Twist2-expressing cells reside in human skeletal muscle and are responsive to aging and resistance exercise training.

Skeletal muscle is maintained and repaired by sub-laminar, Pax7-expressing satellite cells. However, recent mouse investigations have described a second myogenic progenitor population that resides within the myofiber interstitium and expresses the transcription factor Twist2. Twist2-expressing cells exclusively repair and maintain type IIx/b muscle fibers. Currently, it is unknown if Twist2-expressing cells are present in human skeletal muscle and if they function as myogenic progenitors. Here, we perform a combination of single-cell RNA sequencing analysis and immunofluorescence staining to demonstrate the identity and localization of Twist2-expressing cells in human skeletal muscle. Twist2-expressing cells were identified to be anatomically and transcriptionally comparable to fibro-adipogenic progenitors (FAPs) and lack expression of typical satellite cell markers such as Pax7. Comparative analysis revealed that human and mouse Twist2-expressing cells were highly transcriptionally analogous and resided within the same anatomical structures in vivo. Examination of young and aged skeletal muscle biopsy samples revealed that Twist2-positive cells are more prevalent in aged muscle and increase following 12-weeks of resistance exercise training (RET) in humans. However, the quantity of Twist2-positive cells was not correlated with indices of muscle mass or muscle fiber cross-sectional area (CSA) in young or older muscle, and their abundance was surprisingly, negatively correlated with CSA and myonuclear domain size following RET. Taken together, we have identified cells expressing Twist2 in human skeletal muscle which are responsive to aging and exercise. Further examination of their myogenic potential is warranted.

Identification of underexplored mesenchymal and vascular-related cell populations in human skeletal muscle.

Skeletal muscle repair and maintenance are directly and indirectly supported by interstitial cell populations such as vascular cells and fibro-adipogenic progenitors (FAPs), a subset of which express Twist2 and possess direct myogenic potential. Furthermore, work in rodents has highlighted the potential of pericytes to act as progenitor cells, giving rise to muscle cells and transdifferentiating into endothelial cells. However, less is understood about these populations in human skeletal muscle. Here, we performed single-cell RNA sequencing (scRNAseq) on ∼2,000 cells isolated from the human semitendinosus muscle of young individuals. This demonstrated the presence of a vascular-related cell type that expressed pericyte and pan-endothelial genes that we localized to large blood vessels within skeletal muscle cross sections and termed endothelial-like pericytes (ELPCs). RNA velocity analysis indicated that ELPCs may represent a "transition state" between endothelial cells and pericytes. Analysis of published scRNAseq data sets revealed evidence for ELPCs in trunk and heart musculature, which showed transcriptional similarity. In addition, we identified a subset of FAPs expressing TWIST2 mRNA and protein. Human TWIST2-expressing cells were anatomically and transcriptionally comparable to mouse Twist2 cells as they were restricted to the myofiber interstitium, expressed fibrogenic genes but lacked satellite cell markers, and colocalized with the FAPs marker PDGFRα in human muscle cross sections. Taken together, these results highlight the complexity of stromal cells residing in human skeletal muscle and support the utility of scRNAseq for discovery and characterization of poorly described cell populations.

Muscle injury induces a transient senescence-like state that is required for myofiber growth during muscle regeneration.

Cellular senescence is the irreversible arrest of normally dividing cells and is driven by the cell cycle inhibitors Cdkn2a, Cdkn1a, and Trp53. Senescent cells are implicated in chronic diseases and tissue repair through their increased secretion of pro-inflammatory factors known as the senescence-associated secretory phenotype (SASP). Here, we use spatial transcriptomics and single-cell RNA sequencing (scRNAseq) to demonstrate that cells displaying senescent characteristics are "transiently" present within regenerating skeletal muscle and within the muscles of D2-mdx mice, a model of Muscular Dystrophy. Following injury, multiple cell types including macrophages and fibrog-adipogenic progenitors (FAPs) upregulate senescent features such as senescence pathway genes, SASP factors, and senescence-associated beta-gal (SA-ß-gal) activity. Importantly, when these cells were removed with ABT-263, a senolytic compound, satellite cells are reduced, and muscle fibers were impaired in growth and myonuclear accretion. These results highlight that an "acute" senescent phenotype facilitates regeneration similar to skin and neonatal myocardium.

