Alterations of Whole-Body Glucose Metabolism in a Feline SARS-CoV-2 Infection Model.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has been especially devastating to patients with comorbidities, including metabolic and cardiovascular diseases. Elevated blood glucose during SARS-CoV-2 infection increased mortality of COVID-19 patients, although the mechanisms are not well understood. It has been previously demonstrated that glucose transport and utilization is a crucial pathway for other highly infectious RNA viruses. Thus, we hypothesized that SARS-CoV-2 infection could lead to alterations in cellular and whole-body glucose metabolism. Specific pathogen free domestic cats were intratracheally inoculated with USA-WA1/2020 (Wild-type) SARS-CoV-2 or vehicle-inoculated, then sacrificed at 4- and 8-days post-inoculation (dpi). Blood glucose and cortisol concentrations were elevated at 4 and 8 dpi. Blood ketones, insulin, and angiotensin 2 concentrations remained unchanged throughout the experimental timeline. SARS-CoV-2 RNA was detected in the lung and heart, without changes in angiotensin converting enzyme 2 (ACE2) RNA expression. In the lung, SARS-CoV-2 infection increased glucose transporter 1 (GLUT1) protein level at 4 and 8 dpi., while GLUT4 level was only upregulated at 8 dpi. In the heart, GLUT-1 and -4 protein levels remained unchanged. Furthermore, GLUT1 level was upregulated in the skeletal muscle at 8 dpi, and AMPK was activated in the heart of infected cats. SARS-CoV-2 infection increased blood glucose concentration and pulmonary GLUT protein levels. These findings suggest that SARS-CoV-2 infection induces metabolic reprogramming primarily in the lung to support viral replication. Furthermore, this translational feline model mimicked human COVID-19 and could be used to explore novel therapeutic targets to treat metabolic disease during SARS-CoV-2 infection.

An Overview of the Effect of Aging on the Female Urethra.

Urethral function declines by roughly 15% per decade and profoundly contributes to the pathogenesis of urinary incontinence. Individuals with poor urethral function are more likely to fail surgical management for stress incontinence that focus on improving urethral support. The reduced number of intramuscular nerves and the morphologic changes in muscle and connective tissue collectively impact urethral function as women age. Imaging technologies like MRI and ultrasound have advanced our understanding of these changes. However, substantial knowledge gaps remain. Addressing these gaps can be crucial for developing better prevention and treatment strategies, ultimately enhancing the quality of life for aging women.

Correlation between congenital pelvic floor muscle development assessed by magnetic resonance imaging and postoperative defecation.

OBJECTIVE: Children with congenital anorectal malformation (CAM) experience challenges with defecation. This study aims to assess defecation in preschool-age children with CAM and to evaluate the correlation between pelvic floor muscle developed assessed by magnetic resonance imaging (MRI) and postoperative defecation. METHODS: We collected clinical data and MRI results from 89 male children with CAM. The bowel function scores for children with Perineal (cutaneous) fistula, Rectourethral fistula(Prostatic or Bulbar), and Rectovesical fistula were computed. MRI scans were subjected to image analysis of the striated muscle complex (SMC). The association between pelvic floor muscle score and bowel function score was examined using the Cochran-Armitage Trend Test. RESULTS: We observed that 77.4% of the SMC scores by MRI for Perineal fistula were good. The Rectourethral fistula SMC score was 40.6% for moderate and 59.4% for poor. The SMC score for Rectovesical fistula was 100% for moderate. Furthermore, 77.4% of patients with Perineal fistula had bowel function scores (BFS) ≥ 17 points. Among those with Rectourethral fistula and Rectovesical fistula, 12.5% and 0 had BFS ≥ 17 points, respectively. An analysis of muscle development and bowel function in patients with Rectovesical fistula, Rectourethral fistula, and Perineal fistula revealed a correlation between SMC development and BFS. Subgroup analysis showed that the Perineal fistula had statistical significance; however, the Rectourethral fistula and Rectovesical fistula were not statistically significant. CONCLUSION: A correlation exists between pelvic floor muscle development and postoperative defecation in children with Perineal fistula.

