Automatic Clinical Assessment of Swallowing Behavior and Diagnosis of Silent Aspiration Using Wireless Multimodal Wearable Electronics.

Dysphagia is more common in conditions such as stroke, Parkinson's disease, and head and neck cancer. This can lead to pneumonia, choking, malnutrition, and dehydration. Currently, the diagnostic gold standard uses radiologic imaging, the videofluoroscopic swallow study (VFSS); however, it is expensive and necessitates specialized facilities and trained personnel. Although several devices attempt to address the limitations, none offer the clinical-grade quality and accuracy of the VFSS. Here, this study reports a wireless multimodal wearable system with machine learning for automatic, accurate clinical assessment of swallowing behavior and diagnosis of silent aspirations from dysphagia patients. The device includes a kirigami-structured electrode that suppresses changes in skin contact impedance caused by movements and a microphone with a gel layer that effectively blocks external noise for measuring high-quality electromyograms and swallowing sounds. The deep learning algorithm offers the classification of swallowing patterns while diagnosing silent aspirations, with an accuracy of 89.47%. The demonstration with post-stroke patients captures the system's significance in measuring multiple physiological signals in real-time for detecting swallowing disorders, validated by comparing them with the VFSS. The multimodal electronics can ensure a promising future for dysphagia healthcare and rehabilitation therapy, providing an accurate, non-invasive alternative for monitoring swallowing and aspiration events.

In vivo Electroporation of Skeletal Muscle Fibers in Mice.

In vitro models are essential for investigating the molecular, biochemical, and cell-biological aspects of skeletal muscle. Still, models that utilize cell lines or embryonic cells do not fully recapitulate mature muscle fibers in vivo. Protein function is best studied in mature differentiated tissue, where biological context is maintained, but this is often difficult when reliable detection reagents, such as antibodies, are not commercially available. Exogenous expression of tagged proteins in vivo solves some of these problems, but this approach can be technically challenging because either a mouse must be engineered for each protein of interest or viral vectors are required for adequate levels of expression. While viral vectors can infect target cells following local administration, they carry the risk of genome integration that may interfere with downstream analyses. Plasmids are another accessible expression system, but they require ancillary means of cell penetration; electroporation is a simple physical method for this purpose that requires minimal training or specialized equipment. Here, we describe a method for in vivo plasmid expression in a foot muscle following electroporation.

Pharyngeal pathology in a mouse model of oculopharyngeal muscular dystrophy is associated with impaired basal autophagy in myoblasts.

Oculopharyngeal muscular dystrophy (OPMD) is a late-onset dominant disease that primarily affects craniofacial muscles. Despite the fact that the genetic cause of OPMD is known to be expansion mutations in the gene encoding the nuclear polyadenosine RNA binding protein PABPN1, the molecular mechanisms of pathology are unknown and no pharmacologic treatments are available. Due to the limited availability of patient tissues, several animal models have been employed to study the pathology of OPMD. However, none of these models have demonstrated functional deficits in the muscles of the pharynx, which are predominantly affected by OPMD. Here, we used a knock-in mouse model of OPMD, Pabpn1 +/A17 , that closely genocopies patients. In Pabpn1 +/A17 mice, we detected impaired pharyngeal muscle function, and impaired pharyngeal satellite cell proliferation and fusion. Molecular studies revealed that basal autophagy, which is required for normal satellite cell function, is higher in pharynx-derived myoblasts than in myoblasts derived from limb muscles. Interestingly, basal autophagy is impaired in cells derived from Pabpn1 +/A17 mice. Pabpn1 knockdown in pharyngeal myoblasts failed to recapitulate the autophagy defect detected in Pabpn1 +/A17 myoblasts suggesting that loss of PABPN1 function does not contribute to the basal autophagy defect. Taken together, these studies provide the first evidence for pharyngeal muscle and satellite cell pathology in a mouse model of OPMD and suggest that aberrant gain of PABPN1 function contributes to the craniofacial pathology in OPMD.

Fibroadipogenic Progenitors Regulate the Basal Proliferation of Satellite Cells and Homeostasis of Pharyngeal Muscles via HGF Secretion.

