Biology subjects

Rudnicki, M. A.

Publications and source records attributed to Rudnicki, M. A..

8 recordsLinked to original sources

bioRxiv2026

Phosphorylation of CARM1 at Ser572 preserves muscle stem cell self-renewal by restricting premature myogenic commitment

Skeletal muscle regeneration depends on satellite cells, which must balance myogenic commitment with self-renewal to maintain regenerative capacity throughout life. The methyltransferase CARM1 is a key regulator of satellite cell fate, acting through methylation of PAX7 to promote Myf5 expression and myogenic commitment. The dystrophin glycoprotein complex regulates this pathway through p38{gamma}-mediated phosphorylation of CARM1 at Ser572, however the physiological role of this phosphorylation event in vivo remains unknown. Here, we generated a phosphorylation-deficient CARM1 knock-in mouse (CARM1 SA) to determine how CARM1 Ser572 phosphorylation regulates satellite cell fate during regenerative myogenesis. Satellite cell-derived myoblasts from CARM1 SA mice exhibited reduced PAX7 expression, impaired fusion, and abnormal myotube formation despite transcriptional signatures consistent with accelerated myogenic progression. Ex vivo single-myofiber assays revealed precocious acquisition of a committed progenitor identity accompanied by a reduction in the self-renewing satellite cell population. While muscle development and regeneration following a single injury were largely preserved, repeated regenerative challenge resulted in progressive depletion of the satellite cell pool in CARM1 SA mice. Transcriptomic analysis demonstrated minimal effects under homeostatic conditions but identified substantial alterations during regeneration, including reduced expression of extracellular matrix-associated genes, impaired TGF{beta}-SMAD signaling, decreased Tgfbr3 expression, and increased MYC activity, consistent with a loss of self-renewal programs and enhanced activation. Collectively, these findings demonstrate that phosphorylation of CARM1 at Ser572 is a critical mechanism to delay satellite cell commitment, thereby maintaining a balance between self-renewal and commitment to ensure long-term maintenance of the muscle stem cell compartment.

cell biology↗

bioRxiv2026

Regenerative Index reveals declining muscle regeneration in paediatric patients with Duchenne muscular dystrophy

BackgroundDuchenne muscular dystrophy (DMD) is a devastating disease manifested in skeletal muscle by repetitious myonecrosis and regeneration. Because the regenerative process is closely linked to the cumulative severity of muscle damage, which is variably distributed within and between muscle groups, accurately quantifying muscle regeneration has remained a significant challenge. MethodsMyofibers are delineated by immunostaining for laminin, and subsequent image analysis employed to generate a masked outline precisely within each myofiber boundary. Morphometric parameters including minimal Ferets diameter, cross-sectional area, and circularity were measured for each myofiber. In addition, the number of Pax7-expressing satellite cells were quantified. To evaluate regenerative activity, newly formed myofibers were identified by immunostaining for expression of embryonic myosin heavy chain (eMHC). Necrotic myofibers were enumerated by immunofluorescent detection of immunoglobulin G (IgG) infiltration. The Regenerative Index (RI) was calculated as the number of regenerating (eMHC+) myofibers divided by the number of necrotic (IgG+) myofibers. Determination of RI was performed on muscle biopsies from 10 boys with DMD and 3 non-DMD controls of similar age. ResultsA trend toward an increasing minimal Ferets diameter, cross-sectional area and circularity was observed with increasing age in DMD boys, with circularity showing the strongest trend. Furthermore, compared to DMD boys 7- to 8-years old, the boys 9- to 11-years old had significantly increased myofiber circularity. Pax7-expressing cells were significantly elevated in DMD boys compared to control boys of similar ages, without any observation of age-related changes. Notably, the Regenerative Index in DMD boys exhibited a pronounced decline between 7-11 years of age, and a significant inverse correlation between RI and age was observed. ConclusionsUsing eMHC and IgG immunostaining to calculate RI accurately assesses regeneration despite the variation in histopathologic severity between biopsies. This methodology demonstrated a significant negative correlation between RI and age of DMD boys from 7 to 11 years of age.

