Satellite Cells Define the myonuclear domain Describe the satellite cell niche Describe the signaling networks controlling SC proliferation and fusion HGF, FGF, myostatin MRFs, pax

Satellite cell Anatomical definition Mononucleated cells in muscle Oustide the fiber plasma membrane Inside the basal lamina EM of SC on muscle fiber; Dark thymidine-labeled new DNA. Moss & Leblond, 1971

Satellite Cell: Molecular definition Unique c-Met (HGF receptor) M-cadherin (adhesion molecule) Pax3/7 (transcription factor) Overlapping CD34 (hematopoetic) Integrin-a7 (adhesion molecule) Myf5 (transcription factor) Caveolin-1 Laminin M-cadherin Nucelus Irintchev & al 1994

SC Function Quiescent: withdrawn from cell cycle Regeneration SCs “activated” by damage Re-enter cell cycle Fully differentiate Fusemyotubemuscle fiber Hypertrophy/growth Re-enter cell cycle(?) Fusemuscle fiber

Myonuclear domain Volume of cytoplasm supported by one nucleus Conserved during growth Consistent across body size DNA content may limit RNA synthesis and protein content Liu & al., 2008

SC contribution to hypertrophy Adams & al., 2002 Irradiation to silence SC Synergist ablation Irradiation blocks substantial growth MN domain conserved

Signaling during Ir-hypertrophy Early (0-3 days) Muscle IGF-1 Myogenin Persistent MGF/myogenin mTOR effectors Both groups start the same, but Ir deviates after a few days. Fails to maintain trajectory

Another model of SC ablation McCarthy & al 2011 SC-specific, Tamoxifen-inducible diptheria toxin “Cre-Lox” recombination Cre recombinase targets Lox sequences for recombination Tamoxifen-dependent Rosa26/DTA: diptheria toxin preceded by floxed transcription stop sequence Pax7/Cre: satellite-cell specific Cre Start Lox eGFP Stop Lox DTA Stop Start Lox DTA Stop

Pax7/DTA disrupts SC-function BaCl2 kills myofibersregeneration SC-mediated repair is disrupted in pax7/DTA Healthy muscle Injured & repaired muscle (centrally-located nuclei) Injured & not repaired muscle (unstructured fibrosis)

Pax7/DTA synergist ablation DTA fails to block substantial growth MN domain increases

Irradiation vs DTA questions Irradiation kills both SC and bone marrow Is the initial FO response to the injury or to the overload? Most clear DTA results at 2 weeks, when edema dominates protein accretion

Satellite cell niche Niche: physical environment Basal Lamina/ECM Circulating growth factors are only part Physical contacts: myofiber and ECM Paracrine factors: fiber, inflammatory cells Mechanical factors Basal Lamina/ECM Collagen/laminin Heparin sulfate proteoglycans Adhesive substrates Growth factor chelators Receptor cofactors

Contact control of SC Contact with fiber blocks proliferation Contact with BL facilitates proliferation Ground-up muscle helps Dissociate muscle into Fiber-BL chunks Kill fiber with marcaine Count SC (Bischoff, 1990) Satellite cell Basal Lamina shell Killed fiber clot

SC Fate Fiber repair Self renewal Seale & Rudnicki, 2000

Regulatory control of fate Proliferation Cell cycle progression HGF/FGF MyoD/Myf5 Differentiation IGF-1/PGF2a Myogenin/MRF4 Loss of Pax3/7 Charge & Rudnicki, 2004

Activation of quiescent SCs Some signal is released from damaged muscle bFGF? HGF? Cell cycle re-entry (SCadult myoblast) PI3K-mTORgrow the cell ERKcell cycle progression MyoD/myf5be a muscle Negative controls Inhibit differentiation TGF-b family (TGF-b, BMP) Myostatin

Proliferation Mitogens DNA synthesis Cell size integration GF-R (c-Met, FGFR) Mitogens FGF, EGF, HGF DNA synthesis CyD/CDK4 accumulation Degradation of Rb Activation of E2F Cell size integration GSK3 inhibits CDK4 GSK3 inhibits b-Catenin Ras-raf-MEK-ERK Starvation Rb GSK3 E2F Cyclin-A DNA-pol’ase CDK1 DNA synthesis

Differentiation Cell cycle withdrawal Fusion or quiescence M-cadherinb-catenin/TCF MYf5/myogeninterminal differentiation Fusion or quiescence Pax3/7MRF suppressor Cytoskeletal rearrangement Contact recognition of fusion partner

Differentiation bHLH transcription factors Wnt receptor bHLH transcription factors Class A/general TCF Class B/Tissue specific MyoD b-Catenin: bHLH-HDAC deactivator Inhibitor of differentiation (Id) HLH, no b No b = no DNA binding Dishevelled M-cadherin GSK3b B-catenin TCF/LEF MyoD DNA synthesis Myogenin/MHC Myostatin Smad2/3 Id3

Control of fusion Scar/SNS/kette mediated actin foci

Muscle and other stem cells Pluripotency The Side Population Exclude Hoechst dyes via active ABC transporters Subset of many stem-like cell populations Muscle SP cells CD45/Sca1 positive Pluripotent Muscle MP cells Unipotent Asakura & al, 2002

Muscle and other stem cells Hematopoetic stem cells Bone marrow derived Mostly WBC 0.2% SP, pluripotent May contribute to regen in many tissues eg: chimeric neurons after BMT Are muscle SP cells really satellite cells? Do MP and SP cells contribute equally to regeneration? Hypertrophy?

Satellite cell therapies Muscle normally incorporates new genetic material from proliferating cells Grow (and engineer) myoblast population Inject & allow fusion None of them work, yet. Some progress in animal models Skuk & Tremblay, 2003)

Myoblast transplantation (DMD) Correction of genetic defects (Dystrophin) 1e7 donor SC in 100 injections to one muscle CsA as immunosuppressant 6 month follow-up Force gain (CsA) No dystrophin Miller & al., 1997

Satellite cell therapies Cardiovascular support Autograft during LVAD implant 1e7-1e8 cells in 3-30 injections into infarct Some apparent survival > 6 mo Low efficiency (1% @ 18 hr) Endocrine supplement eg, insulin Dib & al., 2005