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Graduate Physiology 503 Muscle Physiology Hurley

MUSCLE PHYSIOLOGY

Part 4: Smooth Muscle Learning objectives:

1. Describe the two mechanisms by which smooth muscle is activated following cellular stimulation.

2. Describe how the force‐velocity and length‐tension curves of smooth muscle are similar and dissimilar to striated muscles.

3. Describe the action potential of smooth muscle 4. Describe how calcium levels are regulated in smooth muscle 5. Describe the latch state 6. Describe how force can be altered by changes in myofilament calcium sensitivity

A. OVERVIEW

The most notable functional requirement of the skeletal musculature is the ability to provide rapid bursts of force generation and/or movement. Not only must the movement itself be rapid, but contractile activity must also be able to be turned on and off with equal rapidity.

Figure 1

Smooth muscles, on the other hand, serve functions in the body which are

Graduate Physiology 503 Muscle Physiology Hurley

more‐or‐less continuous and ongoing, frequently (as in the case of vasoconstriction to maintain arterial pressure or direct blood flow) involving very little motion but rather the maintenance of a steady or ʺtonicʺ force for extended periods of time. In those smooth muscles in which motion is important for function (e.g., intestinal peristalsis or bladder micturition), the movements can be quite large with respect to muscle size but are typically several hundred‐fold slower than in skeletal muscles. Another hallmark of the smooth muscle cell is that it is capable of undergoing phenotypic modulation in response to physiological or pathophysiological stimuli. The predominant phenotype is contractile in which the cell contains a high degree of contractile proteins, contractile receptors, and other components related to contractility. However, the cell can also be stimulated to become a secretory cell with a low content of contractile proteins and a high content of non‐muscle isoforms of myosin, actin, and caldesmon, loss of many receptor subtypes, and an increase in the machinery needed to produce secretory products such as elastin and collagen.

B. FUNCTIONAL ORGANIZATION: THE CONTRACTILE PROTEINS One of the immediately evident differences between skeletal/cardiac muscle cells and smooth muscle cells is in size and general appearance. Smooth muscle cells are among the smallest cells in the body, typically spindle‐shaped fibers 50‐200 μm in length with a broadest diameter of 2‐8 μm. The absence of striations (and hence the name) is due to the absence of the high degree of order which characterizes the contractile filament lattice of skeletal/cardiac muscle. While smooth muscle cells contain approximately 75% of the total amount of contractile protein (actin + myosin + tropomyosin) as other muscle tissues, the distribution of protein types is strikingly different. Actin: Smooth muscle actin, which comprises the thin filament, is chemically similar to skeletal muscle actin but about twice as plentiful. Smooth muscle actin is present as several isoforms of similar molecular weight but differing in isoelectric point (the pH at which the protein has zero charge). The most abundant actin isoform is the alpha smooth muscle specific isoform, followed in content by the beta non‐muscle isoform, and the gamma smooth muscle specific and gamma non‐muscle isoforms. Tropomyosin: Tropomyosin is present in smooth muscle in about the same ratio to actin content as in skeletal muscle (one tropomyosin to seven actins), and is similarly associated with actin filaments. The physiological significance of tropomyosin in smooth muscle has not yet been fully established, although its presence does alter the kinetics of the smooth muscle actin‐myosin interaction, and tropomyosin is known to stabilize actin filaments. Tropomyosin in smooth muscle may transmit the regulatory signal from caldesmon (see below) down the thin filament. Caldesmon: The thin filaments of smooth muscle also contain a Ca2+, calmodulin, actin, and myosin binding protein termed caldesmon. This protein exists in both a high molecular weight isoform which is smooth muscle specific and a low molecular weight isoform

Graduate Physiology 503 Muscle Physiology Hurley

which is found in most cells. The low molecular weight isoform is important in the structural integrity of the cytoskeleton. The precise role of smooth muscle specific high molecular weight caldesmon in the regulation of smooth muscle contraction is controversial. What is known from biochemical studies is that caldesmon inhibits actin‐ activated myosin ATPase activity. This inhibition has been shown to be reversed by either high concentrations of Ca2+ and calmodulin or by phosphorylation catalyzed by several endogenous kinases including protein kinase C, Ca2+/calmodulin dependent protein kinase II, cdc2 protein kinase, casein kinase II, p21 activated protein kinase, or mitogen‐activated protein kinases.

