1Department of Physiology and Cell Biology, College of Medicine and Public Health, Ohio State University, Columbus 43210; 2Department of Molecular and Cellular Physiology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267; and 3Department of Pharmacology and 4Department of Molecular Physiology and Biophysics, University of Vermont, Burlington, Vermont 05405
Submitted 16 January 2004 ; accepted in final form 7 May 2004
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ABSTRACT |
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aorta; mesenteric vessels; calponin; caldesmon
SM-B is the predominant myosin in phasic smooth muscle tissues, including bladder and intestine. This isoform is abundant in neonatal aorta but is replaced by SM-A myosin in the adult (37). Small muscular arteries (e.g., femoral and saphenous arteries; Ref. 8) and arterioles (36) acquire and continue to express substantial amounts of the SM-B isoform in the adult. The expression pattern of SM-B myosin is altered in different pathophysiological conditions (9, 19, 31, 36). However, the functional significance of myosin isoform diversity in smooth muscle physiology is poorly understood.
To determine the role of SM-B myosin in vivo, we generated a mouse model in which the SM-B myosin isoform is replaced by SM-A myosin (3). This results in a significant decrease in velocity of muscle shortening in bladder and mesenteric resistance vessels (3). However, the absence of SM-B myosin does not affect survival, cause any overt smooth muscle pathology, or result in detectable changes in cardiovascular function (3). These results prompted us to hypothesize that loss of SM-B myosin is compensated for by changes in the expression of other thin filament regulatory proteins. To test this hypothesis, we expanded our studies of the contractile properties of aorta and mesenteric vessels to include force generation. We also examined the expression of myosin light chain isoforms and the thin filament proteins calponin and caldesmon, given their potential role in any adaptation that might have occurred. Our results suggest that altered expression of calponin and caldesmon may be involved in the adaptive response required to compensate for the loss of SM-B myosin.
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METHODS |
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Immunohistochemistry. Immunohistochemical staining of aorta and mesenteric vessels from wild-type (WT) and smb/ mice was performed exactly as described previously (23).
Western blot analysis. Muscle proteins were extracted as described previously (9). Briefly, bladder, aorta, or mesenteric vessel tissues from WT and SM-B-null mice were cleaned of connective tissues and powdered in liquid nitrogen. The powdered tissue was homogenized in a buffer containing (in mM) 50 Tris·Cl, pH 6.8, and 10 dithiothreitol with 1% SDS and 10% glycerol. The homogenate was boiled for 5 min, and the supernatant containing myofibrillar proteins was clarified by centrifugation at 13,000 g for 20 min. Protein estimation was done by a modified Lowry method and also quantitated by scanning densitometry of the Coomassie blue-stained gel.
Western blotting was carried out as described previously (3). Briefly, total proteins isolated from bladder, aorta, and mesenteric vessels were separated on 8% for SM1 and SM2, 10% for smooth muscle -actin, light chain-20 isoform (LC20), calponin, caldesmon, and extracellular signal-regulated kinase (ERK)1/2, or 15% for light chain-17 isoform (LC17) SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to nitrocellulose membrane. Membranes were probed with the following primary antibodies: rabbit polyclonal anti-SM1, rabbit polyclonal anti-SM2 (3), mouse monoclonal anti-calponin, mouse monoclonal anti-
-smooth muscle actin, and mouse monoclonal anti-LC20 (all from Sigma), mouse monoclonal anti-caldesmon (a kind gift from Dr. Michael P. Walsh, University of Calgary, Calgary, AB, Canada), mouse monoclonal anti-LC17 (a kind gift from Dr. Kathy Trybus, University of Vermont), and rabbit monoclonal anti-phospho-p44/42 mitogen-activated protein kinase (MAPK; New England Biolabs). Signals were detected by Super Signal West Dura substrate (Pierce), quantitated by densitometry, and analyzed with Image software (version 6.1, National Institutes of Health). Smooth muscle
-actin was used as an internal control to normalize the loading of blots.
Isometric force measurements in aorta. Aortic rings of 5-mm width were dissected with a dissecting microscope and cleaned of loose fat and connective tissue, and endothelium was removed. Rings were mounted between a fixed stainless steel post and a force transducer, the output of which was monitored with a computer-based data collection system (BioPac). The mounted aorta was equilibrated in physiological saline solution (PSS; in mM: 116 NaCl, 5.4 KCl, 1.2 MgSO4, 2.5 NaHCO3, 2.4 CaCl2, 0.5 NaH2PO4, and 11 glucose) and maintained at 37°C and aerated with 95% O2-5% CO2 throughout the course of the experiment. Force measurements were performed as described previously (1). The rings were mounted in a 15-ml organ bath and allowed to equilibrate at 37°C for 1 h, during which the tension was adjusted to 50 mN. Data were acquired with BioPac instrumentation and analyzed with the accompanying AcqKnowledge software. Phenylephrine (105-107 M) concentration-response curves were obtained in a cumulative fashion. Force was normalized to cross-sectional area, using tissue weight and circumference, by the approximation A = 2 x wet wt/circumference.
