Mechanisms of Vascular Smooth Muscle Contraction and the Basis for Pharmacologic Treatment of Smooth Muscle Disorders.

The smooth muscle cell directly drives the contraction of the vascular wall and hence regulates the size of the blood vessel lumen. We review here the current understanding of the molecular mechanisms by which agonists, therapeutics, and diseases regulate contractility of the vascular smooth muscle cell and we place this within the context of whole body function. We also discuss the implications for personalized medicine and highlight specific potential target molecules that may provide opportunities for the future development of new therapeutics to regulate vascular function.

Muscle force and stiffness during activation and relaxation. Implications for the actomyosin ATPase.

Isolated skinned frog skeletal muscle fibers were activated (increasing [Ca2+]) and then relaxed (decreasing [Ca2+]) with solution changes, and muscle force and stiffness were recorded during the steady state. To investigate the actomyosin cycle, the biochemical species were changed (lowering [MgATP] and elevating [H2PO4-]) to populate different states in the actomyosin ATPase cycle. In solutions with 200 microM [MgATP], compared with physiological [MgATP], the slope of the plot of relative steady state muscle force vs. stiffness was decreased. At low [MgATP], cross-bridge dissociation from actin should be reduced, increasing the population of the last cross-bridge state before dissociation. These data imply that the last cross-bridge state before dissociation could be an attached low-force-producing or non-force-producing state. In solutions with 10 mM total Pi, compared to normal levels of MgATP, the maximally activated muscle force was reduced more than muscle stiffness, and the slope of the plot of relative steady state muscle force vs. stiffness was reduced. Assuming that in elevated Pi, Pi release from the cross-bridge is reversed, the state(s) before Pi release would be populated. These data are consistent with the conclusion that the cross-bridges are strongly bound to actin before Pi release. In addition, if Ca2+ activates the ATPase by allowing for the strong attachment of the myosin to actin in an A.M.ADP.Pi state, it could do so before Pi release. The calcium sensitivity of muscle force and stiffness in solutions with 4 mM [MgATP] was bracketed by that measured in solutions with 200 microM [MgATP], where muscle force and stiffness were more sensitive to calcium, and 10 mM total Pi, where muscle force and stiffness were less sensitive to calcium. The changes in calcium sensitivity were explained using a model in which force-producing and rigor cross-bridges can affect Ca2+ binding or promote the attachment of other cross-bridges to alter calcium sensitivity.

Effects of MCI-154 on Ca2+ activation of skinned human myocardium.

We investigated the effects of MCI-154, a new inotropic agent, on tension development in saponin-skinned human trabeculae carneae. The skinned fibers were activated by buffer solutions containing varying concentrations of Ca2+ (10(-8)-10(-4) M). In the sigmoidal tension vs. pCa (-log[Ca2+]M) relationship, the Ca2+ concentration required for half-maximal activation was shifted leftward in the presence of MCI-154. Furthermore, maximal Ca2+-activated tension development was increased in a concentration-dependent manner by MCI-154. Our results suggest that the inotropic effect of MCI-154 may be due, in part, to an increased sensitivity of the myofilaments to Ca2+ and enhancement of maximal Ca2+-activated tension development.

Stepwise shortening: evidence and implications.

The observation that sarcomeres shorten in steps has proved controversial. On the one hand, the phenomenon implies that the contractile process cannot be based on a molecular mechanism that behaves in a random manner: The fact that the steps and pauses characterize the kinetics of large volumes of tissue implies that the elements comprising such volumes must stop and pause synchronously. On the other hand, since current contractile models do not anticipate synchronized behavior, there has been considerable speculation that the phenomenon might not be a genuine feature of contraction, but an instrument-based artifact. We present here a review of observations made with four methods that have been brought to bear on the question. All four show discrete, synchronized contractile behavior. The observation of steps with multiple independent methods implies either that each technique harbors its own " gremlin " that generates spurious steps and pauses of a similar nature, or that the phenomenon is genuine. Finally, some consistent properties of the distribution of step size are considered with respect to possible molecular models.