Structure of mavacamten-free human cardiac thick filaments within the sarcomere by cryoelectron tomography.

Heart muscle has the unique property that it can never rest; all cardiomyocytes contract with each heartbeat which requires a complex control mechanism to regulate cardiac output to physiological requirements. Changes in calcium concentration regulate the thin filament activation. A separate but linked mechanism regulates the thick filament activation, which frees sufficient myosin heads to bind the thin filament, thereby producing the required force. Thick filaments contain additional nonmyosin proteins, myosin-binding protein C and titin, the latter being the protein that transmits applied tension to the thick filament. How these three proteins interact to control thick filament activation is poorly understood. Here, we show using 3-D image reconstruction of frozen-hydrated human cardiac muscle myofibrils lacking exogenous drugs that the thick filament is structured to provide three levels of myosin activation corresponding to the three crowns of myosin heads in each 429Å repeat. In one crown, the myosin heads are almost completely activated and disordered. In another crown, many myosin heads are inactive, ordered into a structure called the interacting heads motif. At the third crown, the myosin heads are ordered into the interacting heads motif, but the stability of that motif is affected by myosin-binding protein C. We think that this hierarchy of control explains many of the effects of length-dependent activation as well as stretch activation in cardiac muscle control.

Sequences in the myosin A rod interact with UNC-89/obscurin and the zinc-finger protein UNC-98 during thick filament assembly and M-line formation in C. elegans striated muscle.

The M-line of striated muscle is a complex structure that anchors myosin-containing thick filaments and also participates in signaling and proteostasis. While the physical associations among many M-line components have been defined, the mechanism of thick filament attachment is not completely understood. In Caenorhabditis elegans, myosin A is essential for viability and forms the site of M-line attachment at the center of the filament, whereas myosin B forms the filament arms. Using a mutant myosin A that forms ectopic filaments, we examined interactions between myosin A and M-line proteins in intact muscle cells. Ectopic myosin A recruits the giant kinase UNC-89/obscurin, a presumed scaffolding protein, in an interaction that requires the zinc-finger protein UNC-98, but not UNC-82/NUAK, UNC-97/PINCH, or UNC-96. In myosin A mutants, UNC-89/obscurin patterning is highly defective in embryos and adults. A chimeric myosin containing 169 residues of the myosin A C-terminal rod, coincident with the UNC-98/ZnF binding site, is sufficient for colocalization of UNC-89/obscurin and UNC-98/ZnF in M-line structures whereas a myosin chimera lacking these residues colocalizes with UNC-89/obscurin in M-lines that lack UNC-98. Thus, at least two myosin A rod regions contribute independently to M-line organization. We hypothesize that these M-line-organizing functions correspond to the essential "filament initiation function" performed by this isoform.

A Case of Liver Cancer with Overlapping Myasthenia Gravis, Myocarditis, Seronegative Autoimmune Autonomic Ganglionopathy, and Myositis Symptoms Induced by Atezolizumab: A Case Report.

An 83-year-old man with hepatocellular carcinoma developed muscle weakness, ptosis, and dyspnea 3 weeks after receiving atezolizumab. Soon after, mechanical ventilation was initiated, which was followed by marked blood pressure spikes. The levels of creatine kinase and troponin-I were significantly elevated, and acetylcholine receptor antibodies were positive. The patient was diagnosed with immune checkpoint inhibitor (ICI)-induced myositis, myasthenia gravis (MG), myocarditis, and suspected autoimmune autonomic ganglionopathy (AAG). After immunotherapy, the serum markers and blood pressure normalized, and he was weaned from the ventilator after five months. To our knowledge, this is the first reported case of AAG secondary to ICI-induced myositis, MG, and myocarditis.

