads to a single nuclear envelope "flare" that protrudes into the cytoplasm. This flare is always associated with the asymmetrically localized nucleolus, which suggests that the site of membrane expansion is spatially confined by an unknown mechanism. Here we show that in spo7Δ cells, mutations in vesicle-trafficking genes lead to multiple flares around the entire nucleus. These mutations also alter the distribution of small nucleolar RNA-associated nucleolar proteins independently of their effect on nuclear shape. Both single- and multi-flared nuclei have increased nuclear envelope surface area, yet they maintain the same nuclear/cell volume ratio as wild-type cells. These data suggest that, upon membrane expansion, the spatial confinement of the single nuclear flare is dependent on vesicle trafficking. Moreover, flares may facilitate maintenance of a constant nuclear/cell volume ratio in the face of altered membrane proliferation.

Unilateral vestibular schwannoma with other neurofibromatosis type 2-related tumors: clinical and molecular study of a unique phenotype.

OBJECT: Although the manifestations of neurofibromatosis Type 2 (NF2) vary, the hallmark is bilateral vestibular schwannomas (VSs). The authors studied the clinical course and genetic basis of unilateral VSs associated with other NF2-related tumors. METHODS: Forty-four adults presenting with unilateral VSs and other NF2-related tumors were identified. A comprehensive review of patient records and cranial imaging was conducted. Molecular analysis of the NF2 locus was performed in available tumors and paired blood specimens. Patient age at symptomatic onset ranged from 11 to 63 years (mean 32 years). Twenty-two patients (50%) presented with eighth cranial nerve dysfunction. Twenty-six presented with multiple lesions. Thirty-eight harbored other intracranial tumors and 27 had spinal tumors, with most lesions situated ipsilateral to the VS. No patient had a relative with NF2, although two of 63 offspring had isolated NF2-related findings. A contralateral VS developed in four patients 3 to 46 years after the symptomatic onset of a unilateral VS, and two of these patients experienced rapid progression to total deafness. Results of a Kaplan-Meier analysis identified actuarial chances of developing contralateral VS: 2.9% (3-17 years after the VS symptomatic onset of unilateral VS), 11% (18-24 years), and 28.8% (25-40 years). Mosaicism for the NF2 gene mutation was proven in eight patients. CONCLUSIONS: The authors describe the clinical features of this unique phenotype--unilateral VS with other NF2-related tumors. Persons with this phenotype should undergo evaluation and monitoring similar to that conducted in patients with NF2, and the possibility of aggressive contralateral VS formation should be considered in their treatment. Molecular genetic analysis is best performed using resected tumor specimens and will enable future studies to determine the genetic risks of individuals with mosaicism.