Apelin stimulation of the vascular skeletal muscle stem cell niche enhances endogenous repair in dystrophic mice.

Impaired skeletal muscle stem cell (MuSC) function has long been suspected to contribute to the pathogenesis of muscular dystrophy (MD). Here, we showed that defects in the endothelial cell (EC) compartment of the vascular stem cell niche in mouse models of Duchenne MD, laminin α2-related MD, and collagen VI-related myopathy were associated with inefficient mobilization of MuSCs after tissue damage. Using chemoinformatic analysis, we identified the 13-amino acid form of the peptide hormone apelin (AP-13) as a candidate for systemic stimulation of skeletal muscle ECs. Systemic administration of AP-13 using osmotic pumps generated a pro-proliferative EC-rich niche that supported MuSC function through angiocrine factors and markedly improved tissue regeneration and muscle strength in all three dystrophic mouse models. Moreover, EC-specific knockout of the apelin receptor led to regenerative defects that phenocopied key pathological features of MD, including vascular defects, fibrosis, muscle fiber necrosis, impaired MuSC function, and reduced force generation. Together, these studies provide in vivo proof of concept that enhancing endogenous skeletal muscle repair by targeting the vascular niche is a viable therapeutic avenue for MD and characterized AP-13 as a candidate for further study for the systemic treatment of MuSC dysfunction.

Clearance of defective muscle stem cells by senolytics restores myogenesis in myotonic dystrophy type 1.

Muscle stem cells, the engine of muscle repair, are affected in myotonic dystrophy type 1 (DM1); however, the underlying molecular mechanism and the impact on the disease severity are still elusive. Here, we show using patients' samples that muscle stem cells/myoblasts exhibit signs of cellular senescence in vitro and in situ. Single cell RNAseq uncovers a subset of senescent myoblasts expressing high levels of genes related to the senescence-associated secretory phenotype (SASP). We show that the levels of interleukin-6, a prominent SASP cytokine, in the serum of DM1 patients correlate with muscle weakness and functional capacity limitations. Drug screening revealed that the senolytic BCL-XL inhibitor (A1155463) can specifically remove senescent DM1 myoblasts by inducing their apoptosis. Clearance of senescent cells reduced the expression of SASP, which rescued the proliferation and differentiation capacity of DM1 myoblasts in vitro and enhanced their engraftment following transplantation in vivo. Altogether, this study identifies the pathogenic mechanism associated with muscle stem cell defects in DM1 and opens a therapeutic avenue that targets these defective cells to restore myogenesis.

Physical Activity and Patient-Reported Outcomes in Monoclonal Plasma Cell Disorders.

INTRODUCTION: Plasma cell disorders (PCD) are a group of conditions characterized by disproportionate proliferation of a single clone of B lymphocytes. Multiple myeloma (MM) is a malignant type of plasma cell disorders. Improvements in MM survival have led patients and physicians to pursue strategies to improve quality of life for those living longer with this disease. Bone disease and instability associated with MM have made physicians reluctant to recommend physical activity (PA) to this patient population. The goal of this study was to examine the relationship between PA and physical and psychosocial patient-reported outcomes in patients with MM and precursor conditions. METHODS: We used a cross-sectional study design. Questionnaires on PA, demographics, fatigue, distress, and other aspects of quality of life were posted on the HealthTree® Cure Hub website, a patient portal through which individuals with MM and related disorders obtain support, track laboratories and other information about their diseases, and participate in research. RESULTS: A total of 794 individuals, including 664 with MM, are included in the current analysis. We observed potential inverse associations between PA and poor quality of life, including problems with sleep, fatigue, neuropathy, distress, and several psychosocial states. On average, patients reported that their PA levels have declined since diagnosis and that they would like to be even more active in the future than they were before their diagnosis. CONCLUSIONS: In our cross-sectional study, regular PA was associated with multiple quality-of-life indicators and other patient-reported outcomes, including better sleep and less fatigue, neuropathy, and distress. The findings of this study can help guide the design of prospective studies of the role of PA in MM survivorship.

