Transcript Calcium Signaling - Georgia Institute of Technology

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 thymidinelabeled 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• Quiescent: withdrawn from cell cycle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
Start
Lox
DTA
Stop Lox
Stop
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
– 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
– FGF, EGF, HGF
• DNA synthesis
– CyD/CDK4 accumulation
– Degradation of Rb
– Activation of E2F
• Cell size integration
– GSK3 inhibits CDK4
– GSK3 inhibits b-Catenin
GF-R
(c-Met,
FGFR)
Ras-rafMEK-ERK
Starvation
Rb
GSK3
E2F
Cyclin-A
DNA-pol’ase
CDK1
DNA synthesis
Differentiation
• Cell cycle withdrawal
– 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
– Class A/general
• TCF
– Class B/Tissue specific
M-cadherin
GSK3b
• MyoD
• b-Catenin: bHLH-HDAC deactivator
• Inhibitor of differentiation (Id)
– HLH, no b
– No b = no DNA binding
Myostatin
Smad2/3
Dishevelled
Id3
B-catenin
MyoD
TCF/LEF
Myogenin/MHC
DNA synthesis
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