SLIDING FILAMENT THEORY Dr. Ayisha Qureshi Assistant Professor MBBS, MPhil

Review of the structure and organization of the skeletal muscle….

Sarcomere: Organization of Fibers

Important: 3-dimensionally, thin filaments are arranged hexagonally around thick filaments and the cross-bridges project from each thick filament in all 6 directions towards the surrounding thin filaments……each thin filament is surrounded by 3 thick filaments.

What are Cross-bridges? With an electron microscope, fine cross bridges can be seen extending from each thick filament to the thin filament. These are formed by the arm and head of the myosin molecules projecting outward from the tail, and pointing towards the thin filaments.

Questions How does cross-bridge interaction between actin and myosin bring about muscle contraction? How does a muscle action potential trigger this contractile process? What is the source of the Ca2 that physically repositions troponin and tropomyosin to permit cross-bridge binding?

SLIDING FILAMENT THEORY Definition: When a muscle cell contracts, the thin filaments slide past the thick filaments, and the sarcomere shortens. This process comprised of several steps is called the Sliding Filament Theory. It is also called the Walk Along Theory or the Ratchet Theory.

After the ATP has bound to the myosin head, the binding of Myosin to Actin molecule takes place:

Once the actin active sites are uncovered, the myosin binds to it:

Power Stroke

POWER STROKE

SLIDING FILAMENT THEORY It has the following steps: Before contraction begins, An ATP molecule binds to the myosin head of the cross-bridges. The ATPase activity of the myosin head immediately cleaves the ATP molecule but the products (ADP+P) remains bound to the head. Now the myosin head is in a high energy state and ready to bind to the actin molecule. When the troponin-tropomyosin complex binds with calcium ions that come from the sarcoplasmic reticulum, it pulls the tropomyosin so that the active sites on the actin filaments for the attachment of the myosin molecule are uncovered. Myosin head binds to the active site on the actin molecule.

SLIDING FILAMENT THEORY (cont) The bond b/w the head of the cross bridges(myosin) & the actin filaments causes a the bridge to change shape bending 45° inwards as if it was on a hinge, stroking towards the centre of the sarcomere, like the stroking of a boat oar. This is called a POWER STROKE. This power stroke pulls the thin filament inward only a small distance. Once the head tilts, this allows release of ADP & phosphate ions. At the site of release of ADP, a new ATP binds. This binding causes the detachment of the myosin head from the actin. A new cycle of attachment-detachment-attachment begins. Repeated cycles of cross-bridge binding, bending and detachment complete the shortening and contraction of the muscle.

Participant Will bind to: 1. Myosin ATP, Actin 2. Actin Myosin, Troponin 3. Tropomyosin  Troponin 4. Troponin Calcium, Actin Tropomyosin 5. ATP Myosin 6. Calcium ions Troponin

Important Through the attachment-detachment-attachment cycle, the myosin heads or cross bridges “walk” along an actin filament to pull it inward relative to the stationary thick filament. Because of the way the myosin molecules are oriented within a thick filament, all the cross-bridges stroke towards the center of the sarcomere. At any time during contraction, part of the cross bridges are attached to the thin filaments and are stroking, while others are returning to their original conformation in preparation for binding with another actin molecule. Thus, some cross-bridges “hold on” while others “let go”. Otherwise, the thin filaments would slip back to their resting position b/w strokes. The detachment of the myosin head from the actin cannot take place until and unless a new ATP does not attach to the myosin head. This is important when death occurs, no more ATP is available and thus, rigor mortis occurs.

Shortening of the Muscle: The thick and thin filaments DO NOT shorten. Contraction is accomplished by the thin filaments from opposite sides of each sarcomere sliding closer together or overlapping the thick filaments further. The H-zone becomes smaller as the thin filaments approach each other. The I band becomes smaller as the thin filaments further overlap the thick filaments. The width of the A band remains unchanged as it depends on the thick filaments and the thick filaments do not change length.

½ I A When muscle contracts, the sarcomere shortens. The I band and H Zone also shorten. But the length of the A band remains the same.

Mechanism of muscle contraction: Width of sarcomere decreases from 2.2 to 2 or less Length of thick and thin filament remain the same Power stroke pull the Z discs towards the center of the sarcomere Results in app. Of the Z discs Cyclic attachment-detachment-attachment of myosin head to actin, till there is complete overlap of thick & thin filaments Decrease in I-band and H-band, A-band stays the same

Neuromuscular junction

NEUROMUSCULAR JUNCTION A NEUROMUSCULAR JUNCTION is an area of contact between a muscle fibre and a neuron. Fig. An electron micrographic sketch of the junction between a single axon terminal and the muscle fiber membrane. The skeletal muscle fibers are innervated by large, myelinated nerve fibers that originate from large motor neurons in the anterior horns of the spinal cord. Each nerve fiber , after entering the muscle belly, normally branches and stimulates from three to several hundred skeletal muscle fibers. Each nerve ending makes a junction, called the neuromuscular junction, with the muscle fiber near its midpoint.