PKC regulates agonist-induced force enhancement in single alpha-toxin-permeabilized vascular smooth muscle cells.

To determine whether activation of protein kinase C (PKC) is involved in the mechanism of agonist-induced force enhancement, force and stiffness were measured in both Ca(2+)- and agonist-stimulated contractions of single isolated alpha-toxin-permeabilized smooth muscle cells. PKC function was inhibited with the pseudosubstrate inhibitor (residues 19-31) of PKC (PKI). For Ca2+ activation, PKI did not change (P > 0.05) steady-state force or stiffness. However, for agonist activation at pCa 7 (n = 13), PKI depressed force by 28.7 +/- 4.5% (P < 0.05), in-phase stiffness by 35.4 +/- 4.0% (P < 0.05), and quadrature stiffness by 25.6 +/- 4.4% (P < 0.05), and for agonist activation at pCa 4 (n = 7), PKI depressed force by 25.8 +/- 2.9% (P < 0.05), in-phase stiffness by 35.6 +/- 5.6% (P < 0.05), and quadrature stiffness by 20.3 +/- 4.1% (P < 0.05). These results suggest that the agonist-induced force enhancement in alpha-toxin-permeabilized smooth muscle is due to the activation of PKC.

Mechanical changes after histamine activation of intact single vascular smooth muscle cells.

Although several different models have been proposed to explain force maintenance in vascular smooth muscle, the molecular mechanism responsible for this phase of contraction has yet to be elucidated. To investigate the molecular mechanism for force maintenance, force and stiffness were measured during (1 microM histamine) activation of single intact arterial smooth muscle cells. After histamine stimulation, the rise in quadrature stiffness preceded force (P < 0.05) and reached a steady state plateau before force (P < 0.05). These data suggest that the number of cycling cross-bridges increases during force activation and then remains constant during force maintenance. In-phase stiffness, on the other hand, continued to increase in amplitude after force had reached a steady state. The increase in the in-phase stiffness during force maintenance suggests that during force maintenance either a population of cross-bridges in an attached nonforce producing state develop or noncross-bridge force bearing structures are formed.

The smooth muscle cross-bridge cycle studied using sinusoidal length perturbations.

The mechanical characteristics of smooth muscle can be broadly defined as either phasic, or fast contracting, and tonic, or slow contracting (, Pharmacol. Rev. 20:197-272). To determine if differences in the cross-bridge cycle and/or distribution of the cross-bridge states could contribute to differences in the mechanical properties of smooth muscle, we determined force and stiffness as a function of frequency in Triton-permeabilized strips of rabbit portal vein (phasic) and aorta (tonic). Permeabilized muscle strips were mounted between a piezoelectric length driver and a piezoresistive force transducer. Muscle length was oscillated from 1 to 100 Hz, and the stiffness was determined as a function of frequency from the resulting force response. During calcium activation (pCa 4, 5 mM MgATP), force and stiffness increased to steady-state levels consistent with the attachment of actively cycling cross-bridges. In smooth muscle, because the cross-bridge states involved in force production have yet to be elucidated, the effects of elevation of inorganic phosphate (P(i)) and MgADP on steady-state force and stiffness were examined. When portal vein strips were transferred from activating solution (pCa 4, 5 mM MgATP) to activating solution with 12 mM P(i), force and stiffness decreased proportionally, suggesting that cross-bridge attachment is associated with P(i) release. For the aorta, elevating P(i) decreased force more than stiffness, suggesting the existence of an attached, low-force actin-myosin-ADP- P(i) state. When portal vein strips were transferred from activating solution (pCa 4, 5 mM MgATP) to activating solution with 5 mM MgADP, force remained relatively constant, while stiffness decreased approximately 50%. For the aorta, elevating MgADP decreased force and stiffness proportionally, suggesting for tonic smooth muscle that a significant portion of force production is associated with ADP release. These data suggest that in the portal vein, force is produced either concurrently with or after P(i) release but before MgADP release, whereas in aorta, MgADP release is associated with a portion of the cross-bridge powerstroke. These differences in cross-bridge properties could contribute to the mechanical differences in properties of phasic and tonic smooth muscle.

