B277 BIOLOGY OF MUSCLEThe molecular motor of muscle

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.

MOLECULAR BASIS OF MUSCLE CONTRACTION

• 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)

We are our proteins

Skeletal muscle differentiation

Originate in somitesWeek 8

Skeletal muscle differentiation(mainly regulated by proteins)

• 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).

Belgian Blue Mutant whippet

Myostatin knockout

Myostatin mutant‘super baby’

GDF-8 mutant NOT found in 65 bodybuilders

Cytology of the Muscle fibre.

Cytology of the skeletal muscle fibre

Special features of the Muscle fibre:-o T-tubules (2 per sarcomere)o Sarcolemma (excitable membrane)o Sarcoplasmic reticulumo Myofibril = sarcomeres

Thick myofilamentsThin myofilamentsAssociated proteins

o (Mitochondria, lipid droplets, glycogen granules)Glycolytic and mitochondrial catabolic enzymesMitochondrial membrane proteins

Structure of the sarcomereMyofilament organisation produces the banding pattern

2.5

MYOFIBRILLAR PROTEINS

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).

H zone

The banding pattern of the sarcomere reflects its molecular organisation.

(myomesin)

(α-actinin)

desmin nebulin

(Myosin)

(actin)

(connectin)

Actin• 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)

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).

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

Myosin• 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

Structure of myosin (thick myofilaments)

Myosin

• ‘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.

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.

Structural proteins• 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)

• H zone shortens• I band shortens• A band remains same

Changes in the banding pattern suggest that filaments slide between each other (basis for the sliding filament theory).

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

How do cross bridges produce force?

• 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.

Biochemistry of contraction

• 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

The cross bridge cycle of filament sliding

The cross bridge cycle

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 Lymn-Taylor model of cross bridge cycling.

Crystal structure of chicken myosin showing ATP and actin binding sites

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

A

Binding of ATP in the ‘rigor state’ opens the actin binding cleft.

SDSU Biology 590 - Actin Myosin Crossbridge 3D Animation

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.

Coupling of sarcolemmaAP to calcium release by DHP and ryanodinereceptors at the T-tubuleSR junction.

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+

Other proteins of mammalian muscle fibres:

Dystrophin and muscular dystrophies

• 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?

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.