MOTOR END-PLATE Definition: It is the specialized portion of a muscle fibre immediately under a terminal nerve fibre. The nerve fibre invaginates a muscle fibre but lies outside the muscle fibre plasma membrane. The entire structure is called the motor end-plate.

NMJ

NEUROMUSCULAR JUNCTION A neuromuscular junction thus consists of: Presynaptic terminal (Nerve fibre) with vesicles containing the NT A synaptic cleft (20-30 nm wide) A synaptic trough or gutter (Muscle fibre) which has numerous folds called subneural clefts. Neuroreceptors for the NT. The NT at an NMJ is ACETYLCHOLINE (Ach). The synaptic cleft contains the enzyme which helps break down Ach and is called Acetylcholinesterase.

Volage-gated Ca channels The presynaptic membrane of the neuron contains linear dense bars. To each side of the dense bars are protein particles penetrating the neural membrane. These are the voltage-gated calcium channels. When an action potential spreads over the terminal, these channels open and allow calcium ions to diffuse from the synaptic space to the interior of the nerve terminal. The vesicles then fuse with the neural membrane and empty their acetylcholine into the synaptic space by the process of exocytosis.

Acetylcholine Receptor: Each Ach receptor complex has a total molecular weight of 275,000. Each receptor complex is composed of 5 subunits: - 2 alpha - 1 beta - 1 gamma - 1 delta. The channels remains closed unless 2 Ach molecules attach to the 2 alpha subunits which open the gate. The opened acetylcholine channel has a diameter of about 0.65 nanometer, which is large enough to allow the important positive ions— Na+, K+ and Ca++ —to move easily through the opening. Why does mostly Na get through, and not K, ca or cl? Because the concentration gradient is the largest for Na …..also, the inside of the muscle is negatively charged and thus, pulls the Na ions inside…. Thus, the conc. Gradient and the pull for Na causes more Na ions than any other ion to pour into the muscle fibre.

The Steps in Neuromuscular Junction An AP reaches the presynaptic terminal of the NMJ. The change in voltage causes the opening of the voltage-gated calcium channels which cause exocytosis of the Ach containing secretory vesicles. The NT Ach is secreted into the synaptic cleft. Ach crosses the synaptic cleft to reach the subneural clefts which contains the Ligand-gated Ach channel. The channels are activated and open allowing the Na+ to move to the inside of the muscle fiber. As long as the Ach is present in the synaptic cleft, it keeps activating the Ach channels which remain open. The influx of Na+ into the muscle lead to the initiation of the END PLATE POTENTIAL (EPP).

END-PLATE POTENTIAL At the motor end-plate, the large influx of the Sodium ions leads to a large number of positive charges pouring into the muscle. This creates a local positive potential change inside the muscle fiber membrane, called the end plate potential. It is usually about 50-75 mv. In turn, this end plate potential initiates an action potential that spreads along the muscle membrane and thus causes muscle contraction.

Degradation of Ach: The Ach present in the synaptic cleft is broken down by the enzyme Acetylcholinesterase, into Acetyl coA+ choline. Both the products are reuptaken by the presynaptic terminal. The Ach is again synthesized by the nerve cell body and then send by anterograde flow to the presynaptic terminal for packaging into secretory vesicles.

Remember: The Neurotransmitter at the NMJ is Acetylcholine. Acetylcholine is degraded by the Acetylcholinestrase. End-plate potential is the name given to the potential generated at the motor end-plate.

Safety Factor at NMJ: Fatigue Each impulse that arrives at the NMJ causes about three times as much end plate potential as that required to stimulate the muscle fiber. Therefore, the normal neuromuscular junction is said to have a high safety factor. However, stimulation of the nerve fiber at rates greater than 100 times per second for several minutes often diminishes the number of acetylcholine vesicles so much that impulses fail to pass into the muscle fiber. This is called fatigue of the neuromuscular junction, and it is the same effect that causes fatigue of synapses in the central nervous system when the synapses are overexcited. Under normal functioning conditions, measurable fatigue of the neuromuscular junction occurs rarely, and even then only at the most exhausting levels of muscle activity. We know that a change of voltage through depolarization of only 25-30 mv is sufficient to trigger an AP. Thus, 3 times as much end-plate potential means 50-65 mv change is sufficient to caused an AP.

