Hyaluronic acid (HA) is an extracellular matrix (ECM) component that has been shown to play a significant role in regulating muscle cell behavior during repair and regeneration. For instance, ECM ...remodeling after muscle injury involves an upregulation in HA expression that is coupled with skeletal muscle precursor cell recruitment. However, little is known about the role of HA during skeletal muscle development. To gain insight into the way in which HA mediates embryonic myogenesis, we first determined the spatial distribution and gene expression of CD44, RHAMM and other HA related proteins in embryonic day (E)10.5 to E12.5 murine forelimbs. While HA and CD44 expression remained high, RHAMM decreased at both the protein (via immunohistochemistry) and RNA (via qPCR) levels. Next, we determined that 4-methylumbelliferone-mediated knockdown of HA synthesis inhibited the migration and proliferation of E11.5/E12.5 forelimb-derived cells. Then, the influence of CD44 and RHAMM on myoblast and connective tissue cell behavior was investigated using antibodies against these receptors. Anti-RHAMM, but not anti-CD44, significantly decreased the total distance myogenic progenitors migrated over 24 h, whereas both inhibited connective tissue cell migration. In contrast, anti-CD44 inhibited the proliferation of connective tissue cells and muscle progenitors, but anti-RHAMM had no effect. However, when myoblasts and connective tissue cells were depleted of CD44 and RHAMM by shRNA, motility and proliferation were significantly inhibited in both cells indicating that blocking cell surface-localized CD44 and RHAMM does not have as pronounced effect as global shRNA-mediated depletion of these receptors. These results show, for the first time, the distribution and activity of RHAMM in the context of skeletal muscle. Furthermore, our data indicate that HA, through interactions with CD44 and RHAMM, promotes myogenic progenitor migration and proliferation. Confirmation of the role of HA and its receptors in directing myogenesis will be useful for the design of regenerative therapies that aim to promote the restoration of damaged or diseased muscle.
•CD44, RHAMM and HA expression temporally varied during forelimb development.•shRNA-mediated depletion of CD44 and RHAMM inhibited proliferation and migration.•Antibody blocking of CD44 and RHAMM had a differential effect than shRNA depletion.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
During embryonic development, IGF‐1 fulfils crucial roles in skeletal myogenesis. However, the involvement of IGF‐1‐induced myoblast proliferation in muscle growth is still unclear. In the present ...study, we have characterised the role of IGF‐1 in myoblast proliferation both in vitro and in vivo and have revealed novel details of how exogenous IGF‐1 influences myogenic genes in chicken embryos. The results show that IGF‐1 significantly induces the proliferation of cultured myoblasts in a dose‐dependent manner. Additionally, the IGF‐1 treatment significantly promoted myoblasts entering a new cell cycle and increasing the mRNA expression levels of cell cycle‐dependent genes. However, these effects were inhibited by the PI3K inhibitor LY294002 and the Akt inhibitor KP372‐1. These data indicated that the pro‐proliferative effect of IGF‐1 was mediated in response to the PI3K/Akt signalling pathway. Moreover, we also showed that exogenous IGF‐1 stimulated myoblast proliferation in vivo. IGF‐1 administration obviously promoted the incorporation of BrdU and remarkably increased the number of PAX7‐positive cells in the skeletal muscle of chicken embryos. Administration of IGF‐1 also significantly induced the upregulation of myogenic factors gene, the enhancement of c‐Myc and the inhibition of myostatin (Mstn) expression. These findings demonstrate that IGF‐1 has strong activity as a promoter of myoblast expansion and muscle fiber formation during early myogenesis. Therefore, this study offers insight into the mechanisms responsible for IGF‐1‐mediated stimulation of embryonic skeletal muscle development, which could have important implications for the improvement of chicken meat production.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
The majority of studies on possible roles for collagen hydrolysates in human health have focused on their effects on bone and skin. Hydroxyprolyl-glycine (Hyp-Gly) was recently identified as a novel ...collagen hydrolysate-derived dipeptide in human blood. However, any possible health benefits of Hyp-Gly remain unclear. Here, we report the effects of Hyp-Gly on differentiation and hypertrophy of murine skeletal muscle C2C12 cells. Hyp-Gly increased the fusion index, the myotube size, and the expression of the myotube-specific myosin heavy chain (MyHC) and tropomyosin structural proteins. Hyp-Gly increased the phosphorylation of Akt, mTOR, and p70S6K in myoblasts, whereas the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 inhibited their phosphorylation by Hyp-Gly. LY294002 and the mammalian target of rapamycin (mTOR) inhibitor rapamycin repressed the enhancing effects of Hyp-Gly on MyHC and tropomyosin expression. The peptide/histidine transporter 1 (PHT1) was highly expressed in both myoblasts and myotubes, and co-administration of histidine inhibited Hyp-Gly-induced phosphorylation of p70S6K in myoblasts and myotubes. These results indicate that Hyp-Gly can induce myogenic differentiation and myotube hypertrophy and suggest that Hyp-Gly promotes myogenic differentiation by activating the PI3K/Akt/mTOR signaling pathway, perhaps depending on PHT1 for entry into cells.