N-acetylneuraminate pyruvate lyase controls sialylation of muscle glycoproteins essential for muscle regeneration and function.

Deleterious variants in N-acetylneuraminate pyruvate lyase (NPL) cause skeletal myopathy and cardiac edema in humans and zebrafish, but its physiological role remains unknown. We report generation of mouse models of the disease: NplR63C, carrying the human p.Arg63Cys variant, and Npldel116 with a 116-bp exonic deletion. In both strains, NPL deficiency causes drastic increase in free sialic acid levels, reduction of skeletal muscle force and endurance, slower healing and smaller size of newly formed myofibers after cardiotoxin-induced muscle injury, increased glycolysis, partially impaired mitochondrial function, and aberrant sialylation of dystroglycan and mitochondrial LRP130 protein. NPL-catalyzed degradation of sialic acid in the muscle increases after fasting and injury and in human patient and mouse models with genetic muscle dystrophy, demonstrating that NPL is essential for muscle function and regeneration and serves as a general marker of muscle damage. Oral administration of N-acetylmannosamine rescues skeletal myopathy, as well as mitochondrial and structural abnormalities in NplR63C mice, suggesting a potential treatment for human patients.

An engineered multicellular stem cell niche for the 3D derivation of human myogenic progenitors from iPSCs.

Fate decisions in the embryo are controlled by a plethora of microenvironmental interactions in a three-dimensional niche. To investigate whether aspects of this microenvironmental complexity can be engineered to direct myogenic human-induced pluripotent stem cell (hiPSC) differentiation, we here screened murine cell types present in the developmental or adult stem cell niche in heterotypic suspension embryoids. We identified embryonic endothelial cells and fibroblasts as highly permissive for myogenic specification of hiPSCs. After two weeks of sequential Wnt and FGF pathway induction, these three-component embryoids are enriched in Pax7-positive embryonic-like myogenic progenitors that can be isolated by flow cytometry. Myogenic differentiation of hiPSCs in heterotypic embryoids relies on a specialized structural microenvironment and depends on MAPK, PI3K/AKT, and Notch signaling. After transplantation in a mouse model of Duchenne muscular dystrophy, embryonic-like myogenic progenitors repopulate the stem cell niche, reactivate after repeated injury, and, compared to adult human myoblasts, display enhanced fusion and lead to increased muscle function. Altogether, we provide a two-week protocol for efficient and scalable suspension-based 3D derivation of Pax7-positive myogenic progenitors from hiPSCs.

Assessment of Muscle Function Following hiPSC-Derived Myoblast Transplantation in Dystrophic Mice.

Muscular dystrophies are caused by genetic variants in genes encoding for proteins important for muscle structure or function, leading to a loss of muscle integrity and muscle wasting. To this day, no cure has been found for these diseases. Different therapeutic approaches are under intensive investigation. Cellular therapy has been extensively studied for diseases such as Duchenne Muscular Dystrophy, a debilitating disease caused by a mutation in the DMD gene, encoding for the dystrophin protein. Healthy myogenic cells transplanted into dystrophic muscles have the potential to engraft at long-term and fuse to donate their nuclei to the dystrophin-deficient myofibers, thereby restoring normal gene expression. Despite promising preclinical studies, the clinical trials had limited success so far due to many technical limitations. The recent technological advances in induced-pluripotent stem cells and genome editing opened new opportunities in this field. One of the keys to efficiently translate these new technologies into clinical benefits is to use relevant endpoints for preclinical studies. Considering that dystrophic muscles are susceptible to contraction-induced injury, the assessment of their resistance to repeated eccentric contractions is an optimal outcome to evaluate their functional recovery following cell transplantation. This protocol describes the procedure to generate induced-pluripotent stem cell-derived myoblasts, transplant these cells into skeletal muscle of immunosuppressed dystrophic mice, and assess muscle function in situ by measuring the resistance of the transplanted muscle to repeated eccentric contractions. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Generation of hiPSC-derived myoblasts. Basic Protocol 2: Transplantation of hiPSC-derived myoblasts in skeletal muscle of dystrophic mice. Basic Protocol 3: Assessment of muscle function in situ.