Monensin poisoning outbreak in free-ranging and captive birds.

Monensin poisoning is uncommon and has been rarely reported in birds. This work aimed to described clinical-pathological aspects of an outbreak of monensin poisoning in captive and free-ranging birds. Thirty-seven of 600 captive birds fed a diet containing 893.19 mg/kg of monensin died within 10 days (mortality 6.17%). There was no ionophore antibiotics on the feed label supplied to captive birds, which established an error in feed production. Necropsies were performed on twelve animals: Muscovy duck (Cairina moschata) (2/12), greater rhea (Rhea americana) (2/12), black-necked swan (Cygnus melancoryphus) (2/12), garganey (Anas querquedula) (1/12), ostrich (Struthio camelus) (1/12), and common pigeon (Columbus livia) (4/12). These four common pigeons were free-ranging birds and died after eating the same contaminated feed. Birds were mainly found dead, however in animals which clinical signs were observed (Columba livia, Rhea americana, Cairina moschata, Anas querquedula, and Struthio camelus), they included incoordination, inability to stand, and intense prostration, that ranged from 24 to 72 h until death. Grossly, five birds had focally extensive pale firm areas in the myocardium and two had in the skeletal muscles, one being concomitant lesions. Histologically, muscle necrosis and degeneration were observed in striated musculature (skeletal and/or heart) in all birds analyzed. Monensin poisoning outbreaks can affect free-ranging birds that are fed on external feeders, as well as captive birds, due to an error in the feed formulation.

Pembrolizumab-induced Myopathy with Anti-striated Muscle Antibodies Successfully Treated by Plasma Exchange.

A 70-year-old woman with advanced endometrial cancer developed right ptosis and muscle weakness in the right quadriceps after pembrolizumab administration. Serum creatine kinase (CK) levels were elevated, and anti-striated muscle antibodies were positive. On magnetic resonance imaging, the right vastus lateral muscle showed an abnormal signal. She was diagnosed with pembrolizumab-induced myopathy. We initiated plasma exchange (PE), and the ptosis immediately resolved. We then introduced oral corticosteroids, which improved her muscle weakness. We were able to rapidly diagnose her with ocular symptoms and serum CK level elevation. The early initiation of PE might prevent the exacerbation of pembrolizumab-induced myopathy.

Dysfunction of the aging female mouse urethra is associated with striated muscle loss and increased fibrosis: an initial report.

The decline of urethral function with advancing age plays a major role in urinary incontinence in women, impairing quality of life and economically burdening the health care system. However, none of the current urinary incontinence treatments address the declining urethral function with aging, and the mechanisms by which aging impacts urethra physiology remain little known or explored. Here, we have compared functional, morphometric, and global gene expression of urethral tissues between young and old female mice. Bladder leak point pressure (LPP) measurement showed that the aged female mice had 26.55% lower LPP compared to younger mice. Vectorized Scale-Invariant Pattern Recognition (VIPR) analysis of the relative abundance of different tissue components revealed that the mid-urethra of old female mice contains less striated muscle, more extracellular matrix/fibrosis, and diminished elastin fibers ratio compared to young mice. Gene expression profiling analysis (bulk RNA-seq of the whole urethra) showed more down-regulated genes in aged than young mice. Immune response and muscle-related (striated and smooth) pathways were predominantly enriched. In contrast, keratinization, skin development, and cell differentiation pathways were significantly downregulated in aged urethral tissues compared to those from young female mice. These results suggest that molecular pathways (i.e., ACVR1/FST signaling and CTGF/TGF-ß signaling) leading to a decreased striated muscle mass and an increase in fibrous extracellular matrix in the process of aging deserve further investigation for their roles in the declined urethral function.

UNC-82/NUAK kinase is required by myosin A, but not myosin B, to assemble and function in the thick filament arms of C. elegans striated muscle.