The Contribution of Innervation to Tissue Repair and Regeneration.

Animals such as amphibians have an incredible capacity for regeneration with some being able to regrow their tail or appendages. Although some mammalian tissues like the skin and bones can repair following injury, there are only a few examples of true multilineage regeneration, including the distal portion of the digit tip. In both amphibians and mammals, however, to achieve successful repair or regeneration, it is now appreciated that intact nerve innervation is a necessity. Here, we review the current state of literature and discuss recent advances that identify axon-derived signals, Schwann cells, and nerve-derived mesenchymal cells as direct and indirect supporters of adult tissue homeostasis and repair. We posit that understanding how nerves positively influence repair and regeneration could lead to targeted regenerative medicine strategies to enhance tissue repair in humans.

Loss of dystrophin expression in skeletal muscle is associated with senescence of macrophages and endothelial cells.

Cellular senescence is the irreversible arrest of normally dividing cells and is driven by cell cycle inhibitory proteins such as p16, p21, and p53. When cells enter senescence, they secrete a host of proinflammatory factors known as the senescence-associated secretory phenotype, which has deleterious effects on surrounding cells and tissues. Little is known of the role of senescence in Duchenne muscular dystrophy (DMD), the fatal X-linked neuromuscular disorder typified by chronic inflammation, extracellular matrix remodeling, and a progressive loss in muscle mass and function. Here, we demonstrate using C57-mdx (8-wk-old) and D2-mdx (4-wk-old and 8-wk-old) mice, two mouse models of DMD, that cells displaying canonical markers of senescence are found within the skeletal muscle. Eight-week-old D2-mdx mice, which display severe muscle pathology, had greater numbers of senescent cells associated with areas of inflammation, which were mostly Cdkn1a-positive macrophages, whereas in C57-mdx muscle, senescent populations were endothelial cells and macrophages localized to newly regenerated myofibers. Interestingly, this pattern was similar to cardiotoxin (CTX)-injured wild-type (WT) muscle, which experienced a transient senescent response. Dystrophic muscle demonstrated significant upregulations in senescence pathway genes [Cdkn1a (p21), Cdkn2a (p16INK4A), and Trp53 (p53)], which correlated with the quantity of senescence-associated ß-galactosidase (SA-ß-Gal)-positive cells. These results highlight an underexplored role for cellular senescence in murine dystrophic muscle.

Mesenchymal Precursor Cells in Adult Nerves Contribute to Mammalian Tissue Repair and Regeneration.

Peripheral innervation plays an important role in regulating tissue repair and regeneration. Here we provide evidence that injured peripheral nerves provide a reservoir of mesenchymal precursor cells that can directly contribute to murine digit tip regeneration and skin repair. In particular, using single-cell RNA sequencing and lineage tracing, we identify transcriptionally distinct mesenchymal cell populations within the control and injured adult nerve, including neural crest-derived cells in the endoneurium with characteristics of mesenchymal precursor cells. Culture and transplantation studies show that these nerve-derived mesenchymal cells have the potential to differentiate into non-nerve lineages. Moreover, following digit tip amputation, neural crest-derived nerve mesenchymal cells contribute to the regenerative blastema and, ultimately, to the regenerated bone. Similarly, neural crest-derived nerve mesenchymal cells contribute to the dermis during skin wound healing. These findings support a model where peripheral nerves directly contribute mesenchymal precursor cells to promote repair and regeneration of injured mammalian tissues.

Aortic Sca-1+ Progenitor Cells Arise from the Somitic Mesoderm Lineage in Mice.

