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CARDIOVASCULAR
Department of Biological Sciences, Marquette University, Milwaukee, Wisconsin (T.J.E., J.M.); Department of Biological Sciences, Cardinal Stritch University, Milwaukee, Wisconsin (D.P.M.); Departments of Biochemistry and Pediatrics, Virginia Commonwealth University, School of Medicine, Richmond, Virginia (A.S.M., P.H.R.); and University of Vermont, Department of Molecular Physiology and Biophysics, Burlington, Vermont (A.S.R.)
Received June 13, 2006; accepted November 6, 2006.
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Abstract |
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80 µM). We found that blebbistatin potently (IC50 3 µM) inhibited the actomyosin ATPase activities of expressed "slow" [smooth muscle myosin IIA (SMA)] and "fast" [smooth muscle myosin IIB (SMB)] smooth muscle myosin II heavy-chain isoforms. Blebbistatin also inhibited the KCl-induced tonic contractions produced by rabbit femoral and renal arteries that express primarily SMA and the weaker tonic contraction produced by the saphenous artery that expresses primarily SMB, with an equivalent potency comparable with that identified for nonmuscle myosin IIA (IC50 5 µM). In femoral and saphenous arteries, blebbistatin had no effect on unloaded shortening velocity or the tonic increase in myosin light-chain phosphorylation produced by KCl but potently inhibited 
-escin permeabilized artery contracted with calcium at pCa 5, suggesting that cell signaling events upstream from KCl-induced activation of cross-bridges were unaffected by blebbistatin. It is noteworthy that KCl-induced contractions of chicken gizzard were less potently inhibited (IC50 20 µM). Adult femoral, renal, and saphenous arteries did not express significant levels of nonmuscle myosin. These data together indicate that blebbistatin is a potent inhibitor of smooth muscle myosin II, supporting the hypothesis that the force-bearing structure responsible for tonic force maintenance in adult mammalian vascular smooth muscle is the cross-bridge formed from the blebbistatin-dependent interaction between actin and smooth muscle myosin II.
; Kovacs et al., 2004; Limouze et al., 2004; Allingham et al., 2005; Shu et al., 2005). The levo-rotatory (–) form of blebbistatin was recently identified as an inhibitor of Dictyostelium myosin II, nonmuscle myosins IIA and IIB, scallop striated muscle myosin II, porcine cardiac muscle myosin II, and rabbit skeletal muscle myosin II, exhibiting IC50 values of between 0.5 and 5 µM for inhibition of myosin ATPase activity (Straight et al., 2003; Limouze et al., 2004). Blebbistatin acts by binding to the ADP-Pi complex at the S1 myosin head and inhibiting Pi release, thus preventing strong binding of cross-bridges and entry into the cross-bridge cycle (Kovacs et al., 2004). Interestingly, the dextrorotatory (+) form of blebbistatin serves as an excellent control because it exhibits no myosin ATPase inhibitory activity even at 100 µM (Straight et al., 2003; Limouze et al., 2004). (–)Blebbistatin does not inhibit unconventional myosins I, V, VII, and X, is a poor inhibitor of Acanthamoeba myosin II, and is at least 16-fold less potent at inhibiting purified avian smooth muscle myosin II (IC50 value for turkey gizzard smooth muscle myosin II 80 µM) (Limouze et al., 2004; Shu et al., 2005). However, there are two isoforms of smooth muscle myosin II that differ in the S1 myosin head region, SMA and SMB, and avian gizzard expresses only SMB (Rovner et al., 1997). Thus, it is conceivable that, although (–) blebbistatin does not inhibit gizzard SMB, it may inhibit SMA. Interestingly, the four amino acid residues identified as the blebbistatin binding site on nonmuscle myosin IIA and smooth muscle myosin II are identical and similar to that for skeletal muscle myosin II, prompting Rayment's group to state that "... the lack of substantial inhibition of smooth muscle myosin II is... more difficult to explain because all of the inner sphere residues are essentially identical to skeletal muscle" (Allingham et al., 2005). We propose that an alternative explanation for the poor inhibition of isolated turkey gizzard smooth muscle myosin II by blebbistatin may be the existence of interfering proteins tightly bound and unique to avian smooth muscle myosin II.
