Fiber Types

1
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

2
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

Single activation
Repeated activation
3
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

4
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
5
Hematoxylin and Eosin

• H metal binding, nuclei
• E alkaline binding, protein

6
Succinate dehydrogenase

• TCA cycle, redox enzyme
• Bound in mitochondria

7
a-glycerophosphate dehydrogenase

• Glycerol-3-phosphate dehydrogenase (GPDH)
• Really looking at glycerol-3-phosphate shuttle
• G-3-p participates in an undesired reaction

Glycerol-1-phosphate (not glycerol-3-phosphate) (n
ot glygeraldehyde-3-phosphate)
Dihydroxyacetonephosphate
Glycerol-3-phosphate
8
a-glycerolphosphate dehydrogenase

• GPDH bound in Z-disks
• More uniform in cross-section than mitochondria

9
Myosin ATPase

• Acid/Alkali-labile
• i.e. acid inactivates fast myosin alkali,
  slow

Myosin ATPase after pH 9.4 preinc
10
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)

11
Spatial distribution

• Section through lower leg stained for SDH and
  acid-stable ATPase

Lateral
SO
FOG
FG
Anterior
Posterior
Medial
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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

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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 cardiacslow fast
• a-actinin
• Tropomyosin

14
Myosin

• 4 conventional isoforms 1(o), 2a(), 2x, 2b
• Limited overlap ()
• Horseradish peroxidase/Diaminobenzidine staining

SC-71 MHC2a
My32 MHC2
A4.84 MHC1
15
Myosin
Slack test
2b
2x

• Heavy chain
• Solution ATPase rate?P0
• ADP affinity?Vmax
• F-actin velocity 2bgt2xgt2agtI
• Light chains (essential/alkali)
• Influence f-actin velocity
• Probably not ATPase

2a
1
MLC3f/MLC2f
Bottinelli et al., 1994
16
SERCA

• 2 dominant isoforms 1a, 2a
• Limited overlap
• Fluorescein indirect fluorescence

SERCA2 (in type 1 fibers)
Ikezoe et al., 2003
De Jonge et al., 2006
17
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
18
Troponin

• 3 subunits w/ fast/slow
• C calcium binding
• T tropomyosin binding
• I Inhibitory
• Provide calcium-dependent activation
• Ca2-free form blocksMHC binding to actin

19
Troponin/Tropomyosin

• Calcium sensitivity
• Slow fibers lower threshold sensitivity
• Fast fibers higher sensitivity

20
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

21
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

22
Fiber types

• Motor unit
• 1 motor neuron
• 10-1000 muscle fibers
• Burke et al., 1973
• Isolate MUs, test functionidentify fibers
• Contractile response
• fibers
• Calcium kinetics
• Myosin kinetics
• ATP turnover

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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

Single activation
Repeated activation
24
Physiological fiber types
Fast Fatiguable Fast Ftg Resistant Slow
Twitch force 20 g 4 g 0.5g
Time to peak tension 30 ms 40 ms 70 ms
"Sag" Yes sometimes No
Fatigue Yes No No
Nerve CV 100 m/s 100 m/s 85 m/s
Myosin ATPase High High Low
DPNHD Low Intermediate High
SDH Low Intermediate High
Lipid Low Low High
a-GPD High High Low
Glycogen High High Low
Characterized by
Correlated with
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Classification correlations

• Rule-of-thumb
• Somewhat species muscle-dependent
• Subject to activity-driven modification
• Staged
• ECC?metabolic?myosin

Functional Metabolic Expression
FF FG 2x/2b
FR FOG 2a/2x
S SO 1
26
Influence of FT on gross performance

• Karatzaferi et al., 2001
• 10-25s maximal cycle ergometer
• ATP in type-identified fibers

27
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

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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