Skeletal Muscle最新文献

Background: Human primary muscle cell (HPMC) lines derived from skeletal muscle biopsies are potentially powerful tools to interrogate the molecular pathways underlying fundamental muscle mechanisms. HPMCs retain their genome in culture, but many endogenous circulating factors are not present in the in vitro environment, or at concentrations that do not mirror physiological levels. To address the assumption that HPMCs are valid models of age and sex-specificity in human muscle research, we examined to what extent differentiated HPMC lines retain their source phenotype in culture.

Methods: Biopsies from the vastus lateralis muscle were collected from ten males aged 18-30, ten females aged 18-30 and ten males aged 60-75 recruited from a healthy population. A portion of the muscle was used for the establishment of 30 individual HMPC lines. The remaining sample was immediately snap frozen and stored for further analysis. RNA was extracted from muscle tissue samples and their corresponding, fully differentiated HMPCs and analysed using RNA Sequencing. To compare their transcriptomic signature, principal component analysis (PCA), differential expression analysis, single-cell deconvolution and pathway enrichment analysis were conducted in R.

Results: A comparison of the transcriptomic signature of 30 human muscle biopsies and their corresponding differentiated HPMCs indicated a near-complete lack of retention of the genes and pathways differentially regulated in vivo when compared to their in vitro equivalent, with the exception of several genes encoded on the Y-chromosome.

Conclusions: The diversity of resident cell populations in muscle tissue and the lack of sex- and age-dependent circulating factors in the cellular milieu likely contribute to these observations, which call for caution when using differentiated HPMCs as an experimental model of human muscle sex or age.

Accurate fibrosis quantification is essential for understanding muscle and cardiac disease, yet current manual and semi‑automated methods remain slow, subjective, and poorly reproducible. We introduce FibroTrack, a standalone deep learning platform with a graphical user interface (GUI) that streamlines fibrosis analysis across Sirius Red (SR), Masson's Trichrome (MT), and immunohistochemistry (IHC) stainings. FibroTrack uniquely integrates LAB (lightness, green-red, blue-yellow) color space normalization with a You Only Look Once version 11 (YOLOv11) segmentation model trained on 2,034 histological images. This approach achieved 99.5% mask precision for muscle segmentation and demonstrated excellent concordance with blinded pathologists (Spearman correlation, r = 0.87-0.96). Automated outputs include segmented images and structured spreadsheets, ensuring high reproducibility and scalability. By combining advanced color analysis with state‑of‑the‑art segmentation in an accessible tool, FibroTrack provides a novel, accurate, and clinically relevant solution for high‑throughput fibrosis quantification in both preclinical research and pathology practice.

Background: Skeletal muscle plays a crucial role in human life, contributing to posture, movement, nutrient storage, and body temperature regulation. Development and regeneration of skeletal muscles rely on embryonic myogenic progenitors and postnatal satellite cells (MuSCs), respectively. Identification of new molecular markers and elucidating their functions in MuSCs will provide better understanding of muscle development and regeneration.

Methods: We surveyed single cell RNA-seq (scRNA-seq) data (Tabula Muris and GSE150366) to identify ASB5 (Ankyrin repeat and Suppressor of cytokine signaling Box containing 5) as a marker of MuSCs. We also used CRISPR-CAS9 genome editing and oviduct electroporation to generate a germline knockout (KO) mouse line of Asb5. We then analyzed the muscle growth and regeneration of the KO mice. We further analyzed proliferation and differentiation of MuSCs attached on myofibers. We finally performed Realtime PCR (qPCR) to examine how Asb5 KO affects gene expression in the skeletal muscle.

Results: Analysis of data publicly available at Tabula Muris identified Asb5 as a specific marker of MuSCs. Further analysis of scRNA-seq data on FACS-purified MuSCs at various regeneration time points revealed that Asb5 is highly expressed in MuSCs and their progenies across various stages of muscle regeneration. We then generated a novel Asb5 KO mouse line through CRISPR-Cas9 deletion of Exon 4. The Asb5-KO mice were born normally and exhibited normal postnatal growth. In addition, Asb5-KO MuSCs proliferated, differentiated and self-renewed normally on myofiber explants. Furthermore, the skeletal muscles of Asb5-KO mice regenerated normally after acute injury. qPCR analysis showed that Asb5 KO reduces the expression levels of Tnfa (Tumor Necrosis Factor Alpha) in the skeletal muscles.

Conclusion: These data together identify ASB5 as an abundantly expressed and specific marker of MuSCs and myogenic progenitors. However, Asb5 loss-of-function has no effects on embryonic development and postnatal growth of skeletal muscles, or behavior and regenerative functions of MuSCs under normal physiological conditions.