Fibro-adipogenic progenitors in skeletal muscle homeostasis, regeneration and diseases.

Skeletal muscle possesses a remarkable regenerative capacity that relies on the activity of muscle stem cells, also known as satellite cells. The presence of non-myogenic cells also plays a key role in the coordination of skeletal muscle regeneration. Particularly, fibro-adipogenic progenitors (FAPs) emerged as master regulators of muscle stem cell function and skeletal muscle regeneration. This population of muscle resident mesenchymal stromal cells has been initially characterized based on its bi-potent ability to differentiate into fibroblasts or adipocytes. New technologies such as single-cell RNAseq revealed the cellular heterogeneity of FAPs and their complex regulatory network during muscle regeneration. In acute injury, FAPs rapidly enter the cell cycle and secrete trophic factors that support the myogenic activity of muscle stem cells. Conversely, deregulation of FAP cell activity is associated with the accumulation of fibrofatty tissue in pathological conditions such as muscular dystrophies and ageing. Considering their central role in skeletal muscle pathophysiology, the regulatory mechanisms of FAPs and their cellular and molecular crosstalk with muscle stem cells are highly investigated in the field. In this review, we summarize the current knowledge on FAP cell characteristics, heterogeneity and the cellular crosstalk during skeletal muscle homeostasis and regeneration. We further describe their role in muscular disorders, as well as different therapeutic strategies targeting these cells to restore muscle regeneration.

Resolvin-D2 targets myogenic cells and improves muscle regeneration in Duchenne muscular dystrophy.

Lack of dystrophin causes muscle degeneration, which is exacerbated by chronic inflammation and reduced regenerative capacity of muscle stem cells in Duchenne Muscular Dystrophy (DMD). To date, glucocorticoids remain the gold standard for the treatment of DMD. These drugs are able to slow down the progression of the disease and increase lifespan by dampening the chronic and excessive inflammatory process; however, they also have numerous harmful side effects that hamper their therapeutic potential. Here, we investigated Resolvin-D2 as a new therapeutic alternative having the potential to target multiple key features contributing to the disease progression. Our in vitro findings showed that Resolvin-D2 promotes the switch of macrophages toward their anti-inflammatory phenotype and increases their secretion of pro-myogenic factors. Moreover, Resolvin-D2 directly targets myogenic cells and promotes their differentiation and the expansion of the pool of myogenic progenitor cells leading to increased myogenesis. These effects are ablated when the receptor Gpr18 is knocked-out, knocked-down, or blocked by the pharmacological antagonist O-1918. Using different mouse models of DMD, we showed that Resolvin-D2 targets both inflammation and myogenesis leading to enhanced muscle function compared to glucocorticoids. Overall, this preclinical study has identified a new therapeutic approach that is more potent than the gold-standard treatment for DMD.

Macrophages Are Key Regulators of Stem Cells during Skeletal Muscle Regeneration and Diseases.

Muscle regeneration is a closely regulated process that involves a variety of cell types such as satellite cells, myofibers, fibroadipogenic progenitors, endothelial cells, and inflammatory cells. Among these different cell types, macrophages emerged as a central actor coordinating the different cellular interactions and biological processes. Particularly, the transition of macrophages from their proinflammatory to their anti-inflammatory phenotype was shown to regulate inflammation, myogenesis, fibrosis, vascularization, and return to homeostasis. On the other hand, deregulation of macrophage accumulation or polarization in chronic degenerative muscle disorders was shown to impair muscle regeneration. Considering the key roles of macrophages in skeletal muscle, they represent an attractive target for new therapeutic approaches aiming at mitigating various muscle disorders. This review aims at summarizing the novel insights into macrophage heterogeneity, plasticity, and functions in skeletal muscle homeostasis, regeneration, and disease.