Sca-1+ progenitor cells in the adult mouse aorta are known to generate vascular smooth muscle cells (VSMCs), but their embryological origins and temporal abundance are not known. Using tamoxifen-inducible Myf5-CreER mice, we demonstrate that Sca-1+ adult aortic cells arise from the somitic mesoderm beginning at E8.5 and continue throughout somitogenesis. Myf5 lineage-derived Sca-1+ cells greatly expand in situ, starting at 4 weeks of age, and become a major source of aortic Sca-1+ cells by 6 weeks of age. Myf5-derived adult aortic cells are capable of forming multicellular sphere-like structures in vitro and express the pluripotency marker Sox2. Exposure to transforming growth factor-ß3 induces these spheres to differentiate into calponin-expressing VSMCs. Pulse-chase experiments using tamoxifen-inducible Sox2-CreERT2 mice at 8 weeks of age demonstrate that ∼35% of all adult aortic Sca-1+ cells are derived from Sox2+ cells. The present study demonstrates that aortic Sca-1+ progenitor cells are derived from the somitic mesoderm formed at the earliest stages of somitogenesis and from Sox2-expressing progenitors in adult mice.

Low-load resistance exercise during inactivity is associated with greater fibre area and satellite cell expression in older skeletal muscle.

BACKGROUND: Age-related sarcopenia is accelerated by physical inactivity. Low-load resistance exercise (LLRE) counters inactivity-induced muscle atrophy in older adults, but changes in muscle fibre morphology are unstudied. We aimed to determine the impact of LLRE during short-term inactivity (step-reduction) on muscle fibre size and capillarity as well as satellite cell (SC) content in older skeletal muscle. METHODS: Fourteen older (~71 years) male adults underwent 14 days of step reduction (<1500 steps/day) while performing six sessions of LLRE (~30% maximal strength) with one leg (SR + EX) while the contralateral leg served as an untrained control (SR). Seven healthy ambulatory age-matched male adults (~69 years) served as a comparator group (COM). Muscle biopsies were taken from the vastus lateralis after 14 days, and immunohistochemical analysis was performed to determine muscle fibre cross-sectional area (CSA), myonuclear content, SC content (PAX7+ cells), and total (C:F) and fibre type-specific (C:Fi) capillary-to-fibre ratios. RESULTS: Type I and II fibre CSA was greater in SR + EX compared with SR. Whereas there were no differences across fibre types between SR + EX and CON, type II fibre CSA was significantly lower in SR compared with COM. Type II myonuclear domain was greater in SR + EX compared with COM and SR. Pax7+ cells associated with type I and II fibres were lower in SR compared with SR + EX. Type II PAX7+ cells were also lower in SR compared with COM with a similar trend for type I fibres. There were trends for a lower C:Fi in SR compared with SR + EX for both fibre types with no differences for each compared with COM. CONCLUSIONS: Minimal LLRE during a period of decreased physical activity is associated with greater muscle fibre CSA, SC content, and capillarization. These results support the use of LLRE as an effective countermeasure to inactivity-induced alterations in muscle morphology with age.

Dedifferentiated Schwann Cell Precursors Secreting Paracrine Factors Are Required for Regeneration of the Mammalian Digit Tip.

Adult mammals have lost multi-tissue regenerative capacity, except for the distal digit, which is able to regenerate via mechanisms that remain largely unknown. Here, we show that, after adult mouse distal digit removal, nerve-associated Schwann cell precursors (SCPs) dedifferentiate and secrete growth factors that promote expansion of the blastema and digit regeneration. When SCPs were dysregulated or ablated, mesenchymal precursor proliferation in the blastema was decreased and nail and bone regeneration were impaired. Transplantation of exogenous SCPs rescued these regeneration defects. We found that SCPs secrete factors that promote self-renewal of mesenchymal precursors, and we used transcriptomic and proteomic analysis to define candidate factors. Two of these, oncostatin M (OSM) and platelet-derived growth factor AA (PDGF-AA), are made by SCPs in the regenerating digit and rescued the deficits in regeneration caused by loss of SCPs. As all peripheral tissues contain nerves, these results could have broad implications for mammalian tissue repair and regeneration.

Exercise-induced mitochondrial p53 repairs mtDNA mutations in mutator mice.