pathology↗

bioRxiv2024

Isolation of small extracellular vesicles from regenerating muscle tissue using Tangential Flow Filtration and Size Exclusion Chromatography

We have recently made the strikingly discovery that upon a muscle injury, Wnt7a is upregulated and secreted from new regenerating myofibers on the surface of exosomes to elicit its myogenerative response distally. Despite recent advances in extracellular vesicle (EVs) isolation from diverse tissues, there is still a lack of specific methodology to purify EVs from muscle tissue. To eliminate contamination with non-EV secreted proteins and cytoplasmic fragments, which are typically found when using classical methodology, such as ultracentrifugation, we adapted a protocol combining Tangential Flow Filtration (TFF) and Size Exclusion Chromatography (SEC). We found that this approach allows simultaneous purification of Wnt7a, bound to EVs (retentate fraction) and free non-EV Wnt7a (permeate fraction). Here we described this optimized protocol designed to specifically isolate EVs from hind limb muscle explants, without cross-contamination with other sources of non-EV bounded proteins. The first step of the protocol is to remove large EVs with sequential centrifugation. Extracellular vesicles are then concentrated and washed in exchange buffer by TFF. Lastly, SEC is performed to remove any soluble protein traces remaining after TFF. Overall, this procedure can be used to isolate EVs from conditioned media or biofluid that contains EVs derived from any cell type or tissue, improving reproducibility, efficiency, and purity of EVs preparations. Our purification protocol results in high purity EVs that maintain structural integrity and thus fully compatible with in vitro and in vivo bioactivity and analytic assays.

cell biology↗

bioRxiv2024

Wnt7a is Required for Regeneration of Dystrophic Skeletal Muscle

Intramuscular injection of Wnt7a has been shown to accelerate and augment skeletal muscle regeneration and to ameliorate dystrophic progression in mdx muscle, a model for Duchenne muscular dystrophy (DMD). However, loss-of-function studies to investigate the requirement for Wnt7a in muscle regeneration has not been evaluated. Here, we assessed muscle regeneration and function in wild type (WT) and mdx mice where Wnt7a was specifically deleted in muscle using a conditional Wnt7a floxed allele and a Myf5-Cre driver. We found that both WT and mdx mice with deletion of Wnt7a in muscle, exhibited marked deficiencies in muscle regeneration at 21 d following cardiotoxin (CTX) induced injury. Unlike WT, deletion of Wnt7a in mdx resulted in a marked decrease in specific force generation prior to CTX injury. However, both WT and mdx muscle lacking Wnt7a displayed decreased specific force generation following CTX injection. Notably the regeneration deficit observed in mdx mice lacking Wnt7a in muscle was rescued by a single tail vein injection of an extracellular vesicle preparation containing Wnt7a (Wnt7a-EVs). Therefore, we conclude that the regenerative capacity of muscle in mdx mice is due to the upregulation of endogenous Wnt7a following injury, and that systemic delivery of Wnt7a-EVs represents a therapeutic strategy for treating DMD.

genetics↗

bioRxiv2023

Wnt binding to Coatomer proteins directs secretion on exosomes independently of palmitoylation

Wnt proteins are secreted hydrophobic glycoproteins that act over long distances through poorly understood mechanisms. We discovered that Wnt7a is secreted on extracellular vesicles (EVs) following muscle injury. Structural analysis identified the motif responsible for Wnt7a secretion on EVs that we term the Exosome Binding Peptide (EBP). Addition of the EBP to an unrelated protein directed secretion on EVs. Disruption of palmitoylation, knockdown of WLS, or deletion of the N-terminal signal peptide did not affect Wnt7a secretion on purified EVs. Bio-ID analysis identified Coatomer proteins as candidates responsible for loading Wnt7a onto EVs. The crystal structure of EBP bound to the COPB2 coatomer subunit, the binding thermodynamics, and mutagenesis experiments, together demonstrate that a dilysine motif in the EBP mediates binding to COPB2. Other Wnts contain functionally analogous structural motifs. Mutation of the EBP results in a significant impairment in the ability of Wnt7a to stimulate regeneration, indicating that secretion of Wnt7a on exosomes is critical for normal regeneration in vivo. Our studies have defined the structural mechanism that mediates binding of Wnt7a to exosomes and elucidated the singularity of long-range Wnt signalling.