Drugs that increase or block transmission at the Neuromuscular Junction:

1. Drugs that block release of Ach. E.g: Botulinum toxin, lack of Ca, excess of Mg. Botulinum prevents the release of Ach by blocking the fusion of Ach containing vesicles with the postsynaptic membrane & thus prevents the exocytosis of these vesicles. It has some therapeutic use to relieve pain of pathological contraction. Lack of Calcium also leads to blocking of exocytosis of the secretory vesicles.

2. Drugs that stimulate the muscle fibre by Ach-like Action Example: methacholine, carbachol, and nicotine They have the same effect on the muscle fiber as does Ach. The difference b/w these drugs and Ach is that the drugs are NOT destroyed by cholinesterase or are destroyed so slowly that their action often persists for many minutes to several hours. The drugs work by causing localized areas of depolarization of the muscle fiber membrane at the motor end plate where the acetylcholine receptors are located. Then, every time the muscle fiber recovers from a previous contraction, these depolarized areas, by virtue of leaking ions, initiate a new action potential. Thus, there is a constant state of muscle spasm.

Neostigmine, physostigmine, and diisopropyl fluorophosphate 3. Drugs That Stimulate the Neuromuscular Junction by Inactivating Acetylcholinesterase. Neostigmine, physostigmine, and diisopropyl fluorophosphate They inactivate the acetylcholinesterase by combining with it in the synaptic cleft so that it no longer hydrolyzes acetylcholine. Therefore, with each successive nerve impulse, additional acetylcholine accumulates and stimulates the muscle fiber repetitively. This causes muscle spasm when even a few nerve impulses reach the muscle. Unfortunately, it can also cause death due to laryngeal spasm, which smothers the person. Neostigmine and physostigmine work for a few hours. Diisopropyl fluorophosphate is effective for weeks. This makes it a particularly lethal poison with great military potential. It is thus used as a powerful “nerve gas poison”.

Nerve Gas

It can have some therapeutic uses: 4. NON-DEPOLARIZING DRUGS: Drugs That Block Transmission at the Neuromuscular Junction. A group of drugs known as curariform drugs e.g. D-tubocurarine can prevent passage of impulses from the nerve ending into the muscle. This is done by competing with the Ach for the receptor sites on the postsynaptic membrane. When this drug is bound to these receptor sites, then Ach cannot act on them, thus preventing sufficient increase in permeability of the muscle membrane channels to initiate an action potential. It can have some therapeutic uses: - used with artificial respiration to control convulsions in tetanus. - used during surgery when complete muscle relaxation is required. From a historical point of view, it is important as it was used by the red indians for as arrow poisons for hunting animals and killing the animals.

MYASTHENIA GRAVIS

The characteristic symptom of myasthenia gravis is fatigability, which means that a muscle that is used repeatedly starts to become weak.  Generally when the patient wakes up in the morning the muscles have had a chance to rest and that is the typically when they are the strongest. As the day goes on they the patients use their muscles more and the muscles tend to get weaker and weaker. The symptoms usually start in the face muscles and spread to the other parts of the body as the disease progresses.  One of the first symptom of myasthenia gravis is drooping of the eye lids eye lids that gets worst as the day goes on; other signs that could develop include double vision, difficulty talking, and difficulty chewing. 

MYASTHENIA GRAVIS It is an autoimmune neuromuscular disorder in which the Neuromuscular junction is blocked. Cause: Auto-antibodies are formed against the Ach receptors on the Motor End Plate. These antibodies completely destroy the receptors. As the receptors are destroyed, the Ach present cannot act upon them and cause an AP. Some patients have other auto-immune disorders as well such as RA, poliomyelitis.

SYMPTOMS: Fatigue is the hallmark of Myasthenia gravis. Fatigue is especially seen with prolonged use of the skeletal muscles. Muscles become progressively weaker during periods of activity and improve after periods of rest. Fatigue is usually more pronounced in the proximal muscles as tongue, occulomotor (eye movements), phryngeal (swallowing), laryngeal muscles (talking), Ptosis (drooping of the eyelids) Diplopia (double vision) Symptoms get better with rest & administration of anti-cholinesterase drugs (drugs that prevent the Acetylcholinesterase from breaking down the Ach). E.g. edrophonium & neostigmine. Patients are usually women in their 30’s.

Presence of autoantibodies in the plasma Nerve conduction study DIAGNOSIS: Presence of autoantibodies in the plasma Nerve conduction study Edrophonium test TREATMENT: Anti-cholinesterase drugs.e.g: Neostigmine Immunosuppressant drugs. E.g: glucocorticoids Thymectomy: removal of thymus helps rebalances the immune system. MYASTHENIC CRISIS: This occurs when the muscles that control breathing weaken to the point that ventilation is inadequate, creating a medical emergency and requiring a respirator for assisted ventilation. In patients whose respiratory muscles are weak, crises - which generally call for immediate medical attention - may be triggered by infection, fever, or an adverse reaction to medication. Another test is called the edrophonium test. This approach requires the intravenous administration of edrophonium chloride or Tensilon(r), a drug that blocks the degradation (breakdown) of acetylcholine and temporarily increases the levels of acetylcholine at the neuromuscular junction. In people with myasthenia gravis involving the eye muscles, edrophonium chloride will briefly relieve weakness. Other methods to confirm the diagnosis include a version of nerve conduction study which tests for specific muscle "fatigue" by repetitive nerve stimulation. This test records weakening muscle responses when the nerves are repetitively stimulated. Repetitive stimulation of a nerve during a nerve conduction study may demonstrate decrements of the muscle action potential due to impaired nerve-to-muscle transmission.