Thin filament regulation of force activation is not essential in single vascular smooth muscle cells.

To investigate thin filament regulation of force activation in smooth muscle, we recorded force and stiffness of alpha-toxin-permeabilized single smooth muscle cells. At pCa 9, the rigor state was characterized by high in-phase stiffness, low force, and low quadrature stiffness, suggesting that the attachment of rigor cross bridges does not depend on either Ca2+ or myosin light chain (MLC) phosphorylation, and cross bridges can enter a rigor state without producing force. At pCa 4, 20 microM ATP increased force, in-phase stiffness, and quadrature stiffness, while 20 microM CTP did not increase any of these parameters, suggesting that although MLC phosphorylation is not required for the formation of rigor cross bridges, MLC phosphorylation is required for detached cross bridges to attach to actin and undergo a force-producing isomerization. These results also suggest that for smooth muscle, force activation is regulated by myosin light-chain kinase. From rigor, 20 microM ATP (pCa 9) increased force and quadrature without changing in-phase stiffness. This force increase could be explained if in rigor solution both actomyosin (AM) and AM.ADP cross bridges exist (2, 32), and ATP-induced detachment of AM cross bridges is accompanied by AM.ADP cross bridges undergoing a force-producing isomerization in combination with cooperative cross-bridge reattachment (36). Thus results of our experiments suggest that thin filament-based regulation of force activation is not essential in smooth muscle, and a population of cross bridges must begin in an attached state for force to be produced in the absence of MLC phosphorylation.

Agonist activation modulates cross-bridge states in single vascular smooth muscle cells.

To determine cross-bridge properties during agonist-stimulated contractions, steady-state force and relative steady-state stiffness were recorded at rest (pCa 9) and during both full (pCa 4) and partial (pCa 7) Ca2+ activations of isolated single alpha-toxin permeabilized vascular smooth muscle cells. For pCa 4 and pCa 7, agonist (1 microM histamine) activation resulted in significant (P < 0.05) increases in both force and stiffness. The agonist-induced increase of steady-state force was significantly (P < 0.05) greater than that of stiffness; at pCa 4, there was a 48% increase for force vs. 17% for stiffness, and, at pCa 7, there was a 160% increase for force vs. 57% for stiffness. The increase in force and stiffness after agonist prestimulation implies that the number of attached cross bridges has increased. However, after agonist prestimulation, we found that the increase of force was greater (P < 0.05) than that of stiffness, resulting in a greater force at any given level of stiffness. Thus these data indicate that agonist activation, presumably via activation of a G protein, increases the relative force per attached cross bridge, possibly by modulating the kinetics of the actomyosin adenosinetriphosphatase to increase in the relative population of cross bridges in force-producing states [actinomyosin (AM) or AM.ADP].

Stimulus-specific changes in mechanical properties of vascular smooth muscle.

To determine whether the mechanical properties of vascular smooth muscle are stimulus specific, force, stiffness, and the unloaded shortening velocity (Vmax) were measured during contractions of aortic smooth muscle strips stimulated with phenylephrine or KCl. After activation, muscle force and stiffness rose to a steady-state plateau where they were maintained. In phenylephrine contractions, Vmax peaked during force development and then fell to a lower steady-state level during force maintenance, whereas in the KCl contractions, Vmax did not decline during sustained contractions. Stimulation with KCl, compared with phenylephrine, produced lower steady-state forces. One possible interpretation is that the muscle formed latch cross-bridges during phenylephrine contractions, but not during KCl depolarizations. The slope of the plot of relative muscle force vs. stiffness for phenylephrine contractions, compared with KCl depolarizations, was reduced. This may imply tht the relative force per attached latch crossbridge could be reduced.

Muscle contraction generates discrete sound bursts.