Upon stimulation, the smooth muscle of elastic arteries produces rapid increases in myosin light-chain phosphorylation, ATP consumption, maximum velocity of cross-bridge cycling, and isometric force (for review, see Kamm and Stull, 1985). When stimulated for a long duration, force can be maintained at high levels despite declines in these other parameters of cross-bridge activation. Molecular explanations for stimulus-induced tonic force maintenance include formation of slowly or noncycling actomyosin cross-bridges, termed latch bridges (Dillon et al., 1981; Hai and Murphy, 1988; Ratz et al., 1989; Khromov et al., 1998); recruitment of nonmuscle myosin II (Morano, 2003; Rhee et al., 2006); formation of caldesmon- or calponin-dependent actin-to-myosin cross-links (Sutherland and Walsh, 1989; Szymanski, 2004); and formation of cytoskeletal force-bearing structures (Rasmussen et al., 1987; Small, 1995). In short, maintenance of strong tonic force may be due to cross-links formed by slowly or noncycling cross-bridges consisting of smooth or nonmuscle myosin II (i.e., latch bridges) or, alternatively, to cross-links formed by other (ancillary) proteins. We recently determined that maintenance of strong tonic force by rabbit femoral artery is consistent with expression of the "slow" smooth muscle myosin II isoform, SMA, and formation of latch bridges (Han et al., 2006). In contrast, the saphenous artery, a major branch of the femoral artery, maintains tonic stress at approximately one half that of femoral artery, does not appear to enter a "latch state," and expresses primarily the "fast" myosin isoform, SMB (Han et al., 2006).
The primary goal of the present work was to determine whether blebbistatin inhibits expressed SMA and SMB isoforms of smooth muscle myosin II and contractions produced by tonic blood vessels that express primarily SMA (renal and femoral arteries) and SMB (saphenous artery). A secondary goal of this study was to use blebbistatin to test the hypothesis that tonic force produced by arterial smooth muscle is caused by actomyosin cross-bridges involving smooth muscle myosin II and not by ancillary proteins distinct from myosin II that cross-link actin and myosin or that form cross-linking cytoskeletal structures other than actomyosin cross-bridges.
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Materials and Methods |
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) and stored in cold (4°C) physiological saline solution (140 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4, 1.6 mM CaCl2, 1.2 mM NaHPO4, 2.0 mM MOPS adjusted to pH 7.4, 0.02 mM Na2-EDTA to chelate heavy metals, and 5.6 mM D-glucose made with high-purity, deionized water). The endothelium of arterial tissues cleaned by microdissection (Olympus SZX12; Tokyo, Japan) was removed by gently rubbing the intimal surface with a metal rod. The large elastic femoral and renal arteries of adult rabbit display comparable lumen diameters and wall thicknesses, contract in a tonic manner, express similar myosin isoforms, and display similar steady-state levels of activated myosin light-chain phosphorylation and velocity of muscle shortening (Ratz, 1993, 1995; Urban et al., 2003; Han et al., 2006; Porter et al., 2006; P. H. Ratz, unpublished observations). Muscles were stretched to their optimum length for muscle contraction using an abbreviated length-tension curve and KCl (physiological saline solution in which 110 mM KCl was substituted iso-osmotically for NaCl) as the stimulus (Herlihy and Murphy, 1973; Ratz and Murphy, 1987), and isometric force was measured as described previously (Ratz, 1993). Unloaded shortening velocity was measured using the slack step method (Eddinger et al., 1986) on intact tissue rings from the femoral and saphenous arteries. The tissues were activated multiple times with KCl with a slack step of variable size introduced at 10 min into the contraction.
Tissue Permeabilization. Tissues were permeabilized with -escin (40 µM) as described previously (Han et al., 2006). -Escin was dissolved in a "relaxing solution" contained 74.1 mM potassium methanesulphonate, 4.0 mM magnesium methanesulphonate, 4 mM Na2ATP, 4 mM EGTA, 5 mM creatine phosphate, 4 mM EGTA, and 30 mM PIPES, neutralized with 1 M KOH to pH 7.1 at 20°C.
