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Graduate Physiology 503 Muscle Physiology Hurley

MUSCLE PHYSIOLOGY

Part 2: Excitation-contraction coupling Learning Objectives:

1. Compare the role of extracellular and intracellular calcium in excitation contraction

coupling in skeletal and cardiac muscle. 2. Describe the role of the T‐tubule and the sarcoplasmic reticulum membrane systems in

excitation‐contraction coupling. 3. Describe how calcium is removed from the cytoplasm for relaxation. 4. Describe how force can be graded in cardiac and skeletal muscles.

A. OVERVIEW Skeletal Muscles are the effector organs of the voluntary locomotor system. The striated appearance of both skeletal and cardiac muscle is because of the ordered arrangement of the contractile elements within the muscle fibers. Skeletal muscle, unlike cardiac muscle, has no intrinsic spontaneous electrical activity and therefore relied upon neural impulses to initiate activity. Most activation of skeletal muscle takes place at specialized nerve ending called motor end plates. There are a few exceptions: facial muscles, for example, are diffusely innervated along the length of the muscle providing multifocal innervation of these skeletal muscles.

B. STRUCTURE OF SKELETAL MUSCLE The neural electrical impulse is amplified at the neuromuscular junction producing an end plate potential that is the first step in muscle contraction.

Figure 1. Key structures of skeletal muscle fiber.

Graduate Physiology 503 Muscle Physiology Hurley

Like neurons, muscle fibers are excitable cells. Their plasma membranes (sarcolemma) express ion channels and pumps necessary to support a very negative resting membrane potential as well as the voltage gated ion channels required to generate an action potential. At any given time, the membrane potential of muscle cells is the result of the net electrochemical gradients of ions that the membrane is permeable to. (Recall Nernst equation; Table 1). The resting membrane potential in skeletal muscle at 37°C is similar to neurons (~-70 – 90 mV). However, there is an importance difference between the ion species that dominate the resting membrane potential in neurons and skeletal muscle.

Question: In contrast to skeletal muscle, what ion species dominates the resting potential of neurons? Explain. Chloride makes a significant contribution to the resting membrane potential of skeletal muscle. The physiological relevance of the Cl- current stems from a need to maintain muscle activity during repeated stimulation. When muscle contracts, there is leakage of K+ from the cell. With repeated activity, there is run-down of the K+ concentration gradient across the sarcolemma. Without the Cl- current to maintain resting membrane potential, the muscle would not repolarize sufficiently to regenerate the active state of the channels responsible for generation of succeeding action potentials.

Table 1 Plasma and cytoplasm concentrations of various ions and the resulting Nernst potential

Ion Plasma (mM)

Cytoplasm (mM)

Ex (mV)

Na+ 145 12 -67 K+ 4 140 -95

Ca2+ 1.2 10-4 +125 Mg2+ 1.5 0.8 +8 Cl- 115 4 -90

A skeletal muscle action potential is generated when the motor endplate potential is sufficient to raise the surrounding sarcolemmal potential above the threshold for activation of the abundant voltage gated Na+ channels throughout the sarcolemma. When these channels are activated, the membrane is rapidly depolarized towards the Nernst potential for Na+ (Table 1). However, the peak potential achieved is approximately +30 mV. The Nernst potential is not achieved for two main reasons. First, because of inactivation of the voltage gated sodium channels. Inactivation is a slower mechanism than activation, so the Na+ current continues to flow for a short period after the onset of inactivation, but not sufficiently to reach the Nernst potential. The second factor limiting the upstroke of the action potential is the voltage activation of rectifying potassium channels. Their activation is initiated also during the upstroke of the action potential but (in a similar way to Na+ channel inactivation) there is a slight delay in channel opening. The resulting K+ current, in addition to limiting the peak of the action potential, is also principally responsible for repolarization.

Graduate Physiology 503 Muscle Physiology Hurley

Once initiated, an action potential spreads over the sarcolemma. Skeletal muscle sarcolemma has invaginations called transverse- or t-tubules that run perpendicular to the surface of the cell and dive deep into its body. The action potential passes down the t- tubular membrane and is carried to the structures responsible for transducing an electrical into a chemical signal that activates the contractile elements. The close proximity of the transverse tubules to the sarcoplasmic reticulum completes the structural pathway for the activation of contraction. Transverse Tubules ‐ An extracellular tubular network comprising an invaginating extension of the sarcolemma winding between and encircling each myofibril within the fiber. Essential for the conduction of action potentials to the innermost myofibrils (Figures 1, 2, and 3). Sarcoplasmic Reticulum ‐ An intracellular tubular network essential for the storage, delivery, and re‐uptake of Ca2+ for the regulation of contraction. The SR consists of the terminal cisternae which stores calcium as a complex with calsequestrin (a calcium binding protein) and releases calcium and the longitudinal tubules which contain a Ca2+-ATPase for the re‐uptake of Ca2+ (Figure 1, 2, and 3).

