B277 BIOLOGY OF MUSCLE The molecular motor of muscle

1. Learning Outcome. Discuss muscle contraction [development, adaptation to training and fatigue]. Learning Objectives. After this lecture and associated reading you will be able to: [Describe myofibrillar and other key proteins of the muscle fibre]. Discuss the molecular interactions that result in myofilament sliding. B277 BIOLOGY OF MUSCLEThe molecular motor of muscle

2. Actin –myosin interactions. Highly organised cytoskeleton Microfilaments (thin myofilaments) of actin (ubiquitous) Thick myofilaments of myosin (myosin ubiquitous but thick filaments characteristic of muscle) Interaction of fixed filaments produces shape change (movement if cell anchored) MOLECULAR BASIS OF MUSCLE CONTRACTION We are our proteins

3. Skeletal muscle differentiation Originate in somites Week 8

4. T4, IGF stimulate myoblast entry to Go and fusion into myotubes. Also withdrawal of FGF, TGF, proliferin. Satellite cells remain for repair. Myo-D regulatory proteins coordinate expression of structural genes. Myofilaments appear at 9 weeks. Myotube formation all pre-natal. Muscle growth is hypertrophy not hyperplasia. Regulated by peptide growth factors such as myostatin (TGF- family; activin type 2 receptor). Skeletal muscle differentiation(mainly regulated by proteins)

5. Belgian Blue Mutant whippet GDF-8 mutant NOT found in 65 bodybuilders Myostatin knockout Myostatin mutant ‘super baby’

6. Cytology of the Muscle fibre.

7. Special features of the Muscle fibre:- T-tubules (2 per sarcomere) Sarcolemma (excitable membrane) Sarcoplasmic reticulum Myofibril = sarcomeres Thick myofilaments Thin myofilaments Associated proteins (Mitochondria, lipid droplets, glycogen granules) Glycolytic and mitochondrial catabolic enzymes Mitochondrial membrane proteins Cytology of the skeletal muscle fibre

8. Structure of the sarcomere Myofilament organisation produces the banding pattern 2.5

9. Analysed following homogenisation in Mg++ATP and EGTA (breaks cross links). Myofilaments (functional; contractile) Thin myofilaments Actin polymers (~330 x G monomers); 6 nm x 1 m. β-actinin cap. Thick myofilaments Polymers of 2 x 150 x Myosin hexamers; 12 nm x 1.6 m Titin (connectin), nebulin, α and β-actinin, myomesin and desmin (structural; stabilising). Tropomyosin and troponin (regulatory). MYOFIBRILLAR PROTEINS

10. The banding pattern of the sarcomere reflects its molecular organisation. (Myosin) (actin) (myomesin) (connectin) (α-actinin) H zone desmin nebulin

11. Globular protein MW42kD (G-actin). Polymerises into double helices (F-actin). Variable length between groups (amphibian<mammalian; 380G in frog sartorius) and even within sarcomere. Associated in light myofilament with Tropomyosin (7 G-actins) Troponin (40 nm spacing; 2 per helix turn) TnC (calcium binding; 4 Ca++ in skeletal, 3 in cardiac) TnT (associated with tropomyosin) TnI (inhibitory; binds actin) Actin

12. A model of the molecular arrangement of troponin (Tn), tropomyosin (Tm), and actin in the skeletal muscle thin filament. Troponin subunits [TnC (red), TnT (yellow; highly asymmetrical), and TnI (green)] lie along the two-stranded tropomyosin (brown, orange) that spans 7 G-actin monomers (grey).

13. Steric change in troponin C with Ca++ binding changes the interaction between actin and tropomyosin.

14. Ubiquitous; myosin 2 in mammalian skeletal muscle (10 classes; ~39 genes). Dimer of 2 x 200kD heavy chains each with 2 x 20kD light chains (ie heterohexamer) RLC (regulatory) ELC (essential) Heavy chain S1, globular, binds ATP and actin S2, fibrous, neck, hinge and tail Myosin

15. Structure of myosin (thick myofilaments)

16. ‘Monomers’ (heterohexamers) spontaneously aggregate in low salt with antiparallel arrangement of heads. Thick myofilament 1.5µ long, 12 nm thick. ~ 300 myosins in thick filament. Myosin

17. Molecular organisation of the sarcomere (Actin might have square array at Z-line) See Millman, B.M. (1998), for review of lattice structure of striated muscle sarcomeres in vertebrates and invertebrates.