Myosin Light-Chain Phosphorylation. Two-dimensional (isoelectric focusing/sodium dodecylsulfate) polyacrylamide gel electrophoresis was performed as described previously (Ratz, 1993; Urban et al., 2003) to measure the degree of myosin light-chain phosphorylation.
Actomyosin ATPase Assay. SMA and SMB heavy meromyosin molecules were expressed in the baculovirus system by swapping equivalent cassettes containing the 25-/50-kDa junction between chicken gizzard and rabbit uterine myosin heavy-chain cDNAs (Rovner et al., 1997). The SMA heavy meromyosin (1169 amino acids) contained a C-terminal hexa-histidine tag and was purified on a nickel-chelate column (Sigma HIS-Select), whereas the SMB heavy meromyosin isoform (1175 residues) was purified via a C-terminal FLAG tag (DYKDDDDK) (Trybus, 2000). ATPase assays as a function of actin were performed at various (–)blebbistatin concentrations using a colorimetric procedure (Trybus, 2000).
Myosin Heavy-Chain Isoform Expression. Myosin heavy-chain (MHC) isoform expression was analyzed as described previously (Han et al., 2006). Tissues were homogenized in 0.125 M Tris, 2% sodium dodecylsulfate (w/v), 20% glycerol, 0.1% bromphenol blue (w/v), and 20 mM dithiothreitol. MHCs were resolved on low cross-linking sodium dodecylsulfate gels (Giulian et al., 1983), and immunoblotting was performed as described previously (Eddinger and Wolf, 1993). Polyclonal antibodies to the SMB (plus seven-amino acid head insert isoform) smooth muscle MHC isoforms were used as described previously. Nonmuscle myosin IIA (NMA)- and NMB-specific nonmuscle-specific antibodies were obtained from Covance (Berkeley, CA). Western immunoblots were reacted as reported previously (Gaylinn et al., 1989).
Statistics. The null hypothesis was examined using Students' t test, and the null hypothesis was rejected at P < 0.05. For each study described, the n value was equal to the number of animals from which tissues were taken or the numbers of independent SMA and SMB preparations prepared.
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), and rabbit femoral and renal arteries (Ratz, 1993, 1995). For example, the control contraction in Fig. 1 displays a rapid phasic phase that peaks within 15 s and a tonic phase that develops within 2 min and is maintained indefinitely. The active isomer of blebbistatin, (–)blebbistatin, produced a dose-dependent inhibition of both the phasic and tonic phases of contractions produced by KCl in the tonic renal artery (Fig. 1A, RA) and femoral artery (data not shown). The inactive enantiomer, (+)blebbistatin, had no effect at 30 µM [Fig. 1A, compare control with 30 µM(+)Bleb]. (+)Blebbistatin is a reliable control for (–)blebbistatin (Shu et al., 2005) because it exhibits no myosin ATPase inhibitory activity even at 100 µM (Straight et al., 2003; Limouze et al., 2004). We found that (+)blebbistatin at 30 µM exerted no effect on actomyosin ATPase activity (n = 1). KCl-induced contractions of the saphenous artery, a muscular branch of the femoral artery that does not appear to enter the latch state and, therefore, produces a stronger phasic than tonic phase of contraction [Fig. 1B, 30 µM(+) Bleb; see Han et al., 2006)] were likewise inhibited by (–)blebbistatin [Fig. 1B, 30 µM (–)Bleb].
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-escin permeabilized femoral artery (Fig. 1, E and F), suggesting that this concentration of myosin II inhibitor was capable of acting directly on smooth muscle myosin II-based cross-bridges to cause inhibition of force. A high (–)blebbistatin concentration (30 µM) nearly abolished contraction of permeabilized femoral artery induced at pCa 6 [%pCa 6, (+)blebbistatin (control) = 85 ± 15, (–)blebbistatin = 10 ± 17, n = 3].