Figure 2. Ultrastructure of the skeletal muscle triad.

Graduate Physiology 503 Muscle Physiology Hurley

Figure 2. Drawing depicting the relative positions of Triad components necessary for ECC.

Triad ‐ A triad consists of the T‐tubule and its two neighboring terminal cisternae. There is a gap of ~1 μm that separates the membranes and the two tubular systems. Spanning this gap are the triadic feet. The triad is the site of coupling excitation of the T‐tubule with the release of calcium from the SR. A voltage‐sensor protein (the dihydropyridine receptor) in the T‐tubule membrane is thought to control the permeability of the Ca2+ release channels (the ryanodine receptor) in the SR. (Figure 1, 2, and 3).

C. EXCITATION CONTRATION COUPLING IN SKELETAL MUSCLE

The term “excitation-contraction coupling” (ECC) encompasses the mechanisms by which the surface membrane action potential leads to the interaction of the intracellular contractile elements. The t-tubular membrane contains the highest density in the body of binding sites for dihydropyridine compounds, such as nifedipine. Although the t-tubular dihydropyridine receptors show marked amino acid homology with L-type voltage gated Ca2+ channels of other tissues, they do not function as Ca2+ channels. Their role in skeletal muscle is that of voltage sensors. When the action potential arrives in the t-tubule, the change in membrane potential leads to a conformational change in the α-subunit of the dihydropyridine receptor. This subunit consists of four transmembrane domains each of six segments. The first and sixth segments of adjacent domains are linked by alternating extracellular and cytoplasmic loops of amino acids. The voltage-induced conformational change results in the projection of the cytoplasmic loop between the second and third transmembrane domains deeper into the cytoplasm. Here, charged amino acid residues of the cytoplasmic loop come into proximity with those of a protein projecting towards the cytoplasmic surface of the t-tubule from the adjacent terminal swelling of the sarcoplasmic reticulum.

Graduate Physiology 503 Muscle Physiology Hurley

Figure 2. Structures involved in ECC. The change in T‐tubule membrane potential causes a conformational change in the αI subunit of the DHPR, in turn resulting in a conformational change in the RyR. This is the calcium release channel of the sarcoplasmic reticulum resulting in the release in stored calcium into the cytoplasm.

Figure 4

Graduate Physiology 503 Muscle Physiology Hurley

Time Course of ECC The release of calcium begins when the depolarization of the sarcolemmal membrane reaches the T‐tubules and ceases with repolarization. There is an important temporal separation of events with the action potential initiating the contraction and contraction occurring after a time lag. Termination of Contraction: Relaxation Myoplasmic calcium concentration is regulated primarily by the active uptake of Ca2+ into the longitudinal SR. The CaATPase pump is always operating and rapidly accumulates Ca2+ into the longitudinal SR. Following the delivery of a pulse of Ca2+ during excitation and binding to TnC, the myoplasmic calcium concentration decreases as a result of active uptake into the SR. Calcium diffuses from the longitudinal SR to the Terminal Cisternae where it is bound to calsequestrin and stored for the next excitatory event. Calcium binding proteins are important in maintaining a low free calcium concentration in the sarcoplasmic reticulum and therefore avoiding transport saturation, saturation occurs at a higher total calcium load, and the efficiency of the CaATPase pump is increased by pumping against a lower free calcium concentration As the myoplasmic calcium concentration decreases, Ca2+ dissociates from TnC, there is restoration of steric blocking and inhibition of actin and myosin interactions by tropomyosin. Actin and myosin interactions decrease producing a decrease in actin‐activated myosin ATPase activity, and the muscle cell relaxes.

Graduate Physiology 503 Muscle Physiology Hurley

D. ECC OF CARDIAC MUSCLE

Cardiac muscle is striated like skeletal muscle but not under voluntary control. Compared to skeletal muscle, cardiac muscle fibers are shorter (~20μm for cardiac muscle fibers versus ~100μm for skeletal muscle fibers) and branched. The smaller cell size means that diffusion of substances from the extracellular space into the myocyte interior occurs more readily in cardiac muscle. The end of neighboring cardiac muscle fibers are connected by irregular transverse thickening of the sarcolemma called intercalated disks. Intercalated disks contain three different types of cell-cell junctions:

Fascia adherens junctions (anchoring junctions): where actin filaments (thin filaments in the sarcomeres) attach to the sarcolemma. Desmosomes: sites of strong cell-to-cell adhesion Gap junctions: provide direct contact between the cardiac cells, allow waves of depolarization to spread rapidly from cell to cell.