BACKGROUND: Human genetic disorders and transgenic mouse models have shown that mitochondrial DNA (mtDNA) mutations and telomere dysfunction instigate the aging process. Epidemiologically, exercise is associated with greater life expectancy and reduced risk of chronic diseases. While the beneficial effects of exercise are well established, the molecular mechanisms instigating these observations remain unclear. RESULTS: Endurance exercise reduces mtDNA mutation burden, alleviates multisystem pathology, and increases lifespan of the mutator mice, with proofreading deficient mitochondrial polymerase gamma (POLG1). We report evidence for a POLG1-independent mtDNA repair pathway mediated by exercise, a surprising notion as POLG1 is canonically considered to be the sole mtDNA repair enzyme. Here, we show that the tumor suppressor protein p53 translocates to mitochondria and facilitates mtDNA mutation repair and mitochondrial biogenesis in response to endurance exercise. Indeed, in mutator mice with muscle-specific deletion of p53, exercise failed to prevent mtDNA mutations, induce mitochondrial biogenesis, preserve mitochondrial morphology, reverse sarcopenia, or mitigate premature mortality. CONCLUSIONS: Our data establish a new role for p53 in exercise-mediated maintenance of the mtDNA genome and present mitochondrially targeted p53 as a novel therapeutic modality for diseases of mitochondrial etiology.

Identification of Drugs that Regulate Dermal Stem Cells and Enhance Skin Repair.

Here, we asked whether we could identify pharmacological agents that enhance endogenous stem cell function to promote skin repair, focusing on skin-derived precursors (SKPs), a dermal precursor cell population. Libraries of compounds already used in humans were screened for their ability to enhance the self-renewal of human and rodent SKPs. We identified and validated five such compounds, and showed that two of them, alprostadil and trimebutine maleate, enhanced the repair of full thickness skin wounds in middle-aged mice. Moreover, SKPs isolated from drug-treated skin displayed long-term increases in self-renewal when cultured in basal growth medium without drugs. Both alprostadil and trimebutine maleate likely mediated increases in SKP self-renewal by moderate hyperactivation of the MEK-ERK pathway. These findings identify candidates for potential clinical use in human skin repair, and provide support for the idea that pharmacological activation of endogenous tissue precursors represents a viable therapeutic strategy.

Direct genesis of functional rodent and human schwann cells from skin mesenchymal precursors.

Recent reports of directed reprogramming have raised questions about the stability of cell lineages. Here, we have addressed this issue, focusing upon skin-derived precursors (SKPs), a dermally derived precursor cell. We show by lineage tracing that murine SKPs from dorsal skin originate from mesenchymal and not neural crest-derived cells. These mesenchymally derived SKPs can, without genetic manipulation, generate functional Schwann cells, a neural crest cell type, and are highly similar at the transcriptional level to Schwann cells isolated from the peripheral nerve. This is not a mouse-specific phenomenon, since human SKPs that are highly similar at the transcriptome level can be made from neural crest-derived facial and mesodermally derived foreskin dermis and the foreskin SKPs can make myelinating Schwann cells. Thus, nonneural crest-derived mesenchymal precursors can differentiate into bona fide peripheral glia in the absence of genetic manipulation, suggesting that developmentally defined lineage boundaries are more flexible than widely thought.

Sox2-mediated regulation of adult neural crest precursors and skin repair.

Nerve-derived neural crest cells are essential for regeneration in certain animals, such as newts. Here, we asked whether they play a similar role during mammalian tissue repair, focusing on Sox2-positive neural crest precursors in skin. In adult skin, Sox2 was expressed in nerve-terminal-associated neural crest precursor cells (NCPCs) around the hair follicle bulge, and following injury was induced in nerve-derived cells, likely dedifferentiated Schwann cell precursors. At later times postinjury, Sox2-positive cells were scattered throughout the regenerating dermis, and lineage tracing showed that these were all neural-crest-derived NCPCs. These Sox2-positive NCPCs were functionally important, since acute deletion of Sox2 prior to injury caused a decrease of NCPCs in the wound and aberrant skin repair. These data demonstrate that Sox2 regulates skin repair, likely by controlling NCPCs, and raise the possibility that nerve-derived NCPCs may play a general role in mammalian tissue repair.

Captopril treatment induces hyperplasia but inhibits myonuclear accretion following severe myotrauma in murine skeletal muscle.