(–)Blebbistatin is reported to be highly selective for inhibition of myosin II, with a less potent inhibition of smooth muscle myosin II compared with other myosin II isoforms (Limouze et al., 2004). The less potent inhibition was based on actomyosin ATPase data from turkey gizzard myosin. However, because avian gizzard expresses only the SMB (fast) smooth muscle myosin isoform (for review, see Babu et al., 2000; Rovner et al., 1997), there exists the possibility that (–)blebbistatin more potently inhibits SMA compared with SMB. We therefore examined the relative potencies of (–)blebbistatin for inhibition of tonic force produced by KCl in mammalian arteries that express primarily the slow myosin II isoform, SMA, and the fast isoform, SMB (Han et al., 2006; see Fig. 4). It is noteworthy that (–)blebbistatin inhibited with the same high potency (4–5 µM) contractions induced by KCl in mammalian femoral (primarily SMA) and saphenous (primarily SMB) arteries (Fig. 2A). Likewise, tonic force induced by KCl in chicken carotid artery (primarily SMA) was inhibited with a high potency of 3 µM (Fig. 2B), and actomyosin ATPase activity measured for expressed SMA and SMB heavy meromyosins was inhibited with an equal potency of 3 µM (Fig. 2C). The peak contraction of chicken gizzard induced by KCl, however, was inhibited less well and displayed a lower potency of 20 µM (Fig. 2B). Moreover, tonic force induced by KCl in mammalian and chicken arteries was nearly abolished by 30 µM(–)blebbistatin (see Fig. 2, A and B, carotid), a concentration that reduced the peak contraction induced in chicken gizzard by only 75%.
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Tonic smooth muscle is characterized by prolonged maintenance of maximal force despite reductions in cross-bridge cycling rates. Two competing models explaining this behavior are the temporal formation of slowly or noncycling actomyosin cross-bridges (latch bridges) versus the temporal formation of cross-linking proteins ancillary to cross-bridges. The development of latch bridges or cross-links increases the internal load against which cycling cross-bridges must act, thereby reducing the cross-bridge cycling rate, measured in intact muscle as a reduction in the maximum rate of muscle shortening (Dillon et al., 1981). Thus, the selective inhibition of latch bridges or cross-links would be expected to abolish or reduce the internal load and increase the rate of muscle shortening. Likewise, the selective inhibition of cross-bridges but not latch bridges or cross-links responsible for the latch state would be expected to reduce the rate of muscle shortening. Three micromolar (–)blebbistatin significantly inhibited force in femoral and saphenous arteries by 20 to 35% but had no effect on unloaded shortening velocity (Vus) in either artery (Fig. 3). Thus, these data suggest that tonic force maintenance in femoral artery was due to actomyosin cross-bridges, not ancillary cross-links. Likewise, because rabbit saphenous artery does not appear to enter the latch state (Han et al., 2006), these data suggest that latch bridges were not selectively inhibited. Moreover, because the rate of cross-bridge cycling is thought to be regulated by cell signaling systems such as those controlling cell calcium (Ratz et al., 1989), the absence of inhibition of Vus supports our conclusion that blebbistatin did not alter cell signaling systems by a nonspecific effect.
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; Limouze et al., 2004; Ekman et al., 2005). We therefore examined the relative expression of smooth and nonmuscle myosin II in rabbit renal, femoral, and saphenous arteries and chicken carotid artery and gizzard. Compared with platelets that express high levels of NMA but low levels of smooth muscle myosin II, all rabbit arteries examined expressed levels of NMA that were barely detectable (and not quantifiable as a third band on Coomassie Blue-stained gels). NM was quantifiable for chicken carotid artery (not gizzard), but the amount expressed was very small, only 5.2 ± 1.5% of total myosin II present in this tissue (Fig. 4).
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Discussion |
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3–5 µM) comparable with the range (0.5–5 µM) published for inhibition of other myosin II isoforms (Limouze et al., 2004). The less potent inhibition of smooth muscle myosin II reported previously and based on actomyosin ATPase activity of purified turkey gizzard (Limouze et al., 2004) agrees with our finding that contraction of chicken gizzard was less potently inhibited by (–)blebbistatin. Thus, an additional protein may uniquely exist in avian gizzard that reduced the effectiveness of (–)blebbistatin.
