B277 BIOLOGY OF MUSCLE
The 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 somites
Week 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 reticulum
o Myofibril = sarcomeres
Thick myofilaments
Thin myofilaments
Associated proteins
o (Mitochondria, lipid droplets, glycogen
granules)
Glycolytic and mitochondrial catabolic enzymes
Mitochondrial membrane proteins
Structure of the sarcomere
Myofilament 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).
The banding pattern of the sarcomere
reflects its molecular organisation.
(Myosin)
(actin)
(myomesin)
(connectin)
H zone
desmin
nebulin
(α-actinin)
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 Zline)
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)
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
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
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 αhelical 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
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 sarcolemma
AP to calcium release by
DHP and ryanodine
receptors at the T-tubule
SR 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.