EXCITATION-CONTRACTION COUPLING

EXCITATION-CONTRACTION COUPLING The process by which depolarization of the muscle fiber initiates muscle contraction is called EXCITATION-CONTRACTION COUPLING

THE TRIAD…. Made up of a t-tubule and the dilated ends called as CISTERNS of Sarcoplasmic Reticulum…..

T-Tubule & the Sarcoplasmic Reticulum The t-tubules have the DHP receptors in their membranes…The DHP is Dihydropyridine Receptor. When this receptor is activated because of an AP, it causes the opening of the voltage gated foot proteins/ Ryanodine channel/ Ca release channel. These channels are present in the cisterns of the Sarcoplasmic Reticulum. The opening of these channels leads to the release of Ca into the Sarcoplasm.

Excitation-contraction coupling….

The t-tubule and the cistern of the SR….

Excitation –Contraction Coupling Steps in contraction: Discharge of motor neuron. Release of NT (Ach) at motor end-plate. Binding of Ach to Ach receptors on the motor end plate. ↑ Na & K conductance in end-plate membrane. Generation of end-plate potential EPP). Remember the Safety Factor! EPP leading to generation of Action Potential (AP). Inward spread of depolarization (as AP) along T tubules Release of Ca2+ from terminal cisterns of SR Binding of Ca2+ to Troponin C Troponin C pulls the tropomyosin off the actin uncovering binding sites on actin. Formation of cross-linkages between actin & myosin. Sliding of thin on thick filaments, producing contraction.

EXCITATION-CONTRACTION COUPLING Steps in relaxation: Ca2+ pumped back into Sarcoplasmic Reticulum (SR) by the ATP-dependant Ca2+ pump in SR membrane. Release of Ca2+ from troponin C. A new ATP binds to the myosin head ↓ interaction between actin and myosin STOPS and RELAXATION of the muscle fiber takes place.

IMPORTANT TERMS The Triad: name given to the structure formed by a single t-tubule and 2 cisterns of SR on its each side. Calsequestrin: the protein present in the SR to which is attached the Calcium. ATPase dependant Calcium Pump: the pump which helps pump the Calcium back into the SR once the contraction is over. DHP: is also called the Dihyropyridine receptor which is present is the membranes of the t-tubule & opens in response to an AP. Ryanodine channel: which is present in the membranes of the cisterns of SR & which open in response to activation of DHP receptor.

RIGOR MORTIS

RIGOR MORTIS Definition: It is one of the recognizable signs of death in which several hours after death, all the muscles of the body go into a state of irreversible rigidity and contracture called Rigor Mortis. The body then becomes difficult to move or manipulate. On Microscopy: Continuous Actin-Myosin interaction. Cause: After death, cellular respiration in organisms ceases to occur, depleting the corpse (dead body) of oxygen used in the making of adenosine triphosphate (ATP). Unlike in normal muscle contraction, after death as ATP is NOT available, the body is unable to complete the contraction cycle and release the coupling b/w actin and myosin. We know that a new molecule of ATP is required to interact with the myosin molecule to cause relaxation at the end of a power stroke. When it is not available, relaxation cannot take place and thus, there is a state of continuous muscular contraction.

RIGOR MORTIS (cont) Mechanism: Absence of ATP→ No reuptake of Ca2+ into the SR as Ca2+ uptake also requires ATP-dependant Ca2+ pump → Ca2+ level of sarcoplasm ↑ →continued binding of Ca2+ to Troponin C →Abnormal, rigid and uninterrupted contraction. No ATP →No relaxation a new molecule of ATP must attach to the myosin head for detachment of actin- myosin interaction →thus, when NO ATP is present, then myosin heads cannot detach themselves from actin.

RIGOR MORTIS (cont.) Time Taken: In humans, it commences after about three to four hours after death, reaches maximum stiffness after 12 hours, and gradually dissipates until approximately 48 to 60 hours (three days) after death. Warm conditions can speed up the process of rigor mortis. When does Rigor Mortis end: when contractile proteins of the muscle like other body tissues undergo autolysis caused by enzymes released by lysosomes.