The mechanisms that ensure proper assembly, activity, and turnover of myosin II filaments are fundamental to a diverse range of cellular processes. In Caenorhabditis elegans striated muscle, thick filaments contain two myosins that are functionally distinct and spatially segregated. Using transgenic double mutants, we demonstrate that the ability of increased myosin A expression to restore muscle structure and movement in myosin B mutants requires UNC-82/NUAK kinase activity. Myosin B function appears unaffected in the kinase-impaired unc-82(e1220) mutant: the recessive antimorphic effects on early assembly of paramyosin and myosin A in this mutant are counteracted by increased myosin B expression and exacerbated by loss of myosin B. Using chimeric myosins and motility assays, we mapped the region of myosin A that requires UNC-82 activity to a 531-amino-acid region of the coiled-coil rod. This region includes the 264-amino-acid Region 1, which is sufficient in chimeric myosins to rescue the essential filament-initiation function of myosin A, as well as two sites that interact with myosin head domains in the Interacting Heads Motif. A specific physical interaction between myosin A and UNC-82::GFP is supported by GFP labeling of ectopic myosin A filaments but not thin filaments. We hypothesize that UNC-82 regulates assembly competence of myosin A during parallel assembly in the filament arms.

Optical EUS Activation to Relax Sensitized Micturition Response.

This study aims to activate the external urethral sphincter (EUS), which plays a critical role in micturition control, through optogenetics and to determine its potential contribution to the stabilization of sensitized micturition activity. The viral vector (AAV2/8-CMV-hChR2(H134R)-EGFP) is utilized to introduce light-gated ion channels (hChR2/H134R) into the EUS of wild-type C57BL/6 mice. Following the induction of sensitized micturition activity using weak acetic acid (0.1%) in anesthetized mice, optical stimulation of the EUS muscle tissue expressing channel rhodopsin is performed using a 473 nm laser light delivered through optical fibers, and the resulting changes in muscle activation and micturition activity are examined. Through EMG (electromyography) measurements, it is confirmed that optical stimulation electrically activates the EUS muscle in mice. Analysis of micturition activity using cystometry reveals a 70.58% decrease in the micturition period and a 70.27% decrease in the voiding volume due to sensitized voiding. However, with optical stimulation, the micturition period recovers to 101.49%, and the voiding volume recovered to 100.22%. Stimulation of the EUS using optogenetics can alleviate sensitized micturition activity and holds potential for application in conjunction with other micturition control methods.

Structure of the Drosophila melanogaster Flight Muscle Myosin Filament at 4.7 Å Resolution Reveals New Details of Non-Myosin Proteins.

Striated muscle thick filaments are composed of myosin II and several non-myosin proteins which define the filament length and modify its function. Myosin II has a globular N-terminal motor domain comprising its catalytic and actin-binding activities and a long α-helical, coiled tail that forms the dense filament backbone. Myosin alone polymerizes into filaments of irregular length, but striated muscle thick filaments have defined lengths that, with thin filaments, define the sarcomere structure. The motor domain structure and function are well understood, but the myosin filament backbone is not. Here we report on the structure of the flight muscle thick filaments from Drosophila melanogaster at 4.7 Å resolution, which eliminates previous ambiguities in non-myosin densities. The full proximal S2 region is resolved, as are the connecting densities between the Ig domains of stretchin-klp. The proteins, flightin, and myofilin are resolved in sufficient detail to build an atomic model based on an AlphaFold prediction. Our results suggest a method by which flightin and myofilin cooperate to define the structure of the thick filament and explains a key myosin mutation that affects flightin incorporation. Drosophila is a genetic model organism for which our results can define strategies for functional testing.

DEHP exposure impairs human skeletal muscle cell proliferation in primary culture conditions: preliminary study.