In cardiac muscle the SR is less extensive than in skeletal muscle with smaller terminal cisternae but larger T‐tubules (Figure 4).

Graduate Physiology 503 Muscle Physiology Hurley

Figure 4

Graduate Physiology 503 Muscle Physiology Hurley

From Bers, DM (2002) Nature 415: 198

Figure 5

ECC of cardiac muscle is in many ways very similar to that of skeletal muscle. However, there are some important and fundamental differences that will be highlighted next. In cardiac muscle, most of the calcium for contraction is derived from the SR as well as some from the extracellular space. In skeletal muscle the contraction is solely dependent on calcium release from the SR, no calcium enters from the extracellular space. 1. Excitation ‐ Calcium enters the cardiac muscle cell during the plateau phase of a ventricular action potential. 2. Time Course ‐ The cardiac action potential has a long duration, ~250 msec;

Graduate Physiology 503 Muscle Physiology Hurley

unlike skeletal muscle, there is considerable overlap in time among the action potential, increase in myoplasmic calcium, and the contraction. 3. Calcium Delivery Calcium channels. Calcium enters the cardiac muscle fiber through calcium channels in the sarcolemma and T‐tubules. The terminal cisternae of the SR is closely opposed to the T‐tubules and forms a dyad (one cisternae and one T‐ tubule) as compared to a triad in skeletal muscle.

Figure 6

Dihydropyridine receptors are located on the sarcolemmal side of the dyad and ryanodine receptors are located on the SR side of the dyad.

Graduate Physiology 503 Muscle Physiology Hurley

In contrast to skeletal muscle, the dihydropyridine receptors do not function as voltage sensors but instead function as calcium influx channels. In response to a depolarizing current, the dihydropyridine receptors open allowing a small influx of calcium, not enough calcium to initiate a contraction. This small amount of calcium binds to the calcium release channel on the SR initiating a large release of the stored SR calcium. This process is called Calcium‐Induced Calcium Release (CICR, see Figure 4 and 5). Neither the small increase in calcium that leads to SR calcium release nor the ʺextraʺ calcium that enters during the plateau phase of the action potential are sufficient to activate the myocardium. The SR is the primary source of activator calcium. Since most of the calcium that enters the cardiac muscle cell during excitation is taken up and stored by the SR, any process that increases the influx of calcium will result in a larger pool of available calcium for subsequent contractile events.

Graduate Physiology 503 Muscle Physiology Hurley

Calcium is removed from the intracellular space (Figure 5 and 6) by CaATPases located in the longitudinal SR and in the sarcolemma. In addition, a Na/Ca exchanger in the sarcolemma provides an important mechanism for calcium extrusion. The Na/Ca exchanger uses energy derived from the Na gradient in the cell which is determined by the Na,K ATPase pump.

E. MODULATION OF CONTRACTION IN CARDIAC MUSCLE Unlike skeletal muscle, cardiac muscle must meet the functional demands of the individual on a beat‐to‐beat basis. Mechanisms have evolved that allow for modulation in the magnitude or rate of force production. Such changes are referred to as changes in contractility. In contrast to the all or none response of skeletal muscle, cardiac muscle responds to an increase in calcium in a graded manner. The rate of force production is also graded in response to the increase in calcium with faster rates of contraction in response to larger concentrations of calcium (see Figure 7).

Graduate Physiology 503 Muscle Physiology Hurley

Figure 8

Because the heart represents an electrical syncytium, with all of the cardiac muscle cells contracting during a single beat, it is not possible to increase the force of contraction by recruiting more muscle cells. Moreover, tetany of the heart would be lethal because it would defeat the critical pumping action of the heart. The heart has therefore developed alternative strategies to increase the force of contraction. It should be noted that the long action potential found in cardiac muscle, which is due to activation of the voltage-gated L-type Ca2+ channel, results in a long refractory period, which in turn prevents tetany. Modulation of Ca2+ influx through L-type Ca2+ channels during an action potential, however, provides the heart with a mechanism to alter cytosolic [Ca2+] and hence the force of contraction.

Graduate Physiology 503 Muscle Physiology Hurley

Calcium availability ‐ Prolongation or attenuation of the plateau phase of the action potential alters the duration of calcium influx and therefore the quantity of calcium released by the SR. If the plateau phase is prolonged, more calcium enters the cell and may increase the amount of force developed as well as the amount of calcium taken up by the SR and available for subsequent contractions. Any change in extracellular calcium concentration will have a profound effect on cardiac muscle calcium metabolism. A decrease in extracellular calcium for example will decrease calcium influx during the action potential and increase calcium efflux due to a decreased concentration gradient. Myofilament calcium response (see Figure 8) ‐ The sensitivity of the contractile proteins of cardiac muscle to calcium can be modulated. Any event that prolongs the ʺlife spanʺ of the TnC conformation that triggers contraction will facilitate contraction.