•Hyp-Gly promotes myogenic differentiation and increases myotube size.•Hyp-Gly induces the phosphorylation of Akt, mTOR, and p70S6K.•Hyp-Gly seems to promote myogenic differentiation by activating the mTOR pathway.•Hyp-Gly-induced phosphorylation of p70S6K is inhibited by histidine.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
The mammalian heart is derived from multiple cell lineages; however, our understanding of when and how the diverse cardiac cell types arise is limited. We mapped the origin of the embryonic mouse ...heart at single-cell resolution using a combination of transcriptomic, imaging, and genetic lineage labeling approaches. This mapping provided a transcriptional and anatomic definition of cardiac progenitor types. Furthermore, it revealed a cardiac progenitor pool that is anatomically and transcriptionally distinct from currently known cardiac progenitors. Besides contributing to cardiomyocytes, these cells also represent the earliest progenitor of the epicardium, a source of trophic factors and cells during cardiac development and injury. This study provides detailed insights into the formation of early cardiac cell types, with particular relevance to the development of cell-based cardiac regenerative therapies.
Skeletal muscle formation occurs through fusion of myoblasts to form multinucleated myofibers. From a genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) loss-of-function ...screen for genes required for myoblast fusion and myogenesis, we discovered an 84–amino acid muscle-specific peptide that we call Myomixer. Myomixer expression coincides with myoblast differentiation and is essential for fusion and skeletal muscle formation during embryogenesis. Myomixer localizes to the plasma membrane, where it promotes myoblast fusion and associates with Myomaker, a fusogenic membrane protein. Myomixer together with Myomaker can also induce fibroblast-fibroblast fusion and fibroblast-myoblast fusion. We conclude that the Myomixer-Myomaker pair controls the critical step in myofiber formation during muscle development.
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BFBNIB, NMLJ, NUK, ODKLJ, PNG, SAZU, UL, UM, UPUK
State of the art techniques have been developed to isolate and analyze cells from various tissues, aiming to capture their in vivo state. However, the majority of cell isolation protocols involve ...lengthy mechanical and enzymatic dissociation steps followed by flow cytometry, exposing cells to stress and disrupting their physiological niche. Focusing on adult skeletal muscle stem cells, we have developed a protocol that circumvents the impact of isolation procedures and captures cells in their native quiescent state. We show that current isolation protocols induce major transcriptional changes accompanied by specific histone modifications while having negligible effects on DNA methylation. In addition to proposing a protocol to avoid isolation-induced artifacts, our study reveals previously undetected quiescence and early activation genes of potential biological interest.