Estimating the dim light melatonin onset of adolescents within a 6-h sampling window: the impact of sampling rate and threshold method.

OBJECTIVE/BACKGROUND: Circadian rhythm sleep-wake disorders (CRSWDs) often manifest during the adolescent years. Measurement of circadian phase such as the dim light melatonin onset (DLMO) improves diagnosis and treatment of these disorders, but financial and time costs limit the use of DLMO phase assessments in clinic. The current analysis aims to inform a cost-effective and efficient protocol to measure the DLMO in older adolescents by reducing the number of samples and total sampling duration. PATIENTS/METHODS: A total of 66 healthy adolescents (26 males) aged 14.8-17.8 years participated in a study; they were required to sleep on a fixed baseline schedule for a week before which they visited the laboratory for saliva collection in dim light (<20 lux). Two partial 6-h salivary melatonin profiles were derived for each participant. Both profiles began 5 h before bedtime and ended 1 h after bedtime, but one profile was derived from samples taken every 30 min (13 samples) and the other from samples taken every 60 min (seven samples). Three standard thresholds (first three melatonin values mean + 2 SDs, 3 pg/mL, and 4 pg/mL) were used to compute the DLMO. An agreement between DLMOs derived from 30-min and 60-min sampling rates was determined using Bland-Altman analysis; agreement between the sampling rate DLMOs was defined as ± 1 h. RESULTS AND CONCLUSIONS: Within a 6-h sampling window, 60-min sampling provided DLMO estimates within ± 1 h of DLMO from 30-min sampling, but only when an absolute threshold (3 or 4 pg/mL) was used to compute the DLMO. Future analyses should be extended to include adolescents with CRSWDs.

A week in the life of full-time office workers: work day and weekend light exposure in summer and winter.

Little is known about the light exposure in full-time office workers, who spend much of their workdays indoors. We examined the 24-h light exposure patterns of 14 full-time office workers during a week in summer, and assessed their dim light melatonin onset (DLMO, a marker of circadian timing) at the end of the working week. Six workers repeated the study in winter. Season had little impact on the workers' schedules, as the timing of sleep, commute, and work did not vary by more than 30 min in the summer and winter. In both seasons, workers received significantly more morning light on workdays than weekends, due to earlier wake times and the morning commute. Evening light in the two hours before bedtime was consistently dim. The timing of the DLMO did not vary between season, and by the end of the working week, the workers slept at a normal circadian phase.

Home lighting before usual bedtime impacts circadian timing: a field study.

Laboratory studies suggest that evening light before bedtime can suppress melatonin. Here, we measured the range of evening light intensity people can generate with their household lights, and for the first time determined if varying home light before usual bedtime can shift circadian phase. This was a 3-week study with two counterbalanced conditions separated by a 5-day break. In a dim week, eight healthy subjects minimized their home light exposure from 4 h before habitual bedtime until a self-selected bedtime. In a bright week, the subjects maximized their home lighting for the same time. The dim light melatonin onset (DLMO) was assessed after each week. On average subjects maximized their lights to approximately 65 lux and minimized their lights to approximately 3 lux. Wrist actigraphy indicated that subjects went to bed slightly later when lights were maximized (average 14 min later, P = 0.05), but wake time did not change. Every subject had a later DLMO after the week of maximum versus minimum light exposure (average 1:03 h later, P < 0.001). These results demonstrate that the light intensity people can generate at home in the few hours before habitual bedtime can alter circadian timing. People should reduce their evening light exposure to lessen circadian misalignment.

Blacks (African Americans) have shorter free-running circadian periods than whites (Caucasian Americans).