Isolated frog sartorius muscles were stimulated to shorten under lightly loaded conditions. A piezoelectric transducer was placed alongside the muscle to record sounds generated during contraction. Shortening was accompanied by the generation of a series of discrete sound bursts. The bursts were found to be moderately repeatable among successive contractions; 44% repeated from contraction to contraction. The duration of each sound burst was on the order of 400 mus, and the temperature dependence of the interval between successive bursts had a Q10 of approximately 2. Sound intensity was variable: average acoustic power ranged from 0.05-0.4 mW/g, or approximately 1% of the heat generated during contraction. The generation of discrete bursts of sound during contraction, rather than continuous sound, implies that contractile behavior may be discontinuous.

The frequency response of smooth muscle stiffness during Ca2+-activated contraction.

To investigate the mechanism of smooth muscle contraction, the frequency response of the muscle stiffness of single beta-escin permeabilized smooth muscle cells in the relaxed state was studied. Also, the response was continuously monitored for 3 min from the beginning of the exchange of relaxing solution to activating solution, and then at 5-min intervals for up to 20 min. The frequency response (30 Hz bandwidth, 0.33 Hz (or 0.2 Hz) resolution) was calculated from the Fourier-transformed force and length sampled during a 3-s (or 5-s) constant-amplitude length perturbation of increasing-frequency (1-32 Hz) sine waves. In the relaxed state, a large negative phase angle was observed, which suggests the existence of attached energy generating cross-bridges. As the activation progressed, the muscle stiffness and phase angle steadily increased; these increases gradually extended to higher frequencies, and reached a steady state by 100 s after activation or approximately 40 s after stiffness began to increase. The results suggest that a fixed distribution of cross-bridge states was reached after 40 s of Ca2+ activation and the cross-bridge cycling rate did not change during the period of force maintenance.

Protein kinase C increases force and slows relaxation in smooth muscle: evidence for regulation of the myosin light chain phosphatase.

To determine if activation of protein kinase C (PKC) participates in the molecular mechanism for agonist induced force enhancement, force was measured in single beta-escin skinned smooth muscle cells stimulated to contract with Ca2+, myosin light chain (MLC) kinase, PKC and microcystin-LR. The constituently active fragment of protein kinase C (PKM) increased both force and MLC phosphorylation in cells previously stimulated to contract at submaximal Ca2+. For cells contracted with saturating Ca2+, PKM stimulation did not increase either force or MLC phosphorylation. For contractions stimulated with both PKM and microcystin-LR, force rose significantly slower than contractions produced by Ca2+ or MLC kinase, suggesting that PKM increases force by a decrease in the rate of myosin dephosphorylation. Consistent with this hypothesis is the finding that the rate of force relaxation was slowed by PKM. This is the first direct demonstration that activation of PKC increases force in smooth muscle, and these results suggest that in smooth muscle, agonist induced activation of PKC plays a role in force regulation via an inhibition of myosin light chain phosphatase activity.

Determinants of the contractile properties in the embryonic chicken gizzard and aorta.

Smooth muscle is generally grouped into two classes of differing contractile properties. Tonic smooth muscles show slow rates of force activation and relaxation and slow speeds of shortening (V(max)) but force maintenance, whereas phasic smooth muscles show poor force maintenance but have fast V(max) and rapid rates of force activation and relaxation. We characterized the development of gizzard and aortic smooth muscle in embryonic chicks to identify the cellular determinants that define phasic (gizzard) and tonic (aortic) contractile properties. Early during development, tonic contractile properties are the default for both tissues. The gizzard develops phasic contractile properties between embryonic days (ED) 12 and 20, characterized primarily by rapid rates of force activation and relaxation compared with the aorta. The rapid rate of force activation correlates with expression of the acidic isoform of the 17-kDa essential myosin light chain (MLC(17a)). Previous data from in vitro motility assays (Rover AS, Frezon Y, and Trybus KM. J Muscle Res Cell Motil 18: 103-110, 1997) have postulated that myosin heavy chain (MHC) isoform expression is a determinant for V(max) in intact tissues. In the current study, differences in V(max) did not correlate with previously published differences in MHC or MLC(17a) isoforms. Rather, V(max) was increased with thiophosphorylation of the 20-kDa regulatory myosin light chain (MLC(20)) in the gizzard, suggesting that a significant internal load exists. Furthermore, V(max) in the gizzard increased during postnatal development without changes in MHC or MLC(17) isoforms. Although the rate of MLC(20) phosphorylation was similar at ED 20, the rate of MLC(20) dephosphorylation was significantly higher in the gizzard versus the aorta, correlating with expression of the M130 isoform of the myosin binding subunit in the myosin light chain phosphatase (MLCP) holoenzyme. These results indicate that unique MLCP and MLC(17) isoform expression marks the phasic contractile phenotype.

