Fiber Types
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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 • NBTformazan • BCIPindigo 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 – AntigenAbAb-enzyme (HRP) – AntigenAbAb-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 rateP0 – ADP affinityVmax – 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 – ECCmetabolicmyosin 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 2b2x2a1 • Cross-reinnervation – MHC 12 – MHC 1masseter; MHC 2bEO • 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