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•Quiescent muscle stem cells undergo major transcriptomic alterations during isolation•Isolation induces histone H3 modifications but leaves intact DNA methylation•In situ fixation allows isolation of quiescent cells in their native, in vivo state•In situ fixation detects quiescence and early activation factors
Machado et al. demonstrate that muscle stem cells undergo changes in transcripts and histone modifications during isolation. The authors develop an in situ fixation-based methodology, which allows capture of cells in their native state. In light of these findings, some high-throughput analyses of tissue extracted cells may need to be revisited.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Abstract Mesenchymal stromal cells (MSCs) secrete paracrine factors that play crucial roles during tissue regeneration. Whether this paracrine function is influenced by the properties of biomaterials ...in general, and those used for cell delivery in particular, largely remains unexplored. Here, we investigated if three-dimensional culture in distinct microenvironments - nanoporous hydrogels (mean pore size ∼5 nm) and macroporous scaffolds (mean pore size ∼120 μm) - affects the secretion pattern of MSCs, and consequently leads to differential paracrine effects on target progenitor cells such as myoblasts. We report that compared to MSCs encapsulated in hydrogel, scaffold seeded MSCs show an enhanced secretion profile and exert beneficial paracrine effects on various myoblast functions including migration and proliferation. Additionally, we show that the heightened paracrine effects of scaffold seeded cells can in part be attributed to N-cadherin mediated cell-cell interactions during culture. In hydrogels, this physical interaction between cells is prevented by the encapsulating matrix. Functionally blocking N-cadherin negatively affected the secretion profile and paracrine effects of MSCs on myoblasts, with stronger effects observed for scaffold seeded compared to hydrogel encapsulated cells. Together, these findings demonstrate that the therapeutic potency of MSCs can be enhanced by biomaterials that promote cell-cell interactions.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
Curcumin exhibits antioxidant properties in normal cells where the uptake is low, unlike in tumor cells where uptake is high and curcumin increases reactive oxygen species (ROS) production and cell ...death. Mitochondria are the main source and primary target of cellular ROS. We hypothesized that curcumin would regulate cellular redox status and mitochondrial function, depending on cell sensitivity and/or curcumin concentration in normal cells. We examined the differences between low and high concentrations of curcumin, with specific attention focused on ROS levels, mitochondrial function, and cell viability in mouse C2C12 myoblast under normal and simulated conditions of diabetes. Cells incubated with high concentrations of curcumin (10–50 μM) resulted in decreased cell viability and sustained robust increases in ROS levels. Mechanistic studies showed that increased ROS levels in cells incubated with 20 μM curcumin induced opening of mitochondrial permeability transition pores and subsequent release of cytochrome c, activation of caspases 9 and 3/7, and apoptotic cell death. Low concentrations of curcumin (1–5 μM) did not affect cell viability, but induced a mild increase in ROS levels, which peaked at 2 hr after the treatment. Incubation with 5 μM curcumin also induced ROS‐dependent increases in mitochondrial mass and membrane potential. Finally, pretreatment with 5 μM curcumin prevented high glucose‐induced oxidative cell injury. Our study suggests that mitochondria respond differentially depending on curcumin concentration‐dependent induction of ROS. The end result is either cell protection or death. Curcumin may be an effective therapeutic target for diabetes and other mitochondrial diseases when used in low concentrations.
Curcumin regulates cellular redox status depending on cell sensitivity and/or curcumin concentration in normal cells. Mitochondria respond differentially depending on curcumin concentration‐dependent induction of ROS. Curcumin may be an effective therapeutic target for diabetes and other mitochondrial diseases when used in low concentrations.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Macroautophagy/autophagy is a degradative process essential for various cellular processes. We previously demonstrated that autophagy-deficiency causes myoblast apoptosis and impairs myotube ...formation. In this study, we continued this work with particular emphasis on mitochondrial remodelling and stress/apoptotic signaling. We found increased (p < 0.05) autophagic (e.g., altered LC3B levels, increased ATG7, decreased SQSTM1) and mitophagic (e.g., BNIP3 upregulation, mitochondrial localized GFP-LC3 puncta, and elevated mitochondrial LC3B-II) signaling during myoblast differentiation. shRNA-mediated knockdown of ATG7 (shAtg7) decreased these autophagic and mitophagic responses, while increasing CASP3 activity and ANXA5/annexin V staining in differentiating myoblasts; ultimately resulting in dramatically impaired myogenesis. Further confirming the importance of mitophagy in these responses, CRISPR-Cas9-mediated knockout of Bnip3 (bnip3
-/-
) resulted in increased CASP3 activity and DNA fragmentation as well as impaired myoblast differentiation. In addition, shAtg7 myoblasts displayed greater endoplasmic reticulum (e.g., increased CAPN activity and HSPA) and mitochondrial (e.g., mPTP formation, reduced mitochondrial membrane potential, elevated mitochondrial 4-HNE) stress. shAtg7 and bnip3
-/-
myoblasts also displayed altered mitochondria-associated signaling (e.g., PPARGC1A, DNM1L, OPA1) and protein content (e.g., SLC25A4, VDAC1, CYCS). Moreover, shAtg7 myoblasts displayed CYCS and AIFM1 release from mitochondria, and CASP9 activation. Similarly, bnip3
-/-
myoblasts had significantly higher CASP9 activation during differentiation. Importantly, administration of a chemical inhibitor of CASP9 (Ac-LEHD-CHO) or dominant-negative CASP9 (ad-DNCASP9) partially recovered differentiation and myogenesis in shAtg7 myoblasts. Together, these data demonstrate an essential role for autophagy in protecting myoblasts from mitochondrial oxidative stress and apoptotic signaling during differentiation, as well as in the regulation of mitochondrial network remodelling and myogenesis.