Figure 2

Calponin: Calponin is an abundant smooth muscle specific protein associated with the thin filaments. Calponin is a very basic (pI ‐ 8.6 ‐ 9.4), 34,000 MW, Ca2+, calmodulin, and actin binding protein. Calponin has been almost exclusively studied in biochemical preparations of smooth muscle contractile proteins which have demonstrated that it inhibits actin‐activated myosin ATPase activity. Whether or not calponin plays any role in regulation of smooth muscle contraction is currently unknown. Myosin: Smooth muscle myosin molecule is morphologically identical to skeletal muscle myosin in the electron microscope, but biochemically quite different; especially in the

Graduate Physiology 503 Muscle Physiology Hurley

enzymatic ATPase activity of the globular ʺheadʺ portion and the associated light chain proteins (20,000 and 18,000 MW). Smooth muscle myosin ATPase activity is approximately 100 times slower than skeletal muscle myosin ATPase activity. Smooth muscle cell myosin content is very low, only about 20% of that in skeletal muscle. The reduced myosin content and lower level of polymerization leads to many fewer thick filaments per cross sectional area relative to skeletal and cardiac muscle. Thick filaments are more‐or‐less uniformly dispersed throughout the cell approximately parallel to the longitudinal axis. The elevated actin content leads to a final ratio of thick to thin filaments of approximately 1:15 in smooth muscle, as compared to 1:2 in skeletal and cardiac muscle. Sarcomere‐like structure (Figure 1): Smooth muscle, as discussed above, does not possess the ʺnormalʺ sarcomeric structure giving striated muscle its name. Instead, several actin filaments may pass through or, more likely, terminate in a protein‐rich ʺdense bodyʺ or ʺdesmosome,ʺ which acts as a point of thin filament anchorage for the transmission of generated force. While some desmosomes appear bound to the plasma membrane, others appear suspended in the cytoplasm. In order to support these cytoplasmic anchorage points, a network of ʺintermediateʺ (10 nm) filaments made from a protein called ʺdesminʺ interconnects membrane and cytoplasmic dense bodies. In some smooth muscle cells desmin is replaced by the protein vimentin while in other cells both can be found. In addition to desmin, the proteins alpha‐actinin and tropomyosin have been identified in smooth muscle dense bodies. Critical Concept Smooth muscle contains NO troponin. Thin filaments are composed of actin, tropomyosin, caldesmon, and calponin. Classical sarcomeres as in striated muscle do not exist in smooth muscle

Figure 3

Graduate Physiology 503 Muscle Physiology Hurley

C. MECHANICAL PERFORMANCE Force‐Length Relation The sliding filament mechanism, as an explanation for the dependence of isometric force generation on muscle length (pre‐load, remember?), applies to smooth muscles as well as skeletal muscles. Without sarcomeric limitations and with inherently longer thick and thin filaments, smooth muscles can develop isometric force over a broader range of muscle lengths and maximally shorten to about 30% of their optimal length. The ramification of this fact on the length/tension relationship is that the plateau of the length/tension curve in smooth muscle is extremely broad. This high degree of shortening also allows for dramatic changes in the diameter of hollow organs such as the bladder or blood vessels. Force‐Velocity Relation (Figure 2): Smooth muscle cells are, on the basis of the inherent slow ATPase activity of smooth muscle myosin, 50 ‐ 100 times slower than skeletal muscles. However, both striated and smooth muscles have qualitatively and mechanistically similar force‐velocity relationships, it is just that smooth muscle velocities are a lot slower.

D. METABOLISM Oxidative Metabolism: The energy metabolism of skeletal and smooth muscle operates on two fundamentally different strategies. In order to permit rapid bursts of activity, skeletal muscles contain large, pre‐formed pools of high energy phosphates (typically 20 ‐ 25 mM ATP + PCr) which can power contraction without being delayed by a need for ongoing glycolytic or oxidative metabolism; although fatigue rapidly ensues if recovery metabolism is blocked. Because smooth muscles frequently operate as ʺtonicʺ muscles generating a continuous level of contractile force, they are designed to be more dependent on simultaneous oxidative metabolism. Pre‐formed high‐energy phosphates are in low concentrations in smooth muscles (typically 2 ‐ 4 mM ATP + PCr). Aerobic Glycolysis: Unlike most other mammalian tissues, even well‐oxygenated vascular smooth muscle (as well as many other smooth muscles) produce large amounts of lactic acid. It has been shown that about 90% of the glucose consumed ends up as lactate and the balance oxidized to CO2. In the absence of exogenous glucose, lactate production remains high, being derived from the large quantities of glycogen stored. In terms of metabolic energy production, some 20 ‐ 30% of total energy production is derived from aerobic glycolysis. The ATP derived from this process however does not appear to be used to power contractile activity. It has been proposed that aerobic

glycolysis is indirectly coupled to Na+ transport in smooth muscle. We will see later in the