The role of ANG II in skeletal muscle and satellite cell regulation is largely unknown. Cardiotoxin (CTX) was used to investigate whether muscle injury activates a local ANG II signaling system. Following injury, immunohistochelmistry (IHC) analysis revealed a robust increase in the intensity of angiotensinogen and angiotensin type 1 (AT(1)) receptor expression. As regeneration proceeded, however, AT(1) and angiotensinogen were downregulated. Nuclear accretion and fiber formation were also assessed during muscle regeneration in mice treated with captopril (an angiotensin-converting enzyme inhibitor). When ANG II formation was blocked through the use of captopril, we observed a significantly reduced accretion of nuclei into myofibers (-25%), while tibialis anterior total fiber number was significantly increased +37%. This phenotype appeared to be due to alterations in satellite cell differentiation kinetics; captopril treatment led to sustained mRNA expression of markers associated with quiescence and proliferation (Myf5, Pax7) and simultaneously delayed or inhibited the expression of myogenin. IHC staining supported these findings, revealing that captopril treatment resulted in a strong trend (P = 0.06) for a decrease in the proportion of myogenin-positive myoblasts. Furthermore, these observations were associated with a delay in muscle fiber maturation; captopril treatment resulted in sustained expression of embryonic myosin heavy chain. Collectively, these findings demonstrate that localized skeletal muscle angiotensin signaling is important to muscle fiber formation, myonuclear accretion, and satellite cell function.

Skeletal muscle myoblasts possess a stretch-responsive local angiotensin signalling system.

A paucity of information exists regarding the presence of local renin-angiotensin systems (RASs) in skeletal muscle and associated muscle stem cells. Skeletal muscle and muscle stem cells were isolated from C57BL/6 mice and examined for the presence of a local RAS using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), immunohistochemistry (IHC), Western blotting and liquid chromatography-mass spectrometry (LC-MS). Furthermore, the effect of mechanical stimulation on RAS member gene expression was analysed. Whole skeletal muscle, primary myoblasts and C2C12 derived myoblasts and myotubes differentially expressed members of the RAS including angiotensinogen, angiotensin-converting enzyme (ACE), angiotensin II (Ang II) type 1 (AT(1)) and type 2 (AT(2)). Renin transcripts were never detected, however, mRNA for the 'renin-like' enzyme cathepsin D was observed and Ang I and Ang II were identified in cell culture supernatants from proliferating myoblasts. AT(1) appeared to co-localise with polymerised actin filaments in proliferating myoblasts and was primarily found in the nucleus of terminally differentiated myotubes. Furthermore, mechanical stretch of proliferating and differentiating C2C12 cells differentially induced mRNA expression of angiotensinogen, AT(1) and AT(2). Proliferating and differentiated muscle stem cells possess a local stress-responsive RAS in vitro. The precise function of a local RAS in myoblasts remains unknown. However, evidence presented here suggests that Ang II may be a regulator of skeletal muscle myoblasts.

Skeletal muscle-endothelial cell cross talk through angiotensin II.

The role of angiotensin II (ANG II) in postnatal vasculogenesis and angiogenesis during skeletal muscle (SKM) regeneration is unknown. We examined the capacity of ANG II to stimulate capillary formation and growth during cardiotoxin-induced muscle regeneration in ACE inhibitor-treated ANG II type 1a receptor knockout (AT1a(-/-)) and C57Bl/6 control mice. Analysis of tibialis anterior (TA) cross-sections revealed 17% and 23% reductions in capillarization in AT1a(-/-) and captopril treated mice, respectively, when compared with controls, 21 days postinjury. Conversely, no differences in capillarization were detected at early time points (7 and 10 days). These results identify ANG II as a regulator of angiogenesis but not vasculogenesis in vivo. In vitro angiogenesis assays of human umbilical vein endothelial cells (HUVECs) further confirmed ANG II as proangiogeneic as 71% and 124% increases in tube length and branch point number were observed following ANG II treatment. Importantly, treatment of HUVECs with conditioned media from differentiated muscle cells resulted in an 84% and 203% increase in tube length and branch point number compared with controls, which was abolished following pretreatment of the cells with an angiotensin-converting enzyme inhibitor. The pro-angiogenic effect of ANG II can be attributed to an enhanced endothelial cell migration because both transwell and under agarose migration assays revealed a 37% and 101% increase in cell motility, respectively. Collectively, these data highlight ANG II as a proangiogenic regulator during SKM regeneration in vivo and more importantly demonstrates that ANG II released from SKM can signal endothelial cells and regulate angiogenesis through the induction of endothelial cell migration.