The length of the free-running period (τ) affects how an animal re-entrains after phase shifts of the light-dark (LD) cycle. Those with shorter periods adapt faster to phase advances than those with longer periods, whereas those with longer periods adapt faster to phase delays than those with shorter periods. The free-running period of humans, measured in temporal isolation units and in forced desychrony protocols in which the day length is set beyond the range of entrainment, varies from about 23.5 to 26 h, depending on the individual and the experimental conditions (e.g., temporal isolation vs. forced desychrony). We studied 94 subjects free-running through an ultradian LD cycle, which was a forced desychrony with a day length of 4 h (2.5 h awake in dim light, ~35 lux, alternating with 1.5 h for sleep in darkness). Circadian phase assessments were conducted before (baseline) and after (final) three 24-h days of the ultradian LD cycle. During these assessments, saliva samples were collected every 30 min and subsequently analyzed for melatonin. The phase shift of the dim light melatonin onset (DLMO) from baseline to final phase assessment gave the free-running period. The mean ± SD period was 24.31 ± .23 h and ranged from 23.7 to 24.9 h. Black subjects had a significantly shorter free-running period than Whites (24.18 ± .23 h, N =20 vs. 24.37 ± .22 h, N = 55). We had a greater proportion of women than men in our Black sample, so to check the τ difference we compared the Black women to White women. Again, Black subjects had a significantly shorter free-running period (24.18 ± .23, N = 17 vs. 24.41 ± .23, N = 23). We did not find any sex differences in the free-running period. These findings give rise to several testable predictions: on average, Blacks should adapt quicker to eastward flights across time zones than Whites, whereas Whites should adjust quicker to westward flights than Blacks. Also, Blacks should have more difficulty adjusting to night-shift work and day sleep, which requires a phase delay. On the other hand, Whites should be more likely to have trouble adapting to the early work and school schedules imposed by society. More research is needed to confirm these results and predictions.

Human phase response curve to intermittent blue light using a commercially available device.

Light shifts the timing of the circadian clock according to a phase response curve (PRC). To date, all human light PRCs have been to long durations of bright white light. However, melanopsin, the primary photopigment for the circadian system, is most sensitive to short wavelength blue light. Therefore, to optimise light treatment it is important to generate a blue light PRC.We used a small, commercially available blue LED light box, screen size 11.2 × 6.6 cm at ∼50 cm, ∼200 µW cm(−2), ∼185 lux. Subjects participated in two 5 day laboratory sessions 1 week apart. Each session consisted of circadian phase assessments to obtain melatonin profiles before and after 3 days of free-running through an ultradian light­dark cycle (2.5 h wake in dim light, 1.5 h sleep in the dark), forced desynchrony protocol. During one session subjects received intermittent blue light (three 30 min pulses over 2 h) once a day for the 3 days of free-running, and in the other session (control) they remained in dim room light, counterbalanced. The time of blue light was varied among subjects to cover the entire 24 h day. For each individual, the phase shift to blue light was corrected for the free-run determined during the control session. The blue light PRC had a broad advance region starting in the morning and extending through the afternoon. The delay region started a few hours before bedtime and extended through the night. This is the first PRC to be constructed to blue light and to a stimulus that could be used in the real world.

Evaluation of microorganisms cultured from injured and repressed tissue regeneration sites in endangered giant aquatic Ozark Hellbender salamanders.

Investigation into the causes underlying the rapid, global amphibian decline provides critical insight into the effects of changing ecosystems. Hypothesized and confirmed links between amphibian declines, disease, and environmental changes are increasingly represented in published literature. However, there are few long-term amphibian studies that include data on population size, abnormality/injury rates, disease, and habitat variables to adequately assess changes through time. We cultured and identified microorganisms isolated from abnormal/injured and repressed tissue regeneration sites of the endangered Ozark Hellbender, Cryptobranchus alleganiensis bishopi, to discover potential causative agents responsible for their significant decline in health and population. This organism and our study site were chosen because the population and habitat of C. a. bishopi have been intensively studied from 1969-2009, and the abnormality/injury rate and apparent lack of regeneration were established. Although many bacterial and fungal isolates recovered were common environmental organisms, several opportunistic pathogens were identified in association with only the injured tissues of C.a. bishopi. Bacterial isolates included Aeromonas hydrophila, a known amphibian pathogen, Granulicetella adiacens, Gordonai terrae, Stenotrophomonas maltophilia, Aerococcus viridans, Streptococcus pneumoniae and a variety of Pseudomonads, including Pseudomonas aeruginosa, P. stutzeri, and P. alcaligenes. Fungal isolates included species in the genera Penicillium, Acremonium, Cladosporium, Curvularia, Fusarium, Streptomycetes, and the Class Hyphomycetes. Many of the opportunistic pathogens identified are known to form biofilms. Lack of isolation of the same organism from all wounds suggests that the etiological agent responsible for the damage to C. a. bishopi may not be a single organism. To our knowledge, this is the first study to profile the external microbial consortia cultured from a Cryptobranchid salamander. The incidence of abnormalities/injury and retarded regeneration in C. a. bishopi may have many contributing factors including disease and habitat degradation. Results from this study may provide insight into other amphibian population declines.

