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 BCIPIndigo Dephos oxidation

5. Hematoxylin and Eosin H: metal binding, nuclei E: alkaline binding, protein

6. Succinate dehydrogenase TCA cycle, redox enzyme – Bound in mitochondria

7.  -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) (not glygeraldehyde-3-phosphate) Dihydroxyacetone phosphate Fructose 1-6 bisphosphateDihydroxyacetone phosphate Glyceraldehyde-3-phosphate Glycerol-3-phosphate

8.  -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 Anterior Lateral Posterior Medial FOG SO FG

12. Enzyme content or activity? Histochemistry measures total reaction – Enzyme content (moles) – Enzyme kinetics (V max, k m ) 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

13. 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  -actinin Tropomyosin

14. Myosin 4 “conventional” isoforms: 1(o), 2a(*), 2x, 2b – Limited overlap (+) – Horseradish peroxidase/Diaminobenzidine staining SC-71 MHC2aMy32 MHC2A4.84 MHC1

15. Myosin Heavy chain – Solution ATPase rate  P 0 – ADP affinity  V max – F-actin velocity 2b>2x>2a>I Light chains (essential/alkali) – Influence f-actin velocity – Probably not ATPase 2b 2x 2a 1 MLC3f/MLC2f Slack test 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 – Ca 2+ -free form blocks MHC binding to actin Tobacman, 1996

19. TnC-slow TM-  TnC-fast TM-  TnC-fast TM-  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- , cardiac-  (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 function identify fibers Contractile response – # fibers – Calcium kinetics – Myosin kinetics – ATP turnover

23. 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 ResistantSlow Twitch force20 g4 g0.5g Time to peak tension30 ms40 ms70 ms "Sag"YessometimesNo FatigueYesNo Nerve CV100 m/s 85 m/s Myosin ATPaseHigh Low DPNHDLowIntermediateHigh SDHLowIntermediateHigh LipidLow High a-GPDHigh Low GlycogenHigh Low Characterized by Correlated with

25. Classification correlations FunctionalMetabolicExpression FFFG2x/2b FRFOG2a/2x SSO1 Rule-of-thumb Somewhat species & muscle-dependent Subject to activity-driven modification – Staged – ECC  metabolic  myosin

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

28. 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