Skeletal muscle stem cells, known as satellite cells (SCs), are quiescent in normal adult limb muscles. Injury stimulates SC proliferation, differentiation, and fusion to regenerate muscle structure. In pharyngeal muscles, which are critical for swallowing foods and liquids, SCs proliferate and fuse in the absence of injury. It is unknown what factors drive increased basal activity of pharyngeal SCs. Here, we determined how niche factors influence the status of pharyngeal versus limb SCs. In vivo, a subset of pharyngeal SCs present features of activated SCs, including large cell size and increased mitochondrial content. In this study, we discovered that the pharyngeal muscle contains high levels of active hepatocyte growth factor (HGF), which is known to activate SCs in mice and humans. We found that fibroadipogenic progenitors (FAPs) are the major cell type providing HGF and are thus responsible for basal proliferation of SCs in pharyngeal muscles. Lastly, we confirmed the critical role of FAPs for pharyngeal muscle function and maintenance. This study gives new insights to explain the distinctive SC activity of pharyngeal muscles.

Sex differences in the involvement of skeletal and cardiac muscles in myopathic Ano5-/- mice.

Limb-girdle muscular dystrophy R12 (LGMD-R12) is caused by recessive mutations in the Anoctamin-5 gene (ANO5, TMEM16E). Although ANO5 myopathy is not X-chromosome linked, we performed a meta-analysis of the research literature and found that three-quarters of patients with LGMD-R12 are males. Females are less likely to present with moderate to severe skeletal muscle and/or cardiac pathology. Because these sex differences could be explained in several ways, we compared males and females in a mouse model of LGMD-R12. This model recapitulates the sex differences in human LGMD-R12. Only male Ano5-/- mice had elevated serum creatine kinase after exercise and exhibited defective membrane repair after laser injury. In contrast, by these measures, female Ano5-/- mice were indistinguishable from wild type. Despite these differences, both male and female Ano5-/- mice exhibited exercise intolerance. Although exercise intolerance of male mice can be explained by skeletal muscle dysfunction, echocardiography revealed that Ano5-/- female mice had features of cardiomyopathy that may be responsible for their exercise intolerance. These findings heighten concerns that mutations of ANO5 in humans may be linked to cardiac disease.

Real-Time Functional Assay of Volumetric Muscle Loss Injured Mouse Masseter Muscles via Nanomembrane Electronics.

Skeletal muscle has a remarkable regeneration capacity to recover its structure and function after injury, except for the traumatic loss of critical muscle volume, called volumetric muscle loss (VML). Although many extremity VML models have been conducted, craniofacial VML has not been well-studied due to unavailable in vivo assay tools. Here, this paper reports a wireless, noninvasive nanomembrane system that integrates skin-wearable printed sensors and electronics for real-time, continuous monitoring of VML on craniofacial muscles. The craniofacial VML model, using biopsy punch-induced masseter muscle injury, shows impaired muscle regeneration. To measure the electrophysiology of small and round masseter muscles of active mice during mastication, a wearable nanomembrane system with stretchable graphene sensors that can be laminated to the skin over target muscles is utilized. The noninvasive system provides highly sensitive electromyogram detection on masseter muscles with or without VML injury. Furthermore, it is demonstrated that the wireless sensor can monitor the recovery after transplantation surgery for craniofacial VML. Overall, the presented study shows the enormous potential of the masseter muscle VML injury model and wearable assay tool for the mechanism study and the therapeutic development of craniofacial VML.

ANO5 ensures trafficking of annexins in wounded myofibers.

Mutations in ANO5 (TMEM16E) cause limb-girdle muscular dystrophy R12. Defective plasma membrane repair is a likely mechanism. Using myofibers from Ano5 knockout mice, we show that trafficking of several annexin proteins, which together form a cap at the site of injury, is altered upon loss of ANO5. Annexin A2 accumulates at the wound to nearly twice the level observed in WT fibers, while annexin A6 accumulation is substantially inhibited in the absence of ANO5. Appearance of annexins A1 and A5 at the cap is likewise diminished in the Ano5 knockout. These changes are correlated with an alteration in annexin repair cap fine structure and shedding of annexin-positive vesicles. We conclude that loss of annexin coordination during repair is disrupted in Ano5 knockout mice and underlies the defective repair phenotype. Although ANO5 is a phospholipid scramblase, abnormal repair is rescued by overexpression of a scramblase-defective ANO5 mutant, suggesting a novel, scramblase-independent role of ANO5 in repair.

