Murine muscle stem cell response to perturbations of the neuromuscular junction are attenuated with aging.

During aging and neuromuscular diseases, there is a progressive loss of skeletal muscle volume and function impacting mobility and quality of life. Muscle loss is often associated with denervation and a loss of resident muscle stem cells (satellite cells or MuSCs); however, the relationship between MuSCs and innervation has not been established. Herein, we administered severe neuromuscular trauma to a transgenic murine model that permits MuSC lineage tracing. We show that a subset of MuSCs specifically engraft in a position proximal to the neuromuscular junction (NMJ), the synapse between myofibers and motor neurons, in healthy young adult muscles. In aging and in a mouse model of neuromuscular degeneration (Cu/Zn superoxide dismutase knockout - Sod1-/-), this localized engraftment behavior was reduced. Genetic rescue of motor neurons in Sod1-/- mice reestablished integrity of the NMJ in a manner akin to young muscle and partially restored MuSC ability to engraft into positions proximal to the NMJ. Using single cell RNA-sequencing of MuSCs isolated from aged muscle, we demonstrate that a subset of MuSCs are molecularly distinguishable from MuSCs responding to myofiber injury and share similarity to synaptic myonuclei. Collectively, these data reveal unique features of MuSCs that respond to synaptic perturbations caused by aging and other stressors.

Breathable, large-area epidermal electronic systems for recording electromyographic activity during operant conditioning of H-reflex.

Operant conditioning of Hoffmann's reflex (H-reflex) is a non-invasive and targeted therapeutic intervention for patients with movement disorders following spinal cord injury. The reflex-conditioning protocol uses electromyography (EMG) to measure reflexes from specific muscles elicited using transcutaneous electrical stimulation. Despite recent advances in wearable electronics, existing EMG systems that measure muscle activity for operant conditioning of spinal reflexes still use rigid metal electrodes with conductive gels and aggressive adhesives, while requiring precise positioning to ensure reliability of data across experimental sessions. Here, we present the first large-area epidermal electronic system (L-EES) and demonstrate its use in every step of the reflex-conditioning protocol. The L-EES is a stretchable and breathable composite of nanomembrane electrodes (16 electrodes in a four by four array), elastomer, and fabric. The nanomembrane electrode array enables EMG recording from a large surface area on the skin and the breathable elastomer with fabric is biocompatible and comfortable for patients. We show that L-EES can record direct muscle responses (M-waves) and H-reflexes, both of which are comparable to those recorded using conventional EMG recording systems. In addition, L-EES may improve the reflex-conditioning protocol; it has potential to automatically optimize EMG electrode positioning, which may reduce setup time and error across experimental sessions.

Dissecting Murine Muscle Stem Cell Aging through Regeneration Using Integrative Genomic Analysis.

During aging, there is a progressive loss of volume and function in skeletal muscle that impacts mobility and quality of life. The repair of skeletal muscle is regulated by tissue-resident stem cells called satellite cells (or muscle stem cells [MuSCs]), but in aging, MuSCs decrease in numbers and regenerative capacity. The transcriptional networks and epigenetic changes that confer diminished regenerative function in MuSCs as a result of natural aging are only partially understood. Herein, we use an integrative genomics approach to profile MuSCs from young and aged animals before and after injury. Integration of these datasets reveals aging impacts multiple regulatory changes through significant differences in gene expression, metabolic flux, chromatin accessibility, and patterns of transcription factor (TF) binding activities. Collectively, these datasets facilitate a deeper understanding of the regulation tissue-resident stem cells use during aging and healing.

All-printed nanomembrane wireless bioelectronics using a biocompatible solderable graphene for multimodal human-machine interfaces.

Recent advances in nanomaterials and nano-microfabrication have enabled the development of flexible wearable electronics. However, existing manufacturing methods still rely on a multi-step, error-prone complex process that requires a costly cleanroom facility. Here, we report a new class of additive nanomanufacturing of functional materials that enables a wireless, multilayered, seamlessly interconnected, and flexible hybrid electronic system. All-printed electronics, incorporating machine learning, offers multi-class and versatile human-machine interfaces. One of the key technological advancements is the use of a functionalized conductive graphene with enhanced biocompatibility, anti-oxidation, and solderability, which allows a wireless flexible circuit. The high-aspect ratio graphene offers gel-free, high-fidelity recording of muscle activities. The performance of the printed electronics is demonstrated by using real-time control of external systems via electromyograms. Anatomical study with deep learning-embedded electrophysiology mapping allows for an optimal selection of three channels to capture all finger motions with an accuracy of about 99% for seven classes.

Superoxide-mediated oxidative stress accelerates skeletal muscle atrophy by synchronous activation of proteolytic systems.