Calculating the dim light melatonin onset: the impact of threshold and sampling rate.

The dim light melatonin onset (DLMO) is the most reliable circadian phase marker in humans, but the cost of assaying samples is relatively high. Therefore, the authors examined differences between DLMOs calculated from hourly versus half-hourly sampling and differences between DLMOs calculated with two recommended thresholds (a fixed threshold of 3 pg/mL and a variable "3k" threshold equal to the mean plus two standard deviations of the first three low daytime points). The authors calculated these DLMOs from salivary dim light melatonin profiles collected from 122 individuals (64 women) at baseline. DLMOs derived from hourly sampling occurred on average only 6-8 min earlier than the DLMOs derived from half-hourly saliva sampling, and they were highly correlated with each other (r ≥ 0.89, p < .001). However, in up to 19% of cases the DLMO derived from hourly sampling was >30 min from the DLMO derived from half-hourly sampling. The 3 pg/mL threshold produced significantly less variable DLMOs than the 3k threshold. However, the 3k threshold was significantly lower than the 3 pg/mL threshold (p < .001). The DLMOs calculated with the 3k method were significantly earlier (by 22-24 min) than the DLMOs calculated with the 3 pg/mL threshold, regardless of sampling rate. These results suggest that in large research studies and clinical settings, the more affordable and practical option of hourly sampling is adequate for a reasonable estimate of circadian phase. Although the 3 pg/mL fixed threshold is less variable than the 3k threshold, it produces estimates of the DLMO that are further from the initial rise of melatonin.

Human phase response curves to three days of daily melatonin: 0.5 mg versus 3.0 mg.

CONTEXT: Phase response curves (PRCs) to melatonin exist, but none compare different doses of melatonin using the same protocol. OBJECTIVE: The aim was to generate a PRC to 0.5 mg of oral melatonin and compare it to our previously published 3.0 mg PRC generated using the same protocol. DESIGN AND SETTING: The study included two 5-d sessions in the laboratory, each preceded by 7-9 d of fixed sleep times. Each session started and ended with a phase assessment to measure the dim light melatonin onset (DLMO). In between were 3 d in an ultradian dim light (<150 lux)/dark cycle (light:dark, 2.5:1.5). PARTICIPANTS: Healthy adults (16 men, 18 women) between the ages of 18 and 42 yr participated in the study. INTERVENTIONS: During the ultradian days of the laboratory sessions, each participant took one pill per day at the same clock time (0.5 mg melatonin or placebo, double blind, counterbalanced). MAIN OUTCOME MEASURE: Phase shifts to melatonin were derived by subtracting the phase shift to placebo. A PRC with time of pill administration relative to baseline DLMO and a PRC relative to midpoint of home sleep were generated. RESULTS: Maximum advances occurred when 0.5 mg melatonin was taken in the afternoon, 2-4 h before the DLMO, or 9-11 h before sleep midpoint. The time for maximum phase delays was not as distinct, but a fitted curve peaked soon after wake time. CONCLUSIONS: The optimal administration time for advances and delays is later for the lower dose of melatonin. When each dose of melatonin is given at its optimal time, both yield similarly sized advances and delays.