From worms to fish to mice.

An multi-species approach can be used to identify small molecules with properties that might prove useful for the treatment of some neuromuscular diseases.

Generation of craniofacial myogenic progenitor cells from human induced pluripotent stem cells for skeletal muscle tissue regeneration.

Craniofacial skeletal muscle is composed of approximately 60 muscles, which have critical functions including food uptake, eye movements and facial expressions. Although craniofacial muscles have significantly different embryonic origin, most current skeletal muscle differentiation protocols using human induced pluripotent stem cells (iPSCs) are based on somite-derived limb and trunk muscle developmental pathways. Since the lack of a protocol for craniofacial muscles is a significant gap in the iPSC-derived muscle field, we have developed an optimized protocol to generate craniofacial myogenic precursor cells (cMPCs) from human iPSCs by mimicking key signaling pathways during craniofacial embryonic myogenesis. At each different stage, human iPSC-derived cMPCs mirror the transcription factor expression profiles seen in their counterparts during embryo development. After the bi-potential cranial pharyngeal mesoderm is established, cells are committed to cranial skeletal muscle lineages with inhibition of cardiac lineages and are purified by flow cytometry. Furthermore, identities of iPSC-derived cMPCs are verified with human primary myoblasts from craniofacial muscles using RNA sequencing. These data suggest that our new method could provide not only in vitro research tools to study muscle specificity of muscular dystrophy but also abundant and reliable cellular resources for tissue engineering to support craniofacial reconstruction surgery.

Myosoft: An automated muscle histology analysis tool using machine learning algorithm utilizing FIJI/ImageJ software.

METHODS: Muscle sections were stained for cell boundary (laminin) and myofiber type (myosin heavy chain isoforms). Myosoft, running in the open access software platform FIJI (ImageJ), was used to analyze myofiber size and type in transverse sections of entire gastrocnemius/soleus muscles. RESULTS: Myosoft provides an accurate analysis of hundreds to thousands of muscle fibers within 25 minutes, which is >10-times faster than manual analysis. We demonstrate that Myosoft is capable of handling high-content images even when image or staining quality is suboptimal, which is a marked improvement over currently available and comparable programs. CONCLUSIONS: Myosoft is a reliable, accurate, high-throughput, and convenient tool to analyze high-content muscle histology. Myosoft is freely available to download from Github at https://github.com/Hyojung-Choo/Myosoft/tree/Myosoft-hub.

A tale of two niches: differential functions for VCAM-1 in satellite cells under basal and injured conditions.

Cell-cell adhesion molecules play key roles in maintaining quiescence or promoting activation of various stem cells in their niche. Muscle stem cells called satellite cells (SC) are critical for skeletal muscle regeneration after injury, but little is known about the role of adhesion molecules in regulating the behavior of these stem cells. Vascular cell adhesion molecule-1 (VCAM-1) is a cell-cell adhesion protein expressed on quiescent and activated SC whose function is unknown in this context. We deleted Vcam1 from SC using an inducible Cre recombinase in young mice. In the injured niche, Vcam1-/- SC underwent premature lineage progression to a more differentiated state as well as apoptosis leading to a transient delay in myofiber growth during regeneration. Apoptosis was also increased in Vcam1-/- SC in vitro concomitant with decreased levels of phosphorylated Akt, a prosurvival signal activated by VCAM-1 signaling in other cell types. During muscle regeneration, we observed an influx of immune cells expressing α4 integrin, a component of the major, high-affinity VCAM-1 ligand, α4ß1 integrin. Furthermore, α4 integrin mRNA and protein were induced in SC 2 days after injury. These results suggest that SC interact with other SC as well as immune cells through α4ß1 integrin in the injured niche to promote expansion of SC. In the uninjured niche, multiple cell types also expressed α4 integrin. However, only basal fusion of Vcam1-/- SC with myofibers was decreased, contributing to decreased myofiber growth. These studies define differential roles for VCAM-1 in SC depending on the state of their niche.