Regulation of muscle satellite cell activation and chemotaxis by angiotensin II.

The role of angiotensin II (Ang II) in skeletal muscle is poorly understood. We report that pharmacological inhibition of Ang II signaling or ablation of the AT1a receptor significantly impaired skeletal muscle growth following myotrauma, in vivo, likely due to impaired satellite cell activation and chemotaxis. In vitro experiments demonstrated that Ang II treatment activated quiescent myoblasts as evidenced by the upregulation of myogenic regulatory factors, increased number of ß-gal+, Myf5-LacZ myoblasts and the acquisition of cellular motility. Furthermore, exogenous treatment with Ang II significantly increased the chemotactic capacity of C2C12 and primary cells while AT1a(-/-) myoblasts demonstrated a severe impairment in basal migration and were not responsive to Ang II treatment. Additionally, Ang II interacted with myoblasts in a paracrine-mediated fashion as 4 h of cyclic mechanical stimulation resulted in Ang II-induced migration of cocultured myoblasts. Ang II-induced chemotaxis appeared to be regulated by multiple mechanisms including reorganization of the actin cytoskeleton and augmentation of MMP2 activity. Collectively, these results highlight a novel role for Ang II and ACE inhibitors in the regulation of skeletal muscle growth and satellite cell function.

Association of interleukin-6 signalling with the muscle stem cell response following muscle-lengthening contractions in humans.

BACKGROUND: The regulation of muscle stem cells in humans in response to muscle injury remains largely undefined. Recently, interleukin-6 (IL-6) has been implicated in muscle stem cell (satellite cell)-mediated muscle hypertrophy in animals; however, the role of IL-6 in the satellite cell (SC) response following muscle-lengthening contractions in humans has not been studied. METHODOLOGY/PRINCIPAL FINDINGS: Eight subjects (age 22+/-1 y; 79+/-8 kg) performed 300 maximal unilateral lengthening contractions (3.14 rad.s(-1)) of the knee extensors. Blood and muscle samples were collected before and at 4, 24, 72, and 120 hours post intervention. IL-6, IL-6 receptor, IL-6R(alpha), cyclin D1, suppressor of cytokine signling-3 (SOCS3) mRNA were measured using quantitative RT-PCR and serum IL-6 protein was measured using an ELISA kit. JAK2 and STAT3 phosphorylated and total protein was measured using western blotting techniques. Immunohistochemical analysis of muscle cross-sections was performed for the quantification of SCs (Pax7(+) cells) as well as the expression of phosphorylated STAT3, IL-6, IL-6R(alpha), and PCNA across all time-points. The SC response, as defined by an amplification of Pax7(+) cells, was rapid, increasing by 24 h and peaking 72 h following the intervention. Muscle IL-6 mRNA increased following the intervention, which correlated strongly (R(2) = 0.89, p<0.002) with an increase in serum IL-6 concentration. SC IL-6R(alpha) protein was expressed on the fiber, but was also localized to the SC, and IL-6(+) SC increased rapidly following muscle-lengthening contractions and returned to basal levels by 72 h post-intervention, demonstrating an acute temporal expression of IL-6 with SC. Phosphorylated STAT3 was evident in SCs 4 h after lengthening contraction, and the downstream genes, cyclin D1 and SOCS3 were significantly elevated 24 hours after the intervention. CONCLUSIONS/SIGNIFICANCE: The increased expression of STAT3 responsive genes and expression of IL-6 within SCs demonstrate that IL-6/STAT3 signaling occurred in SCs, correlating with an increase in SC proliferation, evidenced by increased Pax7(+)/PCNA(+) cell number in the early stages of the time-course. Collectively, these data illustrate that IL-6 is an important signaling molecule associated with the SC response to acute muscle-lengthening contractions in humans.