molecular biology↗

bioRxiv2022

KDR Signaling in Muscle Stem Cells Promotes Asymmetric Division and Progenitor Generation for Efficient Regeneration

The regulation of muscle stem cell (MuSC) asymmetric division plays an essential role in controlling the growth and repair of skeletal muscle. We discover kinase domain receptor (KDR) as a positive modulator of MuSC asymmetric division using an in-niche high-content screen and confirmed its expression in satellite cells by ddPCR and immunofluorescence. Knockdown of KDR significantly reduces the numbers of asymmetric divisions, whereas ligand stimulation of KDR increases the numbers of asymmetric divisions. KDR signaling is impaired in dystrophin-deficient satellite cells and requires a polarized cell environment established by the dystrophin glycoprotein complex (DGC) to direct asymmetric division. Mice lacking KDR in MuSCs exhibit reduced numbers of satellite cells due to precocious differentiation, and deficits in regeneration consistent with impaired asymmetric division and reduced generation of progenitors. Therefore, our experiments identify KDR signaling as playing an essential role in MuSC function in muscle regeneration. HIGHLIGHTSO_LIKDR and VEGFA are expressed in satellite cells C_LIO_LILigand activated KDR stimulates asymmetric satellite stem cell division C_LIO_LIKDR signaling requires the presence of the DGC C_LIO_LIKDR-deficient satellite cells give rise to reduced numbers of progenitors C_LI eTOC blurbChen et al., performed a chemical screen using a novel screening platform to identify modulators of muscle stem cell asymmetric division. They discovered that KDR signalling requires the presence of the dystrophin associated glycoprotein complex and is an important regulator of muscle stem cell asymmetric division.

molecular biology↗

bioRxiv2020

GLI3 Processing by the Primary Cilium Regulates Muscle Stem Cell Entry into GAlert

Satellite cells are required for the growth, maintenance, and regeneration of skeletal muscle. Quiescent satellite cells possess a primary cilium, a structure that regulates the processing of the GLI family of transcription factors. Here we find that GLI3, specifically, plays a critical role in satellite cell activation. Primary cilia-mediated processing of GLI3 is required to maintain satellite cells in a G0 dormant state. Strikingly, satellite cells lacking GLI3 enter GAlert in the absence of injury. Furthermore, GLI3 depletion or inhibition of its processing stimulates symmetrical division in satellite cells and expansion of the stem cell pool. As a result, satellite cells lacking GLI3 display rapid cell-cycle entry, increased proliferation and augmented self-renewal, and markedly enhanced long-term regenerative capacity. Therefore, our results reveal an essential role for primary cilia processing of GLI3 in regulating muscle stem cell activation and fate.

cell biology↗

bioRxiv2020

GREM1 is epigenetically reprogrammed in muscle cells after exercise training and controls myogenesis and metabolism

Exercise training improves skeletal muscle function, notably through tissue regeneration by muscle stem cells. Here, we hypothesized that exercise training reprograms the epigenome of muscle cell, which could account for better muscle function. Genome-wide DNA methylation of myotube cultures established from middle-aged obese men before and after endurance exercise training identified a differentially methylated region (DMR) located downstream of Gremlin 1 (GREM1), which was associated with increased GREM1 expression. GREM1 expression was lower in muscle satellite cells from obese, compared to lean mice, and exercise training restored GREM1 levels to those of control animals. We show that GREM1 regulates muscle differentiation through the negative control of satellite cell self-renewal, and that GREM1 controls muscle lineage commitment and lipid oxidation through the AMPK pathway. Our study identifies novel functions of GREM1 and reveals an epigenetic mechanism by which exercise training reprograms muscle stem cells to improve skeletal muscle function.

cell biology↗