1. 9/27/16 – W5D1H4 Cell Physiology:Muscle Physiology

2. Why This Topic? What would life be like without muscles? We would be trapped and unable to move or even take a breath not to mind our hearts could not beat. Understanding the basic physiological properties of muscle is important for identifying clinical manifestations of various disease and pathological states. Ionic disturbances, toxins, drugs, and congenital issues can all lead to changes in muscle function which will be explored more in depth on W5D2H1-2.

3. 1. Explain the mechanism by which muscle contracts, outlining how the sliding of actin filaments in sarcomeres is driven by ATP-dependent chemo-mechanical cycling of myosin motor proteins. 2. Explain excitation-contraction (EC) coupling and relaxation in skeletal muscle by identifying the roles of the t-tubules, the calcium channels (Cav1.1 [L-type] and Ryanodine receptor [RyR]), the thin filament regulators (troponin and tropomyosin), and the ATP-dependent SERCA pumps in these processes. 3. Understand the differences in EC coupling in skeletal, cardiac, and smooth muscle. Describe the two- stage phospho-regulatory cascade that initiates contraction in smooth muscle. 4. Compare the twitch contractions of slow/type 1 and fast/type 2 skeletal muscle fibers and explain the molecular bases for the differences in twitch behavior. 5. Outline the shift in energy sources in a working skeletal muscle as it goes from rest to extended periods of activity. Compare the energy needs in different muscle types. 6. Define muscle mechanics concepts including contractility, preload, afterload, isotonic and isometric contractions. Compare the mechanics in different muscle types. 7. Explain how smooth, graded contractions of a skeletal muscle are produced by changes in stimulus intensity and by the size principle of motor unit recruitment. 8. Contrast the innervation and location of single-unit vs. multi-unit smooth muscle types. Learning Objectives:

4. LO #1. Explain the mechanism by which muscle contracts, outlining how the sliding of actin filaments in sarcomeres is driven by ATP-dependent chemo-mechanical cycling of myosin motor proteins. Muscle terminology 1)skeletal muscle fibers a.alpha motor neurons b.neuromuscular junctions c.motor endplates d.motor unit e.motor pool. f.upper motor g.lower motor 2)smooth & cardiac muscle a.autonomic motor neurons (postganglionic)

5. LO #1. Explain the mechanism by which muscle contracts, outlining how the sliding of actin filaments in sarcomeres is driven by ATP-dependent chemo- mechanical cycling of myosin motor proteins. (continued) From organ to molecule and back again

6. The “Thin” filament: G actin and F actin (cytoskeletal microfilament)

7. The “Thick” Filament: 300 molecules of myosin II

8. During contraction, the thick and thin filaments slide past one another in this ratcheting process, shortening the length of each sarcomere and consequently the length of the muscle fiber. The sarcomere histology changes, i.e., I-bands will get shorter during contraction with the increasing overlap between thick and thin filaments. contracted sarcomere From Karp, Cell & Molecular Biology relaxed sarcomere LO #1. Explain the mechanism by which muscle contracts, outlining how the sliding of actin filaments in sarcomeres is driven by ATP-dependent chemo-mechanical cycling of myosin motor proteins. (continued)

9. The classic Length/Tension curve for skeletal muscle

10. Molecular Biology of the Cell. 4 th edition

11. Critical Point: No ATP leaves the muscle in a state of rigor (a VERY brief period in a living cell). Critical Point: The hydrolysis of ATP causes myosin to move (a.k.a. “cocking” of the head) into a high energy state. Critical Point: The binding of ATP decreases the affinity of the binding and allows the myosin head to move in the next step.

12. Critical Point: A weak binding at this new location caused Pi release Pi Critical Point: The release of Pi triggers the power stroke. Critical Point: Calcium MUST be present to make binding of myosin to actin possible and for this cycle to occur.

13. During muscle contraction: a. The myosin heads (bound by ADP-P i ) bind to the actin filaments b. Next, the myosin heads undergo a conformational change called a “power stroke” to pull the thin filaments a short distance past the thick filament (ADP released) c. Then the links between actin and myosin break (requires ATP) d. Then the myosin head returns to its original conformation and the process is repeated, as the muscle is contracting. e. ATP is required to break the actin-myosin links to allow another power stroke. From your syllabus:

14. LO 1: Interactive Question (can be made into Poll Everywhere slide): Which of the following statements about the contraction of skeletal muscle is correct? a) The power stroke of skeletal muscle contraction occurs when the myosin head hydrolyses ATP. b) The power stroke of skeletal muscle contraction occurs when the myosin head binds ATP. c) The power stroke of skeletal muscle contraction occurs when the myosin head releases ATP. d) The power stroke of skeletal muscle contraction occurs when the myosin head releases ADP and Pi.

