Transcript Fiber Types - Georgia Institute of Technology

Fiber Types
• Three fiber typing schemes
– Metabolic/biochemical activity of fiber
– Protein/gene expression of fiber
– Functional performance of motor unit
• Explain the relationship between motor unit
types and muscle fiber types
– Burke &al 1973
– Schiaffino & Bottinelli, 2011
Physiological fiber types
• Fast Fatiguable
– Fast force rise
– High force
– Low oxidative cap
• Slow
– Slow force rise
– Low force
– High oxidative capacity
• Fast fatigue Resistant
– High oxidative capacity
Repeated
activation
Single
activation
“Fast” and “Slow” continuum
• White vs red muscle (Ranvier, 1874)
– Not just because of blood content
– Some exceptions: masseter
– Speed correlates with myosin ATPase kinetics
• Heterogeneity within muscles
– Mitochondria: density and activity
– Sarcoplasmic reticulum volume
– Glycogen and Myoglobin content
– Z-disk morphology
• Protein/gene expression
Histochemistry
• Staining: preferential dye binding
• In situ chemical reactions
– Structurally immobilized enzymes
• Transmembrane
• Sarcomeric
– Reaction precipitate retained near enzyme
– Soluble substrates
– Often redox
• NBTformazan
• BCIPindigo
Dephos
oxidation
BCIP
Indigo
Hematoxylin and Eosin
• H: metal binding, nuclei
• E: alkaline binding, protein
Succinate dehydrogenase
• TCA cycle, redox enzyme
– Bound in mitochondria
a-glycerophosphate dehydrogenase
• Glycerol-3-phosphate dehydrogenase (GPDH)
– Really looking at glycerol-3-phosphate shuttle
– G-3-p participates in an undesired reaction
Fructose 1-6 bisphosphate
Glycerol-1-phosphate
(not glycerol-3-phosphate)
(not glygeraldehyde-3-phosphate)
Dihydroxyacetone Glyceraldehyde-3-phosphate
phosphate
Dihydroxyacetone
phosphate
Glycerol-3-phosphate
a-glycerolphosphate dehydrogenase
• GPDH bound in Z-disks
– More uniform in cross-section than mitochondria
Myosin ATPase
• Acid/Alkali-labile
– i.e.: acid inactivates “fast” myosin; alkali, “slow”
Myosin
ATPase after
pH 9.4 preinc
Correlations in histochemistry
• Mitochondrial enzymes
– Succinate
dehydrogenase
– Citrate synthase
– NAD diaphorase
• Myosin
– Acid-stable
• Glycolytic pathway
– GPDH
– Lactate dehydrogenase
– Glycogen
• Myosin
– Kinetic
– Alkali-stable
• Reactions tuned to give binary responses
• Discrete combinations: metabolic fiber types
– Slow, oxidative (SO)
– Fast, glycolytic (FG)
– Fast, oxidative and glycolytic (FOG)
Spatial distribution
• Section through lower leg stained for SDH and acid-stable
ATPase
Lateral
SO
FOG
Posterior
Anterior
FG
Medial
Enzyme content or activity?