The plasticizer di (2-ethylhexyl) phthalate (DEHP) inhibits differentiation, impairs glucose metabolism, and decreases mitochondrial function in murine muscle satellite cells; however, if these effects are translated to human cells is unknown. The goal of this study was to evaluate changes in morphology and proliferation of primary human skeletal muscle cells exposed to DEHP. Rectus abdominis muscle samples were obtained from healthy women undergoing programed cesarean surgery. Skeletal muscle cells were isolated and grown under standard primary culture conditions, generating two independent sample groups of 25 subcultures each. Cells from the first group were exposed to 1 mM DEHP for 13 days and monitored for changes in cell morphology, satellite cell frequency and total cell abundance, while the second group remained untreated (control). Differences between treated and untreated groups were compared using generalized linear mixed models (GLMM). Cell membrane and nuclear envelope boundary alterations, loss of cell volume and presence of stress bodies were observed in DEHP-treated cultures. DEHP-treated cultures also showed a significant reduction in satellite cell frequency compared to controls. Exposure to DEHP reduced human skeletal muscle cell abundance. Statistical differences were found between the GLMM slopes, suggesting that exposure to DEHP reduced growth rate. These results suggest that exposure to DEHP inhibits human skeletal muscle cell proliferation, as evidenced by reduced cell abundance, potentially compromising long-term culture viability. Therefore, DEHP induces human skeletal muscle cell deterioration potentially inducing an inhibitory effect of myogenesis by depleting satellite cells.

Effect of inter-swallow interval on striated esophagus peristalsis; a comparative study with smooth muscle esophagus.

BACKGROUND: Effect of inter-swallow interval on the contractility of smooth muscle esophagus is well-documented. However, the effects on peristalsis of the striated esophagus have not been systematically studied. A better understanding of striated esophagus motor function in health and disease may enhance the interpretation of manometric studies and inform clinical care. The aim of this study was to assess the effect of inter-swallow interval on striated esophagus compared to findings with that of the smooth muscle esophagus. METHODS: We performed two sets of studies to (1) determine the effect of various inter-swallow interval in 20 healthy volunteers and (2) assess the effect of ultra-short swallow intervals facilitated by straw drinking in 28 volunteers. We analyzed variables using ANOVA with Tukey's pairwise comparison and paired t-test. KEY RESULTS: Unlike smooth muscle esophagus, the striated esophagus contractile integral did not change significantly for swallow intervals ranging from 30 to 5 s. On the contrary, striated esophagus demonstrated absent or reduced peristalsis in response to ultra-short (<2 s) intervals during straw-facilitated multiple rapid swallows. CONCLUSIONS AND INFERENCES: Striated esophagus peristalsis is subject to manometrically observed inhibition during swallows with ultra-short intervals. Inter-swallow intervals as short as 5 s that inhibit smooth muscle esophagus peristalsis do not inhibit striated muscle peristalsis. The mechanisms of these observations are unknown but may relate to central or myenteric nervous system influences or the effects of pharyngeal biomechanics.

John Squire and the myosin thick filament structure in muscle.

The structure of the thin, actin-containing filament of muscle is both highly conserved across a broad range of muscle types and is now well understood. The structure of the thick, myosin-containing filaments of striated muscle are quite variable and remained comparatively unknown until recently, particularly in the arrangement of the myosin tails. John Squire played a major role not only in our understanding of thin filament structure and function but also in the structure of the thick filaments. Long before much was known about the structure and composition of muscle thick filaments, he proposed a general model for how myosin filaments were constructed. His role in our current understanding the structure of striated muscle thick filaments and the extent through which his predictions have held true is the topic of this review.

Titin activates myosin filaments in skeletal muscle by switching from an extensible spring to a mechanical rectifier.