The maintenance of skeletal muscle mass depends on the overall balance between the rates of protein synthesis and degradation. Thus, age-related muscle atrophy and function, commonly known as sarcopenia, may result from decreased protein synthesis, increased proteolysis, or simultaneous changes in both processes governed by complex multifactorial mechanisms. Growing evidence implicates oxidative stress and reactive oxygen species (ROS) as an essential regulator of proteolysis. Our previous studies have shown that genetic deletion of CuZn superoxide dismutase (CuZnSOD, Sod1) in mice leads to elevated oxidative stress, muscle atrophy and weakness, and an acceleration in age-related phenotypes associated with sarcopenia. The goal of this study is to determine whether oxidative stress directly influences the acceleration of proteolysis in skeletal muscle of Sod1-/- mice as a function of age. Compared to control, Sod1-/- muscle showed a significant elevation in protein carbonyls and 3-nitrotyrosine levels, suggesting high oxidative and nitrosative protein modifications were present. In addition, age-dependent muscle atrophy in Sod1-/- muscle was accompanied by an upregulation of the cysteine proteases, calpain, and caspase-3, which are known to play a key role in the initial breakdown of sarcomeres during atrophic conditions. Furthermore, an increase in oxidative stress-induced muscle atrophy was also strongly coupled with simultaneous activation of two major proteolytic systems, the ubiquitin-proteasome and lysosomal autophagy pathways. Collectively, our data suggest that chronic oxidative stress in Sod1-/- mice accelerates age-dependent muscle atrophy by enhancing coordinated activation of the proteolytic systems, thereby resulting in overall protein degradation.

Microengineered human blood-brain barrier platform for understanding nanoparticle transport mechanisms.

Challenges in drug development of neurological diseases remain mainly ascribed to the blood-brain barrier (BBB). Despite the valuable contribution of animal models to drug discovery, it remains difficult to conduct mechanistic studies on the barrier function and interactions with drugs at molecular and cellular levels. Here we present a microphysiological platform that recapitulates the key structure and function of the human BBB and enables 3D mapping of nanoparticle distributions in the vascular and perivascular regions. We demonstrate on-chip mimicry of the BBB structure and function by cellular interactions, key gene expressions, low permeability, and 3D astrocytic network with reduced reactive gliosis and polarized aquaporin-4 (AQP4) distribution. Moreover, our model precisely captures 3D nanoparticle distributions at cellular levels and demonstrates the distinct cellular uptakes and BBB penetrations through receptor-mediated transcytosis. Our BBB platform may present a complementary in vitro model to animal models for prescreening drug candidates for the treatment of neurological diseases.

Engineered Heterochronic Parabiosis in 3D Microphysiological System for Identification of Muscle Rejuvenating Factors.

Exposure of aged mice to a young systemic milieu revealed remarkable rejuvenation effects on aged tissues, including skeletal muscle. Although some candidate factors have been identified, the exact identity and the underlying mechanisms of putative rejuvenating factors remain elusive, mainly due to the complexity of in vivo parabiosis. Here, we present an in vitro muscle parabiosis system that integrates young- and old-muscle stem cell vascular niche on a three-dimensional microfluidic platform designed to recapitulate key features of native muscle stem cell microenvironment. This innovative system enables mechanistic studies of cellular dynamics and molecular interactions within the muscle stem cell niche, especially in response to conditional extrinsic stimuli of local and systemic factors. We demonstrate that vascular endothelial growth factor (VEGF) signaling from endothelial cells and myotubes synergistically contribute to the rejuvenation of the aged muscle stem cell function. Moreover, with the adjustable on-chip system, we can mimic both blood transfusion and parabiosis and detect the time-varying effects of anti-geronic and pro-geronic factors in a single organ or multi-organ systems. Our unique approach presents a complementary in vitro model to supplement in vivo parabiosis for identifying potential anti-geronic factors responsible for revitalizing aging organs.

Critical Limb Ischemia Induces Remodeling of Skeletal Muscle Motor Unit, Myonuclear-, and Mitochondrial-Domains.

Critical limb ischemia, the most severe form of peripheral artery disease, leads to extensive damage and alterations to skeletal muscle homeostasis. Although recent research has investigated the tissue-specific responses to ischemia, the role of the muscle stem cell in the regeneration of its niche components within skeletal muscle has been limited. To elucidate the regenerative mechanism of the muscle stem cell in response to ischemic insults, we explored cellular interactions between the vasculature, neural network, and muscle fiber within the muscle stem cell niche. Using a surgical murine hindlimb ischemia model, we first discovered a significant increase in subsynaptic nuclei and remodeling of the neuromuscular junction following ischemia-induced denervation. In addition, ischemic injury causes significant alterations to the myofiber through a muscle stem cell-mediated accumulation of total myonuclei and a concomitant decrease in myonuclear domain size, possibly to enhance the transcriptional and translation output and restore muscle mass. Results also revealed an accumulation of total mitochondrial content per myonucleus in ischemic myofibers to compensate for impaired mitochondrial function and high turnover rate. Taken together, the findings from this study suggest that the muscle stem cell plays a role in motor neuron reinnervation, myonuclear accretion, and mitochondrial biogenesis for skeletal muscle regeneration following ischemic injury.