18. Nebulin Determines length of thin myofilament Titin (= connectin) Anchors thick myofilament to z-line Myomesin Elastic spring in M-line; stabilises lattice β-actinin Terminates thin myofilament α-actinin Cross-links actin and titin in z-line Desmin Connects z-lines in adjacent myofibrils (intermediate filaments) Structural proteins

19. Changes in the banding pattern suggest that filaments slide between each other (basis for the sliding filament theory). • H zone shortens • I band shortens • A band remains same

20. Light micrograph of myofibril fixed in relaxed (above) and contracted (below) state. From Jones et al. (2004).

21. Binding of the myosin head produces a conformational change that stretches the neck. Recoil of the stretched spring moves the thin myofilament past the thick myofilament. How do cross bridges produce force?

22. ATP is involved Mg++-ATP + actomyosin  actomyosin-Mg(ADP+Pi) contraction occurs in vitro in simple solutions. Ca++ is essential too Glycerated muscle fibres produce force and ATPase activity only with Ca++ (NOT in EDTA) Tension only with both ATP and Ca++ Relaxation requires ATP; no Ca++ Ca++ injection into muscle produces contraction Fura-1 illuminates post AP, pre-tension. Hence dissociation of myosin from actin in Mg++-ATP/EGTA Biochemistry of contraction

23. The cross bridge cycle of filament sliding

24. Lymn-Taylor model. (Pollard, TIBS 25 2000) If Ca >10-7 M myosin head binds to actin. Binding promotes release of ADP and Pi and causes power stroke (few pN, ~6nm). Power stroke conformational change allows ATP binding that promotes detachment. Detachment promotes ATP hydrolysis and energises the head. The cross bridge cycle

25. The Lymn-Taylor model of cross bridge cycling.

26. Crystal structure of chicken myosin showing ATP and actin binding sites A Three-dimensional crystal structure of the S-1 portion of the myosin heavy chain molecule. (A) High resolution ribbon diagram of chicken S-1 myosin determined using X-ray crystallography. Labelled are the key features of the molecule, the ATP and actin binding sites, the cleft between them, and the α-helical region to which the myosin light chains bind. (B) Schematic drawing of the myosin S-1 portion based on the ribbon diagram presented in (A) Mike Ferenczi's home page

27. Binding of ATP in the ‘rigor state’ opens the actin binding cleft. SDSU Biology 590 - Actin Myosin Crossbridge 3D Animation

28. Triad proteins and excitation contraction coupling Describe the steps by which a sarcolemmal action potential promotes the release of Ca++ from the SR and results in cross-bridge cycling. The action potential is propagated over the sarcolemma and transmitted throughout the fibre by t-tubules. Voltage sensitive proteins link the t-tubule AP to Ca++ channel opening in the SR membrane.

29. Coupling of sarcolemma AP to calcium release by DHP and ryanodine receptors at the T-tubule SR junction.

30. Environment of the ryanodine receptor channel in skeletal muscle. The t tubule contains tetrads of dihydropyridine receptors (DHPRs). A t-tubule membrane voltage change (VM) activates the DHPR. The t-tubule VM signal is thought to be transmitted to the RyR1 channel by a direct physical DHPR-RyR1 linkage. The t-tubule VM signal triggers the RyR1 channel to open and release the calcium stored inside the sarcoplasmic reticulum (SR). Calsequestrin (CSQ), a low-affinity high-capacity calcium buffer, is found inside the SR lumen near the RyR1 channels. The SR Ca2+-ATPase (SERCA) pumps calcium back into the SR. DHPR Ca2+

31. Duchenne muscular dystrophy; x-linked, 1:3500 males. Lack of Dystrophin. Part of a membrane complex associated with stretch-activated ion channels. Shock absorber? Fibres susceptible to damage; degenerate from ~ 2-5y. Gene therapy? Other proteins of mammalian muscle fibres: Dystrophin and muscular dystrophies

32. The dystrophin-associated protein complex (DPC) in skeletal muscle. Blake, D.J., Weir, A., Newey, S.E. and Davies, K.E. (2002) Function & Genetics of Dystrophin and Dystrophin-Related Proteins in Muscle Physiological Reviews, 82, (2), 291-329.