The maximal velocity of vascular smooth muscle shortening is independent of the expression of calponin.

In smooth muscle, the phosphorylation/dephosphorylation of the 20-kDa regulatory light chain of myosin (MLC20) is known to regulate actomyosin interaction and force. However, a thin filament based regulatory system for actomyosin interaction has been suggested to exist in parallel to MLC20 phosphorylation. Calponin is a thin filament associated protein that in vitro inhibits actomyosin interaction, and has been suggested to reduce maximal shortening velocity (vmax). Using antibodies to h1- and h2-calponin, we demonstrated that calponin was present in smooth muscle from Sprague Dawley (SD) rats, while calponin was not detectable in the smooth muscle from Wistar Kyoto (WKY) rats. vmax determined from the force vs. velocity relationship at maximal Ca2+ activation was not different for either the aorta or the portal vein of SD vs. WKY rats. These results suggest that physiological levels of calponin do not contribute to a thin filament-based secondary regulation to inhibit smooth muscle contraction.

Myosin-based cortical tension in Dictyostelium resolved into heavy and light chain-regulated components.

Cortical tension in most nonmuscle cells is due largely to force production by conventional myosin (myosin II) assembled into the cytoskeleton. Cytoskeletal contraction in smooth muscle and nonmuscle cells is influenced by the degree of myosin filament assembly, and by activation of myosin motor function via regulatory light chain phosphorylation. Recombinant Dictyostelium discoideum cell lines have been generated bearing altered myosin heavy chains, resulting in either constitutive motor function or constitutive assembly into the cytoskeleton. Analysis of these cells allowed stiffening responses to agonists, measured on single cells, to be resolved into an regulatory light chain-mediated component reflecting activation of motor function, and a myosin heavy chain phosphorylation-regulated component reflecting assembly of filaments into the cytoskeleton. These two components can account for all of the cortical stiffening response seen during tested in vivo contractile events.

Fast skeletal muscle troponin T increases the cooperativity of transgenic mouse cardiac muscle contraction.

1. To investigate the functional significance of different troponin T (TnT) isoforms in the Ca2+ activation of muscle contraction, transgenic mice have been constructed with a chicken fast skeletal muscle TnT transgene driven by a cardiac alpha-myosin heavy chain gene promoter. 2. Cardiac muscle-specific expression of the fast skeletal muscle TnT has been obtained with significant myofibril incorporation. Expression of the endogenous cardiac muscle thin filament regulatory proteins, such as troponin I and tropomyosin, was not altered in the transgenic mouse heart, providing an authentic system for the functional characterization of TnT isoforms. 3. Cardiac muscle contractility was analysed for the force vs. Ca2+ relationship in skinned ventricular trabeculae of transgenic mice in comparison with wild-type litter-mates. The results showed unchanged pCa50 values (5.1 +/- 0.04 and 5.1 +/- 0.1, respectively) but significantly steeper slopes (the Hill coefficient was 2.0 +/- 0.2 vs. 1.0 +/- 0.2, P < 0.05). 4. The results demonstrate that the structural and functional variation of different TnT isoforms may contribute to the difference in responsiveness and overall cooperativity of the thin filament-based Ca2+ regulation between cardiac and skeletal muscles.