L14 MSK Physiology 4 Skeletal muscle

Overview

This is Lecture 4 of the nerve and muscle physiology course, following the lectures on neurons, action potentials and the chemical synapse and preceding Lecture 5 on the physical limits to muscle performance and the active control of muscle force. It follows the signal from the motor nerve into the muscle: first the neuromuscular junction and the sequence of events that converts a nerve action potential into a muscle action potential, then the acetylcholine life cycle that supports it, then myasthenia gravis as a case in which that transmission fails. It then turns to the structure of striated muscle, from whole muscle down to the sarcomere and its filaments, and finishes with excitation-contraction coupling and the cross-bridge cycle that turn the muscle action potential into force, and how repeated cycles sum into a twitch.

Stated objectives: be familiar with the neuromuscular junction; case of myasthenia gravis; be familiar with the morphology of striated muscle; understand how excitation-contraction coupling works.

The neuromuscular junction

• The neuromuscular junction (NMJ) is also known as the motor endplate. It is the junction between a motor neuron axon and a muscle fibre.
• Low-power arrangement: a myelinated axon ends in a nerve terminal that is capped by a Schwann cell and sits on the surface of the muscle fibre. In the electron micrograph the motor axon branches over the fibre surface and terminates in an expanded, convoluted endplate.
• Components at the junction itself: axon, part of the Schwann cell, prejunctional membrane, basal lamina, postjunctional membrane, choline uptake, dense projection, ACh receptors, ACh, AChE on the basal lamina, membrane recycling.
• Key structural points: the terminal contains vesicles and mitochondria; the postjunctional membrane is thrown into deep junctional folds; ACh receptors are concentrated at the crests of the folds; acetylcholinesterase sits in the basal lamina within the cleft; released vesicle membrane is recycled back into the terminal.

Transmission at the NMJ: sequence of events

1. Action potential in the axon.
2. Voltage-gated $C{a}^{2+}$ channels open and $C{a}^{2+}$ enters the terminal.
3. $C{a}^{2+}$ triggers vesicle fusion and release of ACh.
4. ACh binds nicotinic ACh receptors (nAChR) on the muscle fibre.
5. The nAChR channel opens and $N{a}^{+}$ enters.
6. Local depolarisation spreads to the extrajunctional membrane.
7. The depolarisation opens voltage-gated $N{a}^{+}$ channels and an action potential is triggered.
8. The action potential propagates down the fibre, running away from the endplate in both directions.
9. ACh is degraded by AChE.

At the endplate the charge reverses: the depolarised region goes from positive outside and negative inside to negative outside and positive inside.

Acetylcholine life cycle

• Choline + acetyl CoA are combined to make ACh in the terminal cytoplasm, catalysed by ChAT.
• ACh is moved into the vesicle by the ACh transporter.
• The vesicle releases ACh into the synaptic cleft, where it acts on ACh receptors on the postsynaptic cell.
• In the cleft, AChE breaks ACh down to choline + acetic acid.
• Choline is carried back into the terminal by the choline transporter, which is coupled to $N{a}^{+}$ entry.

Vesicle machinery of the synaptic bouton (from the CNS synaptic transmission figure): vesicles, actin filaments, $C{a}^{2+}$ channel, endosome, clathrin coat, docking and fusion proteins, dense projection (the active zone), pore to the synaptic cleft, and receptors for neurotransmitter in the postsynaptic membrane opposite the release site. Vesicles move to the active zone, dock and fuse, opening a pore to the cleft; membrane is then retrieved via a clathrin coat into an endosome for recycling.

Myasthenia gravis

• An autoimmune disease of the NMJ: an autoimmune attack on neuromuscular ACh receptors, with antibodies to the muscle nAChR.
• The antibodies reduce receptor number, block the receptor, and provoke an inflammatory response that damages the endplate.
• Reduced NMJ function produces weakness, particularly of frequently used muscles, with muscle weakness (ptosis in particular) and rapid fatigue. Clinical images show unilateral and bilateral ptosis.
• Normal junction versus myasthenic junction: in both, the terminal releases ACh into a cleft facing deeply folded postsynaptic membrane bearing ACh receptors, but in myasthenia gravis the receptors are blocked by antibodies and transmission is reduced.
• Normal transmission tolerates the fall in ACh release that occurs with repetitive activation, because normal receptor numbers ensure a muscle action potential always occurs. In myasthenia gravis the number of functional ACh receptors is reduced, so as ACh release diminishes with repetitive activation, neuromuscular transmission eventually fails.

Why symptoms come on with sustained contraction, and why AChE blockers help

Redundancy in the system:

• A lot of neurotransmitter is released, typically more than is needed.
• With each subsequent action potential the amount of neurotransmitter released drops.
• Not every receptor needs to be activated every time.