Inner Mitochondrial Membrane Disruption Links Apoptotic and Agonist-Initiated Phosphatidylserine Externalization in Platelets.

OBJECTIVE: Phosphatidylserine exposure mediates platelet procoagulant function and regulates platelet life span. Apoptotic, necrotic, and integrin-mediated mechanisms have been implicated as intracellular determinants of platelet phosphatidylserine exposure. Here, we investigate (1) the role of mitochondrial events in platelet phosphatidylserine exposure initiated by these distinct stimuli and (2) the cellular interactions of the procoagulant platelet in vitro and in vivo. APPROACH AND RESULTS: Key mitochondrial events were examined, including cytochrome c release and inner mitochondrial membrane (IMM) disruption. In both ABT-737 (apoptotic) and agonist (necrotic)-treated platelets, phosphatidylserine externalization was temporally correlated with IMM disruption. Agonist stimulation resulted in rapid cyclophilin D-dependent IMM disruption that coincided with phosphatidylserine exposure. ABT-737 treatment caused rapid cytochrome c release, eventually followed by caspase-dependent IMM disruption that again closely coincided with phosphatidylserine exposure. A nonmitochondrial and integrin-mediated mechanism has been implicated in the formation of a novel phosphatidylserine-externalizing platelet subpopulation. Using image cytometry, this subpopulation is demonstrated to be the result of the interaction of an aggregatory platelet and a procoagulant platelet rather than indicative of a novel intracellular mechanism regulating platelet phosphatidylserine externalization. Using electron microscopy, similar interactions between aggregatory and procoagulant platelets are demonstrated in vitro and in vivo within a mesenteric vein hemostatic thrombus. CONCLUSIONS: Platelet phosphatidylserine externalization is closely associated with the mitochondrial event of IMM disruption identifying a common pathway in phosphatidylserine-externalizing platelets. The limited interaction of procoagulant platelets and integrin-active aggregatory platelets identifies a potential mechanism for procoagulant platelet retention within the hemostatic thrombus.

Karyopherin Alpha 1 Regulates Satellite Cell Proliferation and Survival by Modulating Nuclear Import.

Satellite cells are stem cells with an essential role in skeletal muscle repair. Precise regulation of gene expression is critical for proper satellite cell quiescence, proliferation, differentiation and self-renewal. Nuclear proteins required for gene expression are dependent on the nucleocytoplasmic transport machinery to access to nucleus, however little is known about regulation of nuclear transport in satellite cells. The best characterized nuclear import pathway is classical nuclear import which depends on a classical nuclear localization signal (cNLS) in a cargo protein and the heterodimeric import receptors, karyopherin alpha (KPNA) and beta (KPNB). Multiple KPNA1 paralogs exist and can differ in importing specific cNLS proteins required for cell differentiation and function. We show that transcripts for six Kpna paralogs underwent distinct changes in mouse satellite cells during muscle regeneration accompanied by changes in cNLS proteins in nuclei. Depletion of KPNA1, the most dramatically altered KPNA, caused satellite cells in uninjured muscle to prematurely activate, proliferate and undergo apoptosis leading to satellite cell exhaustion with age. Increased proliferation of satellite cells led to enhanced muscle regeneration at early stages of regeneration. In addition, we observed impaired nuclear localization of two key KPNA1 cargo proteins: p27, a cyclin-dependent kinase inhibitor associated with cell cycle control and lymphoid enhancer factor 1, a critical cotranscription factor for ß-catenin. These results indicate that regulated nuclear import of proteins by KPNA1 is critical for satellite cell proliferation and survival and establish classical nuclear import as a novel regulatory mechanism for controlling satellite cell fate. Stem Cells 2016;34:2784-2797.

Pharyngeal Satellite Cells Undergo Myogenesis Under Basal Conditions and Are Required for Pharyngeal Muscle Maintenance.