• Histochemistry measures total reaction
– Enzyme content (moles)
– Enzyme kinetics (Vmax, km)
• Immunohistochemistry identifies proteins
– Immunoglobulin antibodies
– Extremely specific to 10-15 AA epitope
– AntigenAbAb-enzyme (HRP)
– AntigenAbAb-fluorophore
(immunofluorescent histology)
• Biochemistry, if you can purify activity
Muscle proteins with multiple isoforms
• Myosin
– 11 heavy chains
– 5 regulatory, 4 essential light chains
• SERCA
– Fast (1a)
– Slow (2a)
• Troponin
– T: 4 fast, 2 slow
– C, I: cardiac+slow; fast
• a-actinin
• Tropomyosin
Myosin
• 4 “conventional” isoforms: 1(o), 2a(*), 2x, 2b
– Limited overlap (+)
– Horseradish peroxidase/Diaminobenzidine staining
A4.84 MHC1
SC-71 MHC2a
My32 MHC2
Myosin
• Heavy chain
Slack test
2b
2x
2a
1
– Solution ATPase rateP0
– ADP affinityVmax
– F-actin velocity 2b>2x>2a>I
• Light chains (essential/alkali)
– Influence f-actin velocity
– Probably not ATPase
MLC3f/MLC2f
Bottinelli et al., 1994
SERCA
• 2 dominant isoforms: 1a, 2a
– Limited overlap
– Fluorescein indirect fluorescence
SERCA2 (in type 1 fibers)
De Jonge et al., 2006
Ikezoe et al., 2003
Calcium handling
• SERCA
– Faster calcium re-uptake with SERCA-1
– SERCA-2 inhibited by phospholamban
• Parvalbumin
– Only in slow fibers: quenches Ca2+ release
Carroll et al., 1997
Troponin
• 3 subunits w/ fast/slow
– C: calcium binding
– T: tropomyosin binding
– I: Inhibitory
• Provide calcium-dependent activation
– Ca2+-free form blocks
MHC binding to actin
Tobacman, 1996
Troponin/Tropomyosin
• Calcium sensitivity
– Slow fibers: lower threshold & sensitivity
– Fast fibers: higher sensitivity
TnC-slow TM-b
TnC-fast TM-b
TnC-fast TM-a
Expression fiber types
• MHC-centric
– Conventional: 1, 2a, 2x, 2b
– Heart: cardiac-a, cardiac-b (MHC-I)
– Developmental: embryonic, neonatal
– Unconventional: Extra-ocular, Masseter (superfast)
• Humans, no 2b
• Co-expressed regulatory elements
– miRNA
– Antisense intergenic
Functional correlations
• Some muscles seem specialized
– Soleus (cat, some rats): Type SO; high endurance
– Ex Digitorum Longus (rodents): Type FG; low
• Most muscles mixed
• Opportunity for optimization
– High use muscle benefits from oxidative metabolism
– Low use muscle glycolysis minimizes oxygen delivery
Fiber types
• Motor unit
– 1 motor neuron
– 10-1000 muscle fibers
• Burke et al., 1973
– Isolate MUs, test function
identify fibers
• Contractile response
– # fibers
– Calcium kinetics
– Myosin kinetics
– ATP turnover
Three functional categories
• Fast Fatiguable
– Fast force rise
– High force
– Low oxidative cap
• Fast fatigue Resistant
– High oxidative capacity
• Slow
– Slow force rise
– Low force
– High oxidative capacity
Repeated
activation
Single
activation
Correlated with
Characterized by
Physiological fiber types
Twitch force
Time to peak tension
"Sag"
Fatigue
Nerve CV
Myosin ATPase
DPNHD
SDH
Lipid
a-GPD
Glycogen
Fast
Fast Ftg
Fatiguable
Resistant
20 g
4g
30 ms
40 ms
Yes
sometimes
Yes
No
100 m/s
100 m/s
High
High
Low
Intermediate
Low
Intermediate
Low
Low
High
High
High
High
Slow
0.5g
70 ms
No
No
85 m/s
Low
High
High
High
Low
Low
Classification correlations
• Rule-of-thumb
• Somewhat species & muscle-dependent
• Subject to activity-driven modification
– Staged
– ECCmetabolicmyosin
Functional
FF
FR
S
Metabolic
FG
FOG
SO
Expression
2x/2b
2a/2x
1
Influence of FT on gross performance
• Karatzaferi et al., 2001
• 10-25s ‘maximal’ cycle ergometer
• ATP in type-identified fibers
Influence of activity on FT
• Chronic electrical stimulation
– Increase oxidative metabolism
– MHC 2b2x2a1
• Cross-reinnervation
– MHC 12
– MHC 1masseter; MHC 2bEO
• Activity pattern has some influence
• Activity-independent nerve signals have some
influence
Summary
• 3 fiber typing schemes
– Protein isoforms (immunohistology)
– Protein activity (histochemistry)
– Function (force measurements)
• Metabolic and biochemical activity correlates
well with teleological function
• Fiber type is dynamically regulated