But if antibodies are blocking receptors, the pool of available receptors is reduced. During sustained contraction the falling neurotransmitter and the low receptor numbers combine, so the critical number of activated receptors is not reached.

Important

AChE blockers keep the pool of ACh in the synaptic cleft high, giving a greater chance of activating the required number of receptors.

Worked illustration (the slide states these are made-up numbers to illustrate the principle):

Action potential in the train	1	2	3	4
ACh released	100%	95%	90%	85%
Proportion of receptors occupied	100%	95%	90%	85%
Normal number occupied (of 100 receptors)	100	95	90	85
Myasthenic endplate, number occupied (of 60 receptors)	60	57	54	51

If at least 55 occupied receptors are needed to generate sufficient current to depolarise the membrane to threshold, then impulses 3 and 4 fail to activate the muscle fibre.

Organisation of skeletal muscle

• Muscle is arranged into bundles of increasingly small but typically still parallel structures: whole muscle → fasciculus → fibre → myofibril, with the myofibril showing cross-striations.
• Individual muscle cells are called fibres, and a fibre is itself a bundle of myofibrils.
• Each fibre is innervated by a single motor neuron, but a single motor neuron can innervate multiple fibres (developed in Lecture 5).

Inside a single muscle fibre:

• Sarcolemma: the outer membrane of the fibre.
• Myofibrils: multiple cylindrical, cross-striated columns filling the fibre.
• Myofilaments: actin and myosin, making up each myofibril.
• Mitochondria: lying between the myofibrils.
• Sarcoplasmic reticulum: a network wrapped around each myofibril.
• T-tubule: an invagination of the sarcolemma running inwards between the myofibrils and contacting the sarcoplasmic reticulum.

The sarcomere and what changes on contraction

The sarcomere spans between Z discs, where the thin filaments anchor. Regions:

• A band: the full length of the thick filament.
• I band: thin filaments only.
• H zone: thick filaments only.
• M line: the centre of the A band.

Changes with contraction:

• Relaxed: H zone and I band are wide, and the A band spans the thick filaments.
• Partially contracted: the Z discs are closer together, the I band and H zone are narrower, and the A band is unchanged in width.
• Maximally contracted: the I band and H zone have essentially disappeared as the thin filaments have slid fully in and the Z discs abut the thick filaments, leaving only the A band.

Contractile proteins and the calcium switch

• The thick filament is myosin, with projecting heads that act as the cross-bridges. Each myosin head carries actin binding sites and ATP binding sites.
• The thin filament is actin, wound with tropomyosin and studded with troponin.
• Components of the working model: myosin complex (and myosin with ATPase activation), actin helix, tropomyosin, troponin, ATP, ADP, Pi, $C{a}^{2+}$, $M{g}^{2+}$.
• Low sarcoplasmic $[C{a}^{2+}]$: tropomyosin lies over the actin binding sites, so the myosin head cannot bind.
• High sarcoplasmic $[C{a}^{2+}]$: $C{a}^{2+}$ binds troponin, tropomyosin moves off the binding sites, myosin binds actin, and force development follows.

Excitation-contraction coupling

1. The action potential enters the t-tubules.
2. Depolarisation of the t-tubule membrane activates the DHP protein, which opens the RYR channel in the sarcoplasmic reticulum.
3. $C{a}^{2+}$ leaves the sarcoplasmic reticulum down its concentration gradient.
4. Binding of $C{a}^{2+}$ to troponin moves tropomyosin off the binding sites.
5. The cross-bridge cycle is activated and force is generated.
6. $C{a}^{2+}$ is pumped back into the sarcoplasmic reticulum.
7. Tropomyosin moves back onto the binding sites: relaxation.

The cross-bridge cycle

Running through the states of the cycle:

• Energized cross-bridge: the head is cocked with ADP and Pi bound and is not attached, $[A+M\cdot ADP\cdot Pi]$.
• If $[C{a}^{2+}]$ is high, tropomyosin is off the binding sites and myosin binds actin: $[A\cdot M\cdot ADP\cdot Pi]$.
• ADP and Pi are released, the head flexes (power stroke) and force is generated: $[A\cdot M]$.
• ATP binds and the myosin head detaches: $[A+M\cdot ATP]$.
• ATP is hydrolysed to ADP + Pi, re-energizing the head and returning it to the start of the cycle.

Exits from the cycle:

• If $[C{a}^{2+}]$ is low the binding sites are covered and the cycle stops.
• If there is no ATP the head stays attached and the muscle stays stiff: rigor mortis.