The pharyngeal muscles of the nasal, oral, and laryngeal pharynxes are required for swallowing. Pharyngeal muscles are preferentially affected in some muscular dystrophies yet spared in others. Muscle stem cells, called satellite cells, may be critical factors in the development of pharyngeal muscle disorders; however, very little is known about pharyngeal satellite cells (PSC) and their role in pharyngeal muscles. We show that PSC are distinct from the commonly studied hindlimb satellite cells both transcriptionally and biologically. Under basal conditions PSC proliferate, progress through myogenesis, and fuse with pharyngeal myofibers. Furthermore, PSC exhibit biologic differences dependent on anatomic location in the pharynx. Importantly, PSC are required to maintain myofiber size and myonuclear number in pharyngeal myofibers. Together, these results demonstrate that PSC are critical for pharyngeal muscle maintenance and suggest that satellite cell impairment could contribute to pharyngeal muscle pathology associated with various muscular dystrophies and aging.

Mitochondrial complex I deficiency enhances skeletal myogenesis but impairs insulin signaling through SIRT1 inactivation.

To address whether mitochondrial biogenesis is essential for skeletal myogenesis, C2C12 myogenesis was investigated after knockdown of NADH dehydrogenase (ubiquintone) flavoprotein 1 (NDUFV1), which is an oxidative phosphorylation complex I subunit that is the first subunit to accept electrons from NADH. The NDUFVI knockdown enhanced C2C12 myogenesis by decreasing the NAD(+)/NADH ratio and subsequently inactivating SIRT1 and SIRT1 activators (pyruvate, SRT1720, and resveratrol) abolished the NDUFV1 knockdown-induced myogenesis enhancement. However, the insulin-elicited activation of insulin receptor ß (IRß) and insulin receptor substrate-1 (IRS-1) was reduced with elevated levels of protein-tyrosine phosphatase 1B after NDUFV1 knockdown in C2C12 myotubes. The NDUFV1 knockdown-induced blockage of insulin signaling was released by protein-tyrosine phosphatase 1B knockdown in C2C12 myotubes, and we found that NDUFV1 or SIRT1 knockdown did not affect mitochondria biogenesis during C2C12 myogenesis. Based on these data, we can conclude that complex I dysfunction-induced SIRT1 inactivation leads to myogenesis enhancement but blocks insulin signaling without affecting mitochondria biogenesis.

Mitochondrial calcium and reactive oxygen species regulate agonist-initiated platelet phosphatidylserine exposure.

OBJECTIVE: To study the interactions of cytoplasmic calcium elevation, mitochondrial permeability transition pore (mPTP) formation, and reactive oxygen species formation in the regulation of phosphatidylserine (PS) exposure in platelets. METHODS AND RESULTS: mPTP formation, but not the degree of cytoplasmic calcium elevation, was associated with PS exposure in wild-type, cyclophilin D-null, ionomycin-treated, and reactive oxygen species-treated platelets. In the absence of the mPTP regulator cyclophilin D, agonist-initiated mPTP formation and high-level PS exposure were markedly blunted, but cytoplasmic calcium transients were unchanged. Mitochondrial calcium (Ca(2+)(mit)) transients and reactive oxygen species, key regulators of mPTP formation, were examined in strongly stimulated platelets. Increased reactive oxygen species production occurred in strongly stimulated platelets and was dependent on extracellular calcium entry, but not the presence of cyclophilin D. Ca(2+)(mit) increased significantly in strongly stimulated platelets. Abrogation of Ca(2+)(mit) entry, either by inhibition of the Ca(2+)(mit) uniporter or mitochondrial depolarization, prevented mPTP formation and exposure but not platelet aggregation or granule release. CONCLUSIONS: Sustained cytoplasmic calcium levels are necessary, but not sufficient, for high-level PS exposure in response to agonists. Increased Ca(2+)(mit) levels are a key signal initiating mPTP formation and PS exposure. Blockade of Ca(2+)(mit) entry allows the specific inhibition of platelet procoagulant activity.

Platelet biogenesis and functions require correct protein O-glycosylation.