15. LO #2. Explain excitation-contraction (EC) coupling and relaxation in skeletal muscle by identifying the roles of the t-tubules, the calcium channels (Cav1.1 [L-type] and Ryanodine receptor [RyR]), the thin filament regulators (troponin and tropomyosin), and the ATP-dependent SERCA pumps in these processes. VIDEO 2 3 4 5 1 6 Ryanodine receptor SERCA ATP 1.Somatic motor neuron releases ACh at neuromuscular junction (1 impulse = ~60 vesicles of ACh) 2.End plate: Net entry of Na + through nACh receptor channel initiates first an endplate potential (+30-40mV), and then a muscle action potential is produced when nearby voltage-gated channels activated 3.Action potential travels deep into muscle fiber along t-tubules. T-tubles form triad with SR. 4.Voltage change in t-tubules triggers conformational change in Ca 2+ channels (DHP receptors & RyRs) to increase intracellular Ca 2+ 5.Ca 2+ binding to regulatory protein troponin C in thin filaments which displaces tropomyosin to expose myosin binding sites on actin, allowing contraction to begin & myosin binding initiates the cross bridge cycle to generate force. 6.See the SYLLABUS for more detail!! Ryanodine receptor

16. Muscle Action potential (AP) generation ceases as ACh is broken down by acetylcholinesterase (AChE) The SR reabsorbs Ca 2+ via SERCAs and the Ca 2+ concentration in the sarcoplasm decreases. The Na + /Ca 2+ exchanger extrudes Ca 2+ from the sarcoplasm and the Ca 2+ concentration in the sarcoplasm decreases. ATP is necessary for myosin to release from the actin; lack of ATP will cause rigor When Ca 2+ approaches resting levels the troponin and tropomyosin return to their resting positions. This change recovers the actin active sites and prevents further cross-bridge formation Without cross-bridge interactions, further sliding cannot take place and the contraction ends Muscle relaxation occurs, and the muscle returns passively to its resting length LO #2. Explain excitation-contraction (EC) coupling and relaxation in skeletal muscle by identifying the roles of the t-tubules, the calcium channels (Cav1.1 [L-type] and Ryanodine receptor [RyR]), the thin filament regulators (troponin and tropomyosin), and the ATP-dependent SERCA pumps in these processes.

17. Animation of Muscle Contraction Four minute animated review of muscle contraction

18. LO 2: Interactive Question (can be made into Poll Everywhere slide): Phospholamban is a regulatory protein associated with cardiac muscle sarcoplasmic reticulum Ca 2+ ATPase (SERCAs). Phospholamban can become phosphorylated to make it into its active state. Given what you know about skeletal muscle, guess which event in the excitation / contraction coupling pathway for cardiac muscle would most likely be increased if Phospholamban is active? a.Relaxation b.Ca 2+ influx c.Crossbridge cycling d.Electrical conduction e.Acetylcholine release

19. LO #3. Understand the differences in EC coupling in skeletal, cardiac, and smooth muscle. Describe the two- stage phospho- regulatory cascade that initiates contraction in smooth muscle. Cardiac muscle has similar EC coupling as skeletal muscle. Some Important Differences: 1)Cardiac muscle has pacemaker cells that generate current; current can pass between cells through gap junctions 2)Cardiac action potential is broad because of Ca 2+ influx. 3) Diads not triads in cardiac 4)L-type Ca 2+ channels are NOT coupled to ryanodine Ca 2+ release channels as they are in skeletal muscle. Cardiac muscle requires Ca 2+ induced Ca 2+ release (CICR) from SR. 5)Different isoforms of troponin present in cardiac muscle 6)ANS controls the rate of contraction (parasympathetic & sympathetic) and contractility (sympathetic only). 7)During recovery, most Ca 2+ is recovered by the SR, but also extruded by exchanger & Ca 2+ pump

20. The source, spread, and conformation of the AP in cardiac tissue (from your syllabus).

21. EC coupling in cardiac muscle from your syllabus: Please note that this diagram “shows” a triad, but we’ll call it creative license to show both excitation and recovery on one slide.

22. ANS control of cardiac Ca 2+ levels in EC coupling (quick review) The ANS controls both the rate and force which cardiac muscle contracts. Rate is controlled by both parasympathetic and sympathetic innervation of the SA node, but contractility is regulated by the sympathetic nervous system. Sympathetic postganglionic fibers synapse onto the cardiac myocytes and release norepinephrine, which binds to  1 adrenergic receptors to create a signaling cascade (G  s) which leads to increase Ca 2+ and contractility. Circulating epinephrine released from the adrenal medulla has the same effect. The ANS controls rate by modifying the rhythm of pacemaker cells. The pacemaker potential is due to special properties of ion channels in nodal cells, the details of which will be elucidated in the Circulatory systems block. For now, understand that the parasympathetic vagus nerve innervates SA and AV nodes to decrease rate through ACh muscarinic acetylcholine receptor M 2 (G  i) binding which ultimately activates a K + channel to hyperpolarize the cell. Sympathetic nerves (preganglionic originates from T1-T5 level of the spinal cord) innervate SA/AV nodes to increase rate through epi/norepinephrine binding of the  1 receptor which speeds up inactivation of the K + channel and increases the opening probability of Na + and Ca 2+ channels to depolarize the cell.