From cross-bridge cycling to the twitch

• $C{a}^{2+}$ moves troponin off actin, allowing the myosin heads to interact; the head flexes and force is exerted on actin.
• While $C{a}^{2+}$ is present the myosin heads repeatedly engage, flex and release.
• The summation of all these interactions produces a twitch of the muscle.
• The muscle twitch has a long duration (tens to hundreds of milliseconds) compared with the muscle action potential (5 ms).
• The long duration is due to the time taken for release and re-uptake of $C{a}^{2+}$, and to energy stored in muscle elasticity at the beginning of the twitch being released at the end.
• Because the twitch outlasts the action potential, more action potentials can arrive before the twitch has finished, releasing more $C{a}^{2+}$ and giving a bigger twitch: summation. On the plotted traces the action potential is a brief spike, the $[C{a}^{2+}]$ trace is a broad hump, and the force trace is slower and broader still, lagging behind calcium and peaking higher after a second action potential arrives.

Warning

Two transcript flags. The slide on the anatomical composition of skeletal muscle carries a note “(This is slide 11 btw)”, indicating it was moved out of its original numbering, and the identical slide appears again later in the deck. On the myasthenia gravis comparison slide the bottom of the page is cut off: the label under the right-hand panel reads “Reduced transmission” with the second word partly clipped, and further clipped text at the far right edge is illegible.

Self-test

1. Define the neuromuscular junction and give its alternative name.
2. Describe the structural features of the postjunctional membrane at the NMJ and where the ACh receptors and AChE are located.
3. List, in order, the nine steps that take an action potential in the motor axon to a propagating action potential in the muscle fibre.
4. Describe the life cycle of acetylcholine at the nerve terminal, from synthesis to recovery of choline.
5. Explain how the vesicle membrane released during exocytosis is retrieved by the terminal.
6. Explain the innervation rule for skeletal muscle fibres and motor neurons.
7. Describe the mechanism by which antibodies cause weakness in myasthenia gravis.
8. Explain why myasthenic symptoms come on with sustained contraction rather than at rest.
9. Explain why acetylcholinesterase blockers improve neuromuscular transmission in myasthenia gravis.
10. A myasthenic endplate has 60 functional receptors and threshold requires 55 occupied receptors. Given that the proportion of receptors occupied falls to 100%, 95%, 90% and 85% across a train of four impulses, predict which impulses fail and why.
11. A patient reports drooping eyelids that worsen through the day and rapid fatigue of frequently used muscles. Explain the likely mechanism.
12. List the hierarchy of skeletal muscle organisation from whole muscle down to myofibril.
13. List the structures found within a single muscle fibre and state the function or position of the t-tubule and the sarcoplasmic reticulum.
14. Distinguish the A band, the I band and the H zone in terms of which filaments they contain.
15. Describe what happens to the A band, I band, H zone and Z discs as a muscle goes from relaxed to maximally contracted.
16. Explain how sarcoplasmic $[C{a}^{2+}]$ acts as a switch on the thin filament.
17. Describe the seven steps of excitation-contraction coupling.
18. Describe the states of the cross-bridge cycle, naming the chemical state at each stage.
19. Predict what happens to a cross-bridge if ATP is absent, and name the condition this produces.
20. Explain why a muscle twitch lasts much longer than a muscle action potential, and how this permits summation.
21. Integrative: trace the events from an action potential arriving at the motor nerve terminal to force being generated in the sarcomere, naming the key molecules at each transition.