Platelets express a variety of membrane and secreted glycoproteins, but the importance of glycosylation to platelet functions is poorly understood. To explore the importance of O-glycosylation, we generated mice with a targeted deletion of Cosmc in murine endothelial/hematopoietic cells (EHC) (EHC Cosmc(-/y)). X-linked Cosmc encodes an essential chaperone that regulates protein O-glycosylation. This targeted mutation resulted in lethal perinatal hemorrhage in the majority of mice, and the surviving mice displayed severely prolonged tail-bleeding times and macrothrombocytopenia. EHC Cosmc(-/y) platelets exhibited a marked decrease in GPIb-IX-V function and agonist-mediated integrin αIIbß3 activation, associated with loss of interactions with von Willebrand factor and fibrinogen, respectively. Significantly, three O-glycosylated glycoproteins, GPIbα, αIIb, and GPVI normally on platelet surfaces that play essential roles in platelet functions, were partially proteolyzed in EHC Cosmc(-/y) platelets. These results demonstrate that extended O-glycans are required for normal biogenesis of the platelets as well as the expression and functions of their essential glycoproteins, and that variations in O-glycosylation may contribute to altered hemostasis.

Lipid raft proteome reveals that oxidative phosphorylation system is associated with the plasma membrane.

Although accumulating proteomic analyses have supported the fact that mitochondrial oxidative phosphorylation (OXPHOS) complexes are localized in lipid rafts, which mediate cell signaling, immune response and host-pathogen interactions, there has been no in-depth study of the physiological functions of lipid-raft OXPHOS complexes. Here, we show that many subunits of OXPHOS complexes were identified from the lipid rafts of human adipocytes, C2C12 myotubes, Jurkat cells and surface biotin-labeled Jurkat cells via shotgun proteomic analysis. We discuss the findings of OXPHOS complexes in lipid rafts, the role of the surface ATP synthase complex as a receptor for various ligands and extracellular superoxide generation by plasma membrane oxidative phosphorylation complexes.

Mitochondrial oxidative phosphorylation system is recruited to detergent-resistant lipid rafts during myogenesis.

Since detergent-resistant lipid rafts play important roles in the signal transduction for myogenesis, their comprehensive proteomic analysis could provide new insights to understand their function in myotubes. Here, the detergent-resistant lipid rafts were isolated from C2C12 myotubes and analyzed by capillary RPLC/MS/MS. Among the 327 proteins (or protein groups) identified, 28% were categorized to the plasma membrane or raft proteins, 29% to mitochondria, 20% to microsomal proteins, 10% to other proteins, and 13% to unknown proteins. The localization of oxidative phosphorylation (OXPHOS) complexes in the sarcolemma lipid rafts was further confirmed from C2C12 myotubes by cellular fractionation, surface-biotin labeling, immunofluorescence, and lipid raft fractionation. After adding exogenous cytochrome c, the sarcolemma isolated from myotubes had an ability to consume oxygen in the presence of NADH or succinate. The generation of NADH-dependent extracellular superoxide was increased by inhibiting or downregulating OXPHOS I, III, and IV in myotubes, indicating that OXPHOS proteins are major sources for extracellular ROS in skeletal muscle. With all these data, we can conclude that OXPHOS proteins are associated with the sarcolemma lipid rafts during C2C12 myogenesis to generate extracellular ROS.

Ginsenoside Rh2 induces ligand-independent Fas activation via lipid raft disruption.

Lipid rafts are plasma membrane platforms mediating signal transduction pathways for cellular proliferation, differentiation and apoptosis. Here, we show that membrane fluidity was increased in HeLa cells following treatment with ginsenoside Rh2 (Rh2), as determined by cell staining with carboxy-laurdan (C-laurdan), a two-photon dye designed for measuring membrane hydrophobicity. In the presence of Rh2, caveolin-1 appeared in non-raft fractions after sucrose gradient ultracentrifugation. In addition, caveolin-1 and GM1, lipid raft landmarkers, were internalized within cells after exposure to Rh2, indicating that Rh2 might disrupt lipid rafts. Since cholesterol overloading, which fortifies lipid rafts, prevented an increase in Rh2-induced membrane fluidity, caveolin-1 internalization and apoptosis, lipid rafts appear to be essential for Rh2-induced apoptosis. Moreover, Rh2-induced Fas oligomerization was abolished following cholesterol overloading, and Rh2-induced apoptosis was inhibited following treatment with siRNA for Fas. This result suggests that Rh2 is a novel lipid raft disruptor leading to Fas oligomerization and apoptosis.