A recent study employing smooth muscle myosin II knockout mice proposed the intriguing possibility that nonmuscle myosin II plays the prominent role in formation of the latch bridges responsible for force maintenance during contraction of bladder smooth muscle (Morano, 2003). In support of this hypothesis, contractions produced by bladder from newborn mice that express predominantly nonmuscle myosin II, but not adult mice that express predominantly smooth muscle myosin II, are inhibited by 10 µM(–)blebbistatin (Ekman et al., 2005). In contrast, Rhee et al. (2006) recently showed that tonic contractions of adult mouse aorta and bladder are inhibited by (–)blebbistatin, and based on data suggesting that blebbistatin is selective for inhibition of nonmuscle over smooth muscle myosin II (Limouze et al., 2004), these authors concluded that tonic contractions are dependent on nonmuscle myosin. Our data are the first to indicate that expressed smooth muscle myosin II is inhibited by (–)blebbistatin with a potency equivalent to that reported for other myosin II isoforms, including nonmuscle myosin II. Moreover, the adult rabbit arterial smooth muscle examined in the present study does not express significant amounts of nonmuscle myosin II (Han et al., 2006; Fig. 4), and we found that tonic contractions were potently inhibited by (–)blebbistatin. Finally, the amount of nonmuscle myosin II in adult animals is a very small percentage of the total myosin II content (<15% in swine carotid, <10% in rat carotid, and <20% in mouse carotid) (Gaylinn et al., 1989; Eddinger and Murphy, 1991; Eddinger and Wolf, 1993). Although these data cannot prove that nonmuscle myosin II does not contribute to force maintenance, it would seem unlikely that nonmuscle myosin II would be responsible for the maintenance of high stresses in smooth muscles when much larger amounts of smooth muscle myosin II is expressed. Thus, our data suggest that in adult arterial muscle, nonmuscle myosin need not be included in a model describing the regulation of tonic force maintenance.
Our data also support a model that excludes the contribution of proteins ancillary to myosin II as the cross-links directly responsible for bearing force during the tonic phase of contraction in arterial smooth muscle. Caldesmon and calponin can bind both actin and myosin, and an attractive proposal accounting for high force maintenance despite reductions in the rate of cross-bridge cycling is that these, or other proteins, form cross-links between actin and myosin (Sutherland and Walsh, 1989; Szymanski, 2004). In this model, cross-links between actin and myosin are proposed to form with time during muscle stimulation and to be of sufficient strength to permit force maintenance while impeding cross-bridge cycling. (–)Blebbistatin binds myosin in the 50-kDa cleft of the motor domain (Allingham et al., 2005), a location far removed from the sites at which caldesmon or calponin are thought to bind (Ikebe and Reardon, 1988; Hemric and Chalovich, 1990; Szymanski and Tao, 1997; Szymanski, 2004). Thus, (–)blebbistatin is unlikely to alter putative cross-links between actin and myosin formed by proteins such as caldesmon. However, because blebbistatin abolished tonic force produced by KCl, these results do not support the hypothesis that cross-links (other than actomyosin cross-bridges) are involved in maintenance of contraction in arterial smooth muscle. By the same reasoning, cytoskeletal structures distinct from actomyosin cross-bridges (Rasmussen et al., 1987; Small, 1995) and, therefore, unlikely to be inhibited by (–)blebbistatin, do not appear to play a structural role in force maintenance. However, our data do not rule out the possibility that ancillary proteins may regulate actomyosin cross-bridge kinetics (Haeberle, 1994; Obara et al., 1996) and play a role in relaxation (Albrecht et al., 1997; Malmqvist et al., 1997). Or, the ancillary regulatory protein may participate with actin and myosin in controlling force maintenance in mammalian smooth muscle, as in molluscan catch muscle in which twitching-induced force maintenance is regulated by myosin (Butler et al., 2006). In this scenario, a requirement for participation by the ancillary protein is regulation by myosin II. In conclusion, results from the present study show that (–)blebbistatin is a potent inhibitor of smooth muscle myosin II and support the hypothesis that proteins ancillary to actin and myosin probably do not play a structural role in the latch state.
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Acknowledgements |
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Footnotes |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: SMA, smooth muscle myosin IIA; SMB, smooth muscle myosin IIB; MHC, myosin heavy chain; NM, nonmuscle myosin; Vus, unloaded muscle shortening velocity.
Address correspondence to: Dr. Paul H. Ratz, Departments of Biochemistry and Pediatrics, Virginia Commonwealth University, School of Medicine, P.O. Box 980614, 1101 East Marshall Street, Richmond, VA 23298-0614. E-mail: phratz{at}vcu.edu
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