Answers

Reveal answers

1. The junction between a motor neuron axon and a muscle fibre; it is also known as the motor endplate.
2. The postjunctional membrane is thrown into deep junctional folds. ACh receptors are concentrated at the crests of the folds, and AChE sits in the basal lamina within the cleft. The terminal opposite contains vesicles and mitochondria and is capped by a Schwann cell.
3. Action potential in the axon; voltage-gated $C{a}^{2+}$ channels open and $C{a}^{2+}$ enters; $C{a}^{2+}$ triggers vesicle fusion and ACh release; ACh binds nAChR on the muscle fibre; the nAChR channel opens and $N{a}^{+}$ enters; local depolarisation spreads to the extrajunctional membrane; the depolarisation opens voltage-gated $N{a}^{+}$ channels and an action potential is triggered; the action potential propagates down the fibre in both directions from the endplate; ACh is degraded by AChE.
4. Choline + acetyl CoA are combined into ACh by ChAT in the terminal cytoplasm; the ACh transporter loads ACh into vesicles; the vesicle releases ACh into the cleft, where it acts on ACh receptors on the postsynaptic cell; AChE in the cleft breaks ACh into choline + acetic acid; the choline transporter, coupled to $N{a}^{+}$ entry, carries choline back into the terminal.
5. Vesicles move to the dense projection (active zone), dock and fuse via docking and fusion proteins, opening a pore to the cleft; membrane is then retrieved with a clathrin coat into an endosome for recycling.
6. Each muscle fibre is innervated by a single motor neuron, but one motor neuron can innervate multiple fibres.
7. Antibodies to the muscle nAChR reduce receptor number, block the receptor, and provoke an inflammatory response that damages the endplate, so NMJ function is reduced.
8. There is normally redundancy: more neurotransmitter is released than needed and not every receptor must be activated. Release falls with each successive action potential. When antibodies have reduced the available receptor pool, the falling transmitter and low receptor number combine during sustained contraction so the critical number of activated receptors is not reached and transmission fails.
9. They keep the pool of ACh in the synaptic cleft high, giving a greater chance of activating the required number of receptors.
10. Occupancy is 60, 57, 54 and 51 receptors across the four impulses. Impulses 3 and 4 fall below the 55-receptor threshold, so there is insufficient current to depolarise the membrane to threshold and those impulses fail to activate the fibre.
11. An autoimmune attack on neuromuscular ACh receptors (myasthenia gravis): antibodies reduce and block the receptors, so with repetitive activation the falling ACh release and reduced receptor pool cause transmission to fail, producing weakness, ptosis and rapid fatigue, particularly in frequently used muscles.
12. Whole muscle → fasciculus → fibre (the individual muscle cell) → myofibril, which shows cross-striations.
13. Sarcolemma (the outer membrane), myofibrils, myofilaments (actin and myosin), mitochondria between the myofibrils, sarcoplasmic reticulum and t-tubules. The sarcoplasmic reticulum forms a network wrapped around each myofibril; the t-tubule is an invagination of the sarcolemma that runs inwards between myofibrils and contacts the sarcoplasmic reticulum.
14. A band: the full length of the thick filament. I band: thin filaments only. H zone: thick filaments only.
15. The Z discs move closer together, the I band and H zone narrow and then essentially disappear as the thin filaments slide fully in and the Z discs abut the thick filaments; the A band stays the same width throughout.
16. At low sarcoplasmic $[C{a}^{2+}]$ tropomyosin lies over the actin binding sites so the myosin head cannot bind. At high $[C{a}^{2+}]$, $C{a}^{2+}$ binds troponin, tropomyosin moves off the binding sites, myosin binds actin and force develops.
17. Action potential enters the t-tubules; t-tubule depolarisation activates the DHP protein, which opens the RYR channel in the SR; $C{a}^{2+}$ leaves the SR down its concentration gradient; $C{a}^{2+}$ binds troponin and moves tropomyosin off the binding sites; the cross-bridge cycle is activated and force is generated; $C{a}^{2+}$ is pumped back into the SR; tropomyosin moves back onto the binding sites and the muscle relaxes.
18. Energized cross-bridge with ADP and Pi bound, unattached $[A+M\cdot ADP\cdot Pi]$; if $[C{a}^{2+}]$ is high, myosin binds actin $[A\cdot M\cdot ADP\cdot Pi]$; ADP and Pi are released, the head flexes and force is generated $[A\cdot M]$; ATP binds and the head detaches $[A+M\cdot ATP]$; ATP is hydrolysed to ADP + Pi, re-energizing the head. If $[C{a}^{2+}]$ is low the binding sites are covered and the cycle stops.
19. Without ATP the head cannot detach, so it stays attached in the $[A\cdot M]$ state and the muscle stays stiff: rigor mortis.
20. The twitch lasts tens to hundreds of milliseconds against a 5 ms action potential because of the time needed for release and re-uptake of $C{a}^{2+}$ and because energy stored in muscle elasticity at the start of the twitch is released at the end. Since the twitch outlasts the action potential, further action potentials can arrive before it finishes and release more $C{a}^{2+}$, producing a larger twitch (summation).
21. The axonal action potential opens voltage-gated $C{a}^{2+}$ channels; $C{a}^{2+}$ entry triggers vesicular ACh release; ACh binds nAChR, $N{a}^{+}$ entry depolarises the endplate and opens voltage-gated $N{a}^{+}$ channels, generating a muscle action potential that propagates along the sarcolemma and into the t-tubules; t-tubule depolarisation activates DHP, which opens RYR and releases $C{a}^{2+}$ from the sarcoplasmic reticulum; $C{a}^{2+}$ binds troponin, tropomyosin moves off the actin binding sites, and myosin heads cycle through attachment, power stroke and ATP-dependent detachment, generating force which sums into a twitch.