Titin is a molecular spring in parallel with myosin motors in each muscle half-sarcomere, responsible for passive force development at sarcomere length (SL) above the physiological range (>2.7 µm). The role of titin at physiological SL is unclear and is investigated here in single intact muscle cells of the frog (Rana esculenta), by combining half-sarcomere mechanics and synchrotron X-ray diffraction in the presence of 20 µM para-nitro-blebbistatin, which abolishes the activity of myosin motors and maintains them in the resting state even during activation of the cell by electrical stimulation. We show that, during cell activation at physiological SL, titin in the I-band switches from an SL-dependent extensible spring (OFF-state) to an SL-independent rectifier (ON-state) that allows free shortening while resisting stretch with an effective stiffness of ~3 pN nm-1 per half-thick filament. In this way, I-band titin efficiently transmits any load increase to the myosin filament in the A-band. Small-angle X-ray diffraction signals reveal that, with I-band titin ON, the periodic interactions of A-band titin with myosin motors alter their resting disposition in a load-dependent manner, biasing the azimuthal orientation of the motors toward actin. This work sets the stage for future investigations on scaffold and mechanosensing-based signaling functions of titin in health and disease.

The Enigmas of Lymphatic Muscle Cells: Where Do They Come From, How Are They Maintained, and Can They Regenerate?

Lymphatic muscle cell (LMC) contractility and coverage of collecting lymphatic vessels (CLVs) are integral to effective lymphatic drainage and tissue homeostasis. In fact, defects in lymphatic contractility have been identified in various conditions, including rheumatoid arthritis, inflammatory bowel disease, and obesity. However, the fundamental role of LMCs in these pathologic processes is limited, primarily due to the difficulty in directly investigating the enigmatic nature of this poorly characterized cell type. LMCs are a unique cell type that exhibit dual tonic and phasic contractility with hybrid structural features of both vascular smooth muscle cells (VSMCs) and cardiac myocytes. While advances have been made in recent years to better understand the biochemistry and function of LMCs, central questions regarding their origins, investiture into CLVs, and homeostasis remain unanswered. To summarize these discoveries, unexplained experimental results, and critical future directions, here we provide a focused review of current knowledge and open questions related to LMC progenitor cells, recruitment, maintenance, and regeneration. We also highlight the high-priority research goal of identifying LMC-specific genes towards genetic conditional- inducible in vivo gain and loss of function studies. While our interest in LMCs has been focused on understanding lymphatic dysfunction in an arthritic flare, these concepts are integral to the broader field of lymphatic biology, and have important potential for clinical translation through targeted therapeutics to control lymphatic contractility and drainage.

Synaptopodin-2 Isoforms Have Specific Binding Partners and Display Distinct, Muscle Cell Type-Specific Expression Patterns.

Synaptopodin-2 (SYNPO2) is a protein associated with the Z-disc in striated muscle cells. It interacts with α-actinin and filamin C, playing a role in Z-disc maintenance under stress by chaperone-assisted selective autophagy (CASA). In smooth muscle cells, SYNPO2 is a component of dense bodies. Furthermore, it has been proposed to play a role in tumor cell proliferation and metastasis in many different kinds of cancers. Alternative transcription start sites and alternative splicing predict the expression of six putative SYNPO2 isoforms differing by extended amino- and/or carboxy-termini. Our analyses at mRNA and protein levels revealed differential expression of SYNPO2 isoforms in cardiac, skeletal and smooth muscle cells. We identified synemin, an intermediate filament protein, as a novel binding partner of the PDZ-domain in the amino-terminal extension of the isoforms mainly expressed in cardiac and smooth muscle cells, and demonstrated colocalization of SYNPO2 and synemin in both cell types. A carboxy-terminal extension, mainly expressed in smooth muscle cells, is sufficient for association with dense bodies and interacts with α-actinin. SYNPO2 therefore represents an additional and novel link between intermediate filaments and the Z-discs in cardiomyocytes and dense bodies in smooth muscle cells, respectively. In pathological skeletal muscle samples, we identified SYNPO2 in the central and intermediate zones of target fibers of patients with neurogenic muscular atrophy, and in nemaline bodies. Our findings help to understand distinct functions of individual SYNPO2 isoforms in different muscle tissues, but also in tumor pathology.