23. LO #3. Understand the differences in EC coupling in skeletal, cardiac, and smooth muscle. Describe the two-stage phospho-regulatory cascade that initiates contraction in smooth muscle.

24. Important points in smooth muscle EC coupling. 1.Action potential (AP) / nervous system not required; all that is necessary is increase in Ca 2+ through a variety of mechanisms 2.Ca 2+ influx in myocyte can lead to calcium-induced calcium release (CICR) 3.Ca 2+ binds to calmodulin (CaM) 4.Smooth muscle lacks troponin; for actin and myosin to interact, the light chain of myosin must be activated by myosin light chain kinase (MLCK), which is activated by CaM 5.Active MLCK phosphorylates light chain myosin heads and increases myosin ATPase activity 6.Active myosin crossbridges slide across actin to create muscle tension (no sarcomeres; cell contracts like “corkscrew”) 7.Relaxation occurs when Ca 2+ levels renormalize via Ca 2+ ATPases & Na + /Ca 2+ on membrane, SERCAs 8.Crossbridge cycling continues as long as ATP is present; relaxation is dependent on myosin phosphatase, which dephosphorylates MLCK to deactivate it. 9.Relaxation of smooth muscle in some vasculature can also occur through a nitric oxide (NO)-mediated pathway

25. LO 3: Interactive Question (can be made into Poll Everywhere slide): Which of the following would cause a smooth muscle to contract more forcefully? a.Blocking troponin b.Increased IP 3 c.Increased myosin phosphatase d.Blocking gap junctions e.Decreased Calmodulin activation

26. LO #4. Compare the twitch contractions of slow/type I and fast/type II skeletal muscle fibers and explain the molecular bases for the differences in twitch behavior. Three types of skeletal muscle fibers: Type I (slow), Type IIa (fast oxidative), and Type IIb (fast glycolytic). Fiber types are defined based on: velocity, myosin ATPase isoform, and biochemical profile.

27. LO #4. Compare the twitch contractions of slow/type 1 and fast/type 2 skeletal muscle fibers and explain the molecular bases for the differences in twitch behavior. A twitch contraction is when tension is generated in a motor unit in response to a single stimulus (like an AP). A twitch has three phases: latent, contraction & relaxation periods Latent period is not variable, but contraction & relaxation are determined by the fiber types in any given muscle.

28. Figure 18. Mean absolute power comparison between Type I and IIa muscle fibers in female ~74 yr old vastus lateralis muscles pre- and post- progressive resistance training.

29. LO #5. Outline the shift in energy sources in a working skeletal muscle as it goes from rest to extended periods of activity. Compare the energy needs in different muscle types. Four sources of energy (ATP): 1.Stored ATP (about 5 mmol/kg muscle; only about 3-5 s of max power before stores are depleted, but they NEVER do get depleted!) 2.Phosphocreatine (about 25 to 30 mmol/kg muscle; about 30 s max power until depleted) 3.Anaerobic glycolysis (peaks in as early as 30 s but can result in [Lac] blood of 15-20 mmol/L). 4.Aerobic respiration (limited at the muscle by mitochondrial capacity)

30. Byproducts 1.Heat 2.CO 2 and H 2 O 3.Lactic acid Stores 1.Myoglobin stores O 2 2.Glycogen – Primary source of glucose Vmax for ATP production 1.CK reaction and myokinase reaction are the fastest 2.Anaerobic glycolysis is about 2.5x faster than aerobic pathway but harvests only about 5% as much ATP per C 6 H 12 O 6 and can’t use fat! LO #5. Outline the shift in energy sources in a working skeletal muscle as it goes from rest to extended periods of activity. Compare the energy needs in different muscle types.