Graduate Physiology 503 Muscle Physiology Hurley

hour that Na+ transport is critically important in the regulation of smooth muscle. Critical Concept Smooth muscle is highly economical. It is designed to maintain high levels of force for long periods of time with very little expenditure of energy.

E. MEMBRANE POTENTIALS AND ACTION POTENTIALS IN SMOOTH MUSCLE

Membrane potential The membrane potential depends, as in all excitable cells, on the momentary condition of the muscle and its varying permeability to ionic species. At normal resting conditions, the intracellular potential is typically around -50 to -60 mV. The smooth muscle membrane expresses many more voltage gated calcium channels than skeletal muscle and only a few voltage gated sodium channels. Therefore, the action potentials in smooth muscle are mainly calcium based. The slower kinetics of calcium channels in terms of their activation and inactivation accounts for the electrical behavior observed in smooth muscle cells during action potentials. Action Potentials Action potentials occur in unitary smooth muscle either as spike potentials or action potentials with pronounced plateaus. Spike potentials occur commonly in unitary smooth muscle with a duration of about 10 – 50 ms. These spike potentials can be evoked by electrical stimuli or the actions of endocrine signals or neurotransmitters, stretch, or they may be generated spontaneously. Action potentials with plateaus have a similar depolarization phase as spike potentials but repolarization of the membrane potential is significantly delayed ( by around 1 sec) allowing for sustained periods of contraction. Action potentials arise spontaneously within smooth muscle and they often exhibit a slow wave rhythm… essentially a characteristic oscillation of the smooth muscle’s membrane potential.

Graduate Physiology 503 Muscle Physiology Hurley

Figure 4 Typical smooth muscle action potentials. (A) Action potential elicited by external stimuli. (B) Repetitive spike potentials riding on top of slow wave depolarizations. (C) Action potential with pronounced plateau phase, recorded from uterine smooth muscle.

Graduate Physiology 503 Muscle Physiology Hurley

One important point is that an action potential is NOT required for an increase in force in many smooth muscles. An action potential will produce a more robust contraction, but smaller non‐propagating decreases in membrane potential can produce graded increases in force in many smooth muscle tissues. This form of cellular activation is called Electromechanical Coupling, shown below in Figure 5

Figure 5. Various mechanism for regulating calcium in smooth muscle.

F. EXCITATION‐CONTRACTION COUPLING As with all muscles, the key signal in activating contractile activity in smooth muscle is an increase in intracellular free [Ca2+] from less than l0‐7 M to something sometimes in excess of l0‐6 M. The two major differences between skeletal/cardiac muscle and smooth muscle in this regard are: (1) Where activator Ca2+ comes from, and (2) The mechanism by which elevated Ca2+ turns on the contractile apparatus. Calcium metabolism: In contrast to striated muscle, smooth muscle contains very little SR. Therefore, activator Ca2+ in smooth muscle arises from both inside the cell (released from the SR) and outside the cell. Calcium enters from outside the cell as a transmembrane flux through specific Ca2+ channels which may be opened by depolarization of the membrane potential (voltage operated channels, VOCʹs, dihydropyridine receptors) or by an agonist‐receptor interaction (receptor operated channels, ROCʹs), see Figure 5. Calcium influx through membrane channels is believed to be the primary but not sole source of calcium