Calcium‐sensitizing drugs increase the response of the myofilaments to calcium without a change in the cytoplasmic calcium concentration. Although not yet clinically available, development of calcium sensitizers has been a long term goal of many pharmaceutical firms. It is now known that phosphorylation of the troponin protein can change myofilament calcium sensitivity. Protein kinase A phosphorylates cardiac TnI which increases the rate of cardiac muscle relaxation. TnI phosphorylation decreases the affinity of TnC for calcium and increases the off‐rate for dissociation of calcium. Protein kinase A also phosphorylates a protein called phospholamban on the SR and increases CaATPase activity, thus also facilitating relaxation.

Graduate Physiology 503 Muscle Physiology Hurley

Any event that increases the interaction of actin and myosin will increase the level of force produced at any given calcium concentration, such as a shift to the optimal length‐tension relationship.

F. MODULATION OF CONTRACTION IN SKELETAL MUSCLE The Motor Unit and Motor Pool

Figure 9. Motor units and motor pools for skeletal muscle.

Force production in skeletal muscle is largely under neural control. Each skeletal muscle fiber is electrically and mechanically independent; fibers are electrically isolated and mechanical events are transmitted through tendons. Whole skeletal muscle is organized into motor units. A motor unit is defined as a motor nerve and the skeletal muscle fibers that it innervates. When a motor unit is stimulated, action potentials are conducted through branches to each muscle fiber in the motor unit. Force in skeletal muscle can be graded by three mechanisms. Gradation by Frequency of Nerve Stimulation (Figure 10) Twitch ‐ the response to a single action potential is called a twitch, which is a transient increase in force followed by a relaxation.

Graduate Physiology 503 Muscle Physiology Hurley

Summation of force ‐ Calcium transients from two or more closely spaced action potentials will sum, producing more force. A single action potential does NOT release ALL of the SR calcium. So multiple action potentials can produce more force by releasing more total calcium, how much depends on the timing. Timing of action potentials can also release ʺrecycledʺ calcium that had been taken back up by the SR from a previous action potential. The additional force derives from the activation of more cross‐bridges that add to those that remain active from the proceeding stimulus (remember that force lasts much longer than the calcium response which lasts much longer than the action potential). Repetitive stimulation of the muscle at a frequency that does not allow complete relaxation results in summation of twitch responses.

Figure 10. Modulating tension in skeletal muscle

Tetanus ‐ Repetitive stimulation at an appropriately high frequency can lead to a smooth increase in force, this is tetanus. Tetanus force is the result of maximum activation of the muscle. This rarely occurs physiologically.

Graduate Physiology 503 Muscle Physiology Hurley

The frequency of mechanical activation is limited by the electrical properties of the muscle membrane. The maximum frequency at which a membrane can be excited is set by the duration of its refractory period. The temporal relationship between the muscle action potential and its contractile response determine whether or not a muscle can undergo a tetanic contraction. In skeletal and smooth muscles, the electrical events are brief and temporally separate from mechanical events. The refractory period of the action potential is over well before peak force is reached ‐ thus these muscles can be tetanized. In cardiac muscle, the electrical and mechanical events follow a similar time course, thus cardiac muscle cannot be tetanized. Gradation by Number of Motor Units The force produced by a motor unit is the arithmetic sum of the force produced by each muscle fiber. The total force of a muscle will depend on the number of motor units activated. The increase in the number of motor neurons firing and activating motor units is termed recruitment. Gradation by Activation of Larger Motor Units. The number of fibers in a motor unit determines its force output. Small motor units contain only a few muscle fibers such as the extrinsic muscle of the eye which contain only 3‐6 muscle fibers. Large motor units such as those in the leg muscles may contain hundreds of muscle fibers. Large motor units are innervated by nerves having larger diameter cell bodies and axons than small motor units. The conduction velocity of large motor units is faster. The Henneman Size Principle is a hypothesis that states that recruitment of motor units within a muscle takes place in an invariant order. Motor neurons with the smallest cell bodies and are the easiest to bring to firing threshold and will be recruited first.

Graduate Physiology 503 Muscle Physiology Hurley

These will have the smallest number of muscle fibers per muscle unit. As more units are recruited, the larger and more forceful the contraction. The distribution of motor units is such that the percent increase in force that each additional unit adds to the total force is constant ‐ thus ensuring a smooth gradation of force as recruitment proceeds.