Background: Malignant Hyperthermia (MH) is characterized by life-threatening whole-body hyperthermic reactions triggered by volatile anesthetics in susceptible individuals. MH susceptibility (MHS) and exertional heat stroke (EHS) are linked to mutations in the type I ryanodine receptor (RYR1). However, the discordance between the estimated prevalence of RYR1 MHS mutations in the general population (1:625-1,075) and the occurrence of MH episodes during anesthesia (1:100,000) suggests additional factors beyond genetics.

Methods: Using an established MHS mouse model (Y524S or YS mice), 10-12-week-old WT and YS mice were exercised on a treadmill (20° incline, 15 m/min, 60 min) and/or exposed to heat (15 min at 37℃) or 2% isoflurane either in a low or high humid environment. A second cohort of 10-12-week-old WT and YS mice were exposed to 2% isoflurane under a low or high humid environment. Vehicle or dantrolene (4 mg/kg) was administered during isoflurane exposure once the rectal temperature reached 39℃. Rectal temperature was continuously monitored under all conditions. In addition, historical humidity and temperature data were collected from dates on which MHS individuals either experienced or did not experience a defined MH episode.

Results: In young, but not older, YS male mice, survival rates significantly decreased during heat challenge (HR Dry/Humid = 0.1621, P = 0.018) and isoflurane exposure in a humid environment (HR Dry/Humid = 0.2273, P = 0.035). Dantrolene improved survival (HR DMSO/Dantrolene ranging from 4.959 to 8.667 in all conditions) and slowed the rate of core temperature increase in young YS mice regardless of humidity. Humidity was similarly associated with increased MH occurrence (AUC = 0.8104, P = 0.0026) in male MHS patients.

Conclusions: Absolute humidity was associated with worsened outcomes during heat and isoflurane exposure in male MHS mice and an increase in MH occurrence in male MHS patients. These results support maintaining humidity levels within operating suites at lower levels of current guidelines, especially where dantrolene is not available.

Background: Pluripotent stem cell-derived myogenic progenitors change from an embryonic to a postnatal molecular signature upon engrafting as satellite cells, which coincides with upregulation of Notch3. Since a role for Notch3 in skeletal muscle maturation is unknown, here we investigate whether Notch3 is required for this in vivo molecular maturation switch.

Results: Our results show that lack of Notch3 in transplanted progenitors (N3KO) does not impact degree of engraftment, but leads to increased numbers of embryonic myofibers. Conversely, transplantation of Notch3 overexpressing (N3OE) myogenic progenitors results in lower numbers of embryonic myofibers, but diminished muscle grafts when compared to empty vector (EV) controls. Secondary transplantation studies confirmed these effects, whereby Notch3 overexpression significantly reduced secondary engraftment. Further characterization of N3OE donor-derived satellite cells revealed reduced proliferation and downregulation of cell cycle genes. Importantly, secondary grafts from N3KO satellite cells had increased numbers of embryonic myofibers compared to N3OE and EV controls.

Conclusions: Taken together, these findings demonstrate that Notch3 signaling is required for myofiber maturation, and that constant activation of Notch3 impairs proliferation and muscle regeneration. Transcriptional profiles of N3OE donor-derived satellite cells suggest that dampened regeneration may be driven by inhibitory alterations in cell cycle regulation.

Background: Skeletal muscle dysfunction and elevated CO₂ in the blood, or hypercapnia, are both associated with higher mortality in acute and chronic pulmonary diseases. Hypercapnia-, aging and autophagy dysfunction-induced skeletal muscle phenotypes are highly overlapping. While DNA methylation regulates aging-associated cellular processes, no comparative study of CO₂- and age-induced changes in skeletal muscle has ever been conducted. Moreover, while previously reported skeletal muscle DNA methylation analyses involve about 1% of the genomic areas susceptible to this epigenetic modification, hypercapnia- and age-induced DNA methylation changes have never been investigated at the whole genome level.

Methods: C57BL/6 mice previously exposed to normo- and hypercapnia were compared with room air-maintained aged animals of similar background. Muscles from these mice were processed for whole genome methylation sequencing (WGMS) and RNA sequencing. The overlap between hypercapnia- and age-induced DNA methylation and transcript expression levels were established. Skeletal muscle-specific autophagy genetic ablation and mass spectrometry analyses were conducted to investigate the potential mechanisms regulating hypercapnia-induced DNA methylation changes.

Results: Hypercapnic mice demonstrate aberrant DNA methylation patterns in comparison to animals never exposed to elevated CO₂. These animals also elicit changes in myofiber type composition and protracted muscle mass deterioration even after returning to normocapnia. While aging leads to consistent DNA methylation changes over time, these epigenetic changes do not overlap with CO₂-induced differential methylation. Hypercapnia does not regulate the methylome via autophagy or substrate imbalance-related mechanisms.

Conclusion: Hypercapnia causes durable muscle wasting that persists even after regaining ambient air environment, which is associated with a perturbed methylome landscape. High CO₂-induced DNA methylation changes do not overlap with age-induced differentially methylation positions and are independent of substrate imbalances and autophagy regulation.