VDCC

PKC AC

GsDAG Gq PI-PLC - R

R cAMP

PKA IP3

Ca2+ GC

CM cGMP

PKG

CONTRACTION

IP3-R SERCA

SR Ca2+

Graduate Physiology 503 Muscle Physiology Hurley

for sustained contraction. And unfortunately for students, this is the third type of dihydropyridine that you will need to learn about. The good thing is that at least in muscle, it is the last one also. This particular dihydropyridine receptor is a real calcium channel that allows sufficient calcium into the cell to initiate contraction. Unlike skeletal muscle, it is NOT a voltage sensor and unlike cardiac muscle it allows ʺlotsʺ of calcium to enter the cell. Intracellular calcium (SR bound) is released by a complex series of events initiated by agonist/receptor binding. Receptors on smooth muscle membranes are coupled to heterotrimeric G‐proteins. Agonist binding to a receptor activates this G‐protein which in turn activates an enzyme called phospholipase C. Phospholipase C catalyzes the release of diacylglycerol (DAG) and inositol trisphosphate (IP3) from phosphatidylinositol. DAG is an activator of an extremely important cellular enzyme called protein kinase C. The multiple actions of protein kinase C are beyond the scope of this course. In terms of calcium metabolism, the important molecule is IP3. IP3 diffuses through the cytosol and binds to specific IP3 receptors on the SR. The IP3‐receptor (IP3R) is in fact a calcium channel and when bound to IP3, it opens and releases SR Ca2+ into the cellular space. This form of cellular activation is called Pharmacomechanical Coupling. In addition to the IP3R, ryanodine receptors are present in the SR of most smooth muscles. Remember ryanodine receptors are the ones responsible for Calcium‐Induced Calcium Release from the cardiac muscle SR. However, with one single exception (rabbit urinary bladder smooth muscle), the physiological relevance of ryanodine receptors in smooth muscle is unknown. Upon withdrawal of the agonist or membrane repolarization, intracellular Ca2+ is reduced principally by active uptake into the SR and extrusion from the cell by a plasma membrane Ca2+‐pump. In addition to a true CaATPase, there exists a Na+/ Ca2+ exchange mechanism which uses the energy derived from the Na+ leak into the cell down its electrochemical gradient to drive the outward movement of Ca2+ up its gradient ‐ hence the need for a direct supply of energy for the Na/K pump. Physiological regulators of calcium metabolism include the class of compounds known as cyclic nucleotides. Cyclic AMP and cyclic GMP acting through their respective cyclic nucleotide‐dependent protein kinases (PKA and PKG) decrease levels of calcium by inhibiting influx of calcium through membrane channels and increasing the activities of the CaATPases in the SR and plasma membrane. Critical Concept Initial increase in cellular Ca2+ results primarily but not solely from release of SR stores; steady state increases in cellular Ca2+ results primarily but not exclusively from influx from extracellular spaces.

Graduate Physiology 503 Muscle Physiology Hurley

Activation of the contractile proteins (Figures 6 and 7 ): In striated muscle, Ca2+ binds to troponin C, which removes the inhibition of myosin binding to actin by causing (through interactions mediated by troponin I and T) steric alterations in tropomyosin, revealing actin binding sites. Troponin is NOT present in smooth muscle. In contrast to the ʺdisinhibitionʺ regulatory system present in striated muscle, smooth muscle contains a true ʺactivationʺ system.

Figure 6

At resting levels of calcium, smooth muscle myosin is inactive and cannot interact with actin. When the intracellular Ca2+ concentration rises, Ca2+ binds to a calcium binding protein called calmodulin forming a ʺCa‐calmodulin complexʺ. Ca‐calmodulin then binds to an enzyme called the myosin light chain kinase (MLC kinase). Ca‐calmodulin binding to the MLC kinase activates the enzyme which in turn catalyzes the phosphorylation of the 20,000 dalton myosin light chain (MLC) of myosin. It is this step, phosphorylation of the 20,000 dalton MLC (also called the regulatory or phosphorylatable light chain) which activates myosin and allows interaction with actin. The 20 kDa MLC is positioned along the S‐2 or hinge portion of the myosin molecule. It is believed that phosphorylation of this MLC causes a conformational change in the S‐2 allowing the ʺbusinessʺ end of the myosin molecule, the S‐1, to interact with actin.

Graduate Physiology 503 Muscle Physiology Hurley

The cycle is terminated when the myosin light chain is dephosphorylated by a myosin light chain phosphatase (MLC phosphatase). Upon reduction of intracellular Ca2+, Ca2+ - calmodulin complex dissociates, MLC kinase activity is reduced, and myosin is progressively dephosphorylated by the phosphatase until the muscle is relaxed. Critical Concept Unlike striated muscle which is a disinhibition system, smooth muscle is regulated by an activation system. Smooth muscle myosin must be activated by phosphorylation in order to interact with actin and produce force or shortening.