31. Energy sources and ATP utilization 1.Short high intensity activities like weight lifting or sprinting rely on ATP and phosphocreatine stores. Burst-like activities like tennis rely on glycolysis and respiration. Endurance activities depend on respiration using both glucose and fatty acids. As long as a muscle has oxygen (and it always does unless blood flow or PO 2 is restricted) it will form ATP by aerobic pathways. But if the exercise demands are great, the cells will resort to anaerobic pathways for ATP. 2.Energy sources for cardiac and smooth muscles are generally aerobic. The heart primarily uses fatty acids (60-80 %) as energy sources. Limited anaerobic pathways (2 % ATP derived from glycolysis) means that the heart requires high amounts of O 2 and if it becomes limited, irreversible hypoxic muscle damage ensues (myocardial infarction). 3.Smooth muscle has slow ATPase activity so the ATP depletion is not usually an issue. LO #5. Outline the shift in energy sources in a working skeletal muscle as it goes from rest to extended periods of activity. Compare the energy needs in different muscle types. (continued)

32. 1)Contractility is the ability to shorten forcibly when adequately stimulated. 2)Most skeletal muscles exhibit some amount of tension, or muscle tone even if they are not consciously being contracted. 3)A muscle is typically stretched to some length (preload) such that the overlap between actin filaments and myosin heads is optimized; too little or too much preload will lead to less contraction in skeletal muscle because fewer crossbridges can form. This is referred to as the length-tension relationship. 4)Skeletal and Cardiac muscle have very different length-tension curves. 5)Innumerable crossbridge cycles occur during muscle contraction, but the amount of force they generate is finite. The force resisting further shortening after the muscle is stimulated to contract is called the afterload. In skeletal muscle, the load is the weight of the object being moved. IN cardiac muscle, afterload in mean arterial blood pressure.

33. LO #6. Define muscle mechanics concepts including contractility, preload, afterload, isotonic and isometric contractions. Compare the mechanics in different muscle types. Two types of muscle contractions: 1.Isotonic: occur when a muscle shortens but maintains a constant tension – not common in normal use. 2.Isometric: when the afterload is too heavy to lift and the muscle cannot shorten even though crossbridge cycling continues to generate tension. Cardiac muscle mechanics: Myocardial excitation always involves all fibers, and so all fibers are involved in generating force as a unit. In cardiac, there is no option for recruiting motor units, so if the force is to be changed, the ANS will regulate Ca 2+ permeability. 1.Contractility, preload, and afterload should wait until the cardiovascular class in the next block! They are the major determinants of stroke volume! Contractility considers the force of contraction at a given cell length and is changed by changes in calcium availability. Preload involves stretching the myocytes and is changed by ventricular filling. Afterload involved the degree of pressure in the aorta which is affected by MAP. Smooth muscle mechanics: Smooth muscle can contract rapidly, and then completely relax, but in other cases, smooth muscle can maintain a low level of active tension for long periods without cyclic contraction and relaxation.

34. LO 4-6: Interactive Question (can be made into Poll Everywhere slide): What would happen if a skeletal muscle were stretched to the point where thick and thin filaments no longer overlapped? a. Cross bridge attachment would be optimum because of all the free binding sites on actin. b. Maximum force production would result since the muscle has a maximum range of travel. c. No muscle tension could be generated by nerve stimulation. d. ATP consumption would increase since the sarcomere is "trying" to contract. e. More motor units would be recruited to generate maximum force.

35. LO #8: Explain how smooth, graded contractions of a skeletal muscle are produced by changes in stimulus intensity (frequency) and by the size principle of motor unit recruitment. MUSCLE ACTIVITY VARIES! How can more or less tension be generated? This is done through graded muscle responses. In general muscle contraction can be graded in two ways: 1.Temporal summation - changing the frequency of stimulation by the alpha motor neuron 2.Spatial summation – changing the number of motor units recruited (a.k.a. the size principle)

36. Temporal summation in muscle: Increased frequency of stimulation generates more force; tetanus results when maximum force is produced.

37. Motor units are recruited by size: as motor units with larger and larger muscle fibers ae excited, contractile strength increases (size principle). Size principle allows for force increases to occur in small steps. Rarely are all motor units recruited; typically they are recruited asynchronously and “take turns” to delay fatigue. Spatial Summation of Motor Units

38. Smooth muscle has two main functional types: 1.Multiunit, tonic: Individual myocytes function independently and allows for fine control. capable of maintaining sustained contractions. Found in e.g. large airways, ciliary & iris muscle of eye, sphincters, vasculature, arrector pili (the small muscles that raise hairs, and goose-bumps, on skin). 2.Visceral, phasic, single-unit, unitary: Gap junctions between neighboring cells allow for current to spread across the muscle cells, so they act together as a syncytium. Some smooth muscle cells contain pacemaker cells but autonomic neurons do synapse on a few of the cells in the muscle to help regulate contraction. Phasic smooth muscle contracts transiently when stimulated. Found in e.g. GI tract and urogenital walls However, most smooth muscles are a blend of phasic and tonic, which allows them to respond to a range of stimuli. LO #8: Contrast the innervation and location of single-unit vs. multi-unit smooth muscle types.