Abbreviations: 3MA: 3-methyladenine; 4-HNE: 4-hydroxynonenal; ACT: actin; AIFM1/AIF: apoptosis-inducing factor, mitochondrion-associated 1; ANXA5: annexin V; ATG7: autophagy related 7; AU: arbitrary units; BAX: BCL2-associated X protein; BCL2: B cell leukemia/lymphoma 2; BECN1: beclin 1, autophagy related; BNIP3: BCL2/adenovirus E1B interacting protein 3; CAPN: calpain; CASP: caspase; CASP3: caspase 3; CASP8: caspase 8; CASP9: caspase 9; CASP12: caspase 12; CAT: catalase; CQ: chloroquine; CYCS: cytochrome c, somatic; DCF; 2',7'-dichlorofluorescein; DNM1L/DRP1: dynamin 1-like; DM: differentiation media; DMEM: Dulbecco's modified Eagle's medium; ER: endoplasmic reticulum; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescent protein; GM: growth media; p-H2AFX: phosphorylated H2A histone family, member X; H2BFM: H2B histone family, member M; HBSS: Hanks balanced salt solution; HSPA/HSP70: heat shock protein family A; JC-1: tetraethylbenzimidazolylcarbocyanine iodide; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; mPTP: mitochondrial permeability transition pore; MYH: myosin heavy chain; MYOG: myogenin; OPA1: OPA1, mitochondrial dynamin like GTPase; PI: propidium iodide; PINK1: PTEN induced putative kinase 1; PPARGC1A/PGC1α: peroxisome proliferative activated receptor, gamma, coactivator 1 alpha; ROS: reactive oxygen species; SLC25A4/ANT1: solute carrier family 25 (mitochondrial carrier, adenine nucleotide translocator), member 4; SOD1: superoxide dismutase 1, soluble; SOD2: superoxide dismutase 2, mitochondrial; SQSTM1/p62: sequestosome 1; VDAC1: voltage-dependent anion channel 1
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BFBNIB, GIS, IJS, KISLJ, NUK, PNG, UL, UM, UPUK
Muscle satellite cells are resistant to cytotoxic agents, and they express several genes that confer resistance to stress, thus allowing efficient dystrophic muscle regeneration after ...transplantation. However, once they are activated, this capacity to resist to aggressive agents is diminished resulting in massive death of transplanted cells. Although cell immaturity represents a survival advantage, the signalling pathways involved in the control of the immature state remain to be explored. Here, we show that incubation of human myoblasts with retinoic acid impairs skeletal muscle differentiation through activation of the retinoic-acid receptor family of nuclear receptor. Conversely, pharmacologic or genetic inactivation of endogenous retinoic-acid receptors improved myoblast differentiation. Retinoic acid inhibits the expression of early and late muscle differentiation markers and enhances the expression of myogenic specification genes, such as
PAX7
and
PAX3
. These results suggest that the retinoic-acid-signalling pathway might maintain myoblasts in an undifferentiated/immature stage. To determine the relevance of these observations, we characterised the retinoic-acid-signalling pathways in freshly isolated satellite cells in mice and in siMYOD immature human myoblasts. Our analysis reveals that the immature state of muscle progenitors is correlated with high expression of several genes of the retinoic-acid-signalling pathway both in mice and in human. Taken together, our data provide evidences for an important role of the retinoic-acid-signalling pathway in the regulation of the immature state of muscle progenitors.
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EMUNI, FZAB, GEOZS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NUK, OBVAL, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
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