Figure 7

The Latch State (Figure 8): One of the more interesting aspects of smooth muscle regulation is a unique state that has been termed the ʺLatch Stateʺ. It is this state that allows smooth muscle to maintain high levels of tone or force for prolonged periods of time with very little energy utilization. This is especially important for smooth muscle cells in sites such as the aorta which must maintain vessel integrity against a high pulsatile pressure for the entire life of the individual. The Latch State is formed in response to all known modes of cellular activation in all smooth muscle tissues examined to date. Therefore it is a fundamental property of the cell. Upon tissue stimulation, intracellular calcium increases which is followed with a short time lag by an increase in MLC phosphorylation and finally force development. However, with continued tissue stimulation, cytosolic calcium concentration and MLC

Graduate Physiology 503 Muscle Physiology Hurley

phosphorylation values return to basal or near basal values while force is maintained at high levels. The maintenance of high levels of force during conditions of low or basal values of calcium and MLC phosphorylation is the Latch State. How the Latch State is initiated, how it is regulated, and how it is ʺturned‐offʺ is not known. But what is known is that phosphorylated crossbridges (20 kDa MLC phosphorylation) have a high cycling rate and therefore a high shortening velocity. Dephosphorylated crossbridges (phosphorylated 20 kDa MLC that have been dephosphorylated by the MLC phosphatase) are called Latchbridges and have a low cycling rate and therefore a slow shortening velocity. It has been proposed that the phosphorylation level of the 20 kDa MLC not only activates the myosin molecule but due to its importance in determining whether a crossbridge is a rapidly cycling phosphorylated crossbridge or a dephosphorylated slowly cycling latchbridge ‐ it also determines shortening velocity. Critical Concept: High levels of force can be maintained in smooth muscle without proportional levels of myosin light chain phosphorylation, the latch state. Force is maintained by dephosphorylated latchbridges. This allows for a highly energy efficient muscle.

Figure 8

Graduate Physiology 503 Muscle Physiology Hurley

Modulation of contractile protein Ca2+ sensitivity (Figure 9): In addition to the phenomena described by the latch hypothesis, it is also known that the sensitivity of the contractile apparatus to Ca2+ can be modulated. Stimulation of smooth muscle by an agonist produces more force for any given level of cytosolic calcium as compared to stimulation by a mechanism that only depolarizes the membrane. As can be seen in Figure 8, at each level of activator calcium, more force is developed following agonist/receptor/G‐protein mediated contraction than by a simple change in membrane potential. The mechanism responsible for the increased force per calcium appears to involve agonist‐ dependent activation of the small GTP‐binding protein, RhoA. The active GTP‐bound form of RhoA binds to and activates Rho kinase. Rho kinase has been shown to have two major substrates in smooth muscle.

Figure 9

First, Rho kinase can directly phosphorylate the MLC thus acting synergistically with the MLC kinase in activating the contractile proteins. Second, Rho kinase inhibits the activity of the MLC phosphatase by phosphorylating the myosin binding sub‐unit. Since the net level of MLC phosphorylation is the sum of the forward kinase reaction and the back phosphatase reaction, then any decrease in the back phosphatase reaction will increase net levels of MLC phosphorylation and hence increase force.

Graduate Physiology 503 Muscle Physiology Hurley

In addition, protein kinase C (remember activation of the receptor associated G‐protein activates phospholipase C which releases diacyglycerol which activates protein kinase C) has been shown to be involved in increasing myofilament calcium sensitivity. Protein kinase C phosphorylates a small 17 kDa protein called CPI‐17. Phosphorylated CPI‐17 binds to the MLC phosphatase and inhibits its activity. Similar to the actions of Rho kinase, this also results in an increase in the net levels of MLC phosphorylation. More and more now, scientists are finding that phosphatases are a major site of cellular regulation. It makes sense actually in terms of maximizing the use and content of cellular ATP stores. Rather than increasing kinase activity which would increase ATP utilization, decreasing phosphatase activity saves ATP as well as making the kinase work more efficiently. Putting it all together: Figure 10 provides a cartoon depicting most of what we have discussed about the regulation of a smooth muscle cell.

Figure 10