Force-velocity measurements. Segments of aorta prepared as described above were equilibrated in MOPS-PSS (in mM: 140 NaCl, 4.7 KCl, 1.2 NaH2PO4, 20 MOPS, 0.02 EDTA, 1.2 MgSO4, 2.5 CaCl2, and 11.1 glucose, pH 7.4) at 37°C for 15 min. MOPS-PSS was used in this case to avoid noise attributed to bubbling with 5% CO2, required for PSS. Passive tension was adjusted to a force that was calculated to be equivalent to 100 mmHg. This was previously shown to place the muscle at a length for optimal force development (1). Force-velocity relationships for each aorta were measured as described previously (28). Briefly, aortas were stimulated (50 mM KCl), and after steady-state isometric force was developed, ramp shortenings for a duration of 500 ms at 1-min intervals were imposed with an ergometer (Aurora Scientific). Six different velocities were imposed, and the force maintained at 200 ms was recorded and used for constructing force-velocity relations. Similar measurements were made under unstimulated conditions, and these forces were subtracted from the total tension to create the active force-velocity relations. These data were fit to the hyperbolic Hill equation with Origin software for each aorta. The fitted parameters, maximum isometric force, maximum velocity, and curvature parameter for each aorta were averaged and reported as means ± SE.
Mechanical measurements in mesenteric vessels. The mechanical properties of mesenteric vessel were measured as previously described (5). Briefly, mice were euthanized, and small mesenteric arteries (130200 µm internal diameter) were removed and placed in cold PSS (in mM: 119 NaCl, 4.7 KCl, 24 NaHCO3, 0.2 KH2PO4, 1.1 EDTA, 1.2 MgSO4, 1.6 CaCl2, and 10.6 glucose, pH 7.4). Arterial segments (2 mm in length) were then mounted in a resistance artery myograph and perfused with warmed (37°C), gassed (95% O2-5% CO2, pH 7.4) PSS. Arteries were then stretched to a preload of 300-mg force, which in preliminary experiments was determined to be the optimal preload for maximal force development. After 30-min equilibration, the arteries were contracted by exposure to bath solution containing 10 µM phenylephrine and 120 mM K+ (equimolar substitution of KCl for NaCl). The rate of isometric contraction velocity was calculated as the time required to reach half-maximal contraction, to confirm prior results (3). Vessel length and media thickness of the arterial segments were measured directly in the mounted vessels by phase-contrast microscopy (Nikon TMS microscope). Media stress was calculated as F/2L x M, where F is maximum active force (mg), L is segment length (mm), and M is media thickness (mm).
Statistical analysis. Data are presented as means ± SE or means ± SD of at least three independent experiments. Statistical analysis was performed with two-tailed ANOVA. Significance was assigned at P < 0.05.
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RESULTS |
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DISCUSSION |
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Although SM-B is present in the mesenteric vessels, the pattern of SM-B expression is heterogeneous, as we (23) and others (2, 10, 17) previously reported, becoming more homogeneous in smaller vessels. Individual cells may contain both head isoforms as heterodimers (10), which have unique mechanical properties (29), although the existence of heterodimers in vivo remains to be determined. This, together with the unique contractile properties of SM-B itself, provides one basis for the changes in contractile properties we observed in these vessels. Adult aorta contains SM-A myosin, although the presence of small amounts of SM-B has not been excluded. Nonetheless, both shortening velocity and force are affected in the smb/ aorta. It is possible that the absence of SM-B myosin in developing aorta, when it normally is present (37), leads to subsequent functional changes in the adult. Additionally, although the levels are too low to be easily detected, the presence of even very small amounts of SM-B conceivably could have functional consequences. In any case, the reduction in shortening velocity at the tissue level was predictable from studies of purified SM-B myosin (8, 20, 21, 30).
Our previous studies (3) demonstrated that loss of SM-B leads to a decrease in force generation in bladder. Force generation, however, is increased in mesenteric vessels and aorta from smb/ animals. Clearly, these two smooth muscle types have substantially different functions. Bladder is distinctly phasic, and aorta has a tonic function, whereas mesenteric vessels also have phasiclike activity critical for regulation of blood pressure and distribution (6).
The unexpected changes in force we observed caused us to look for alterations in other contractile and regulatory proteins that might accompany the loss of SM-B (32). Changes we found included changes in the LC17 ratio, the expression of calponin and caldesmon, and the level of activity of the ERK signaling pathway.
Increased expression of LC17b might contribute to the reduction in velocity (16, 24) we observed in aorta. Upregulation of calponin expression would do likewise, although the effect of this change on force remains controversial (13, 27, 34). Increased expression of calponin together with the activation of ERK in the SM-B-null mice suggests that calponin also may be involved in the activation of ERK signaling, again modifying contractile function (18, 22, 25). Decreased caldesmon expression might minimize the loss of velocity (35).
The question arises as to why changes have occurred in aorta, in which there is little or no SM-B in WT animals. The aforementioned argument that SM-B expression during development somehow is an essential determinant of the contractile properties of adult aorta is one possibility that could be explored further by studies of contractile properties in developing aorta in SM-B-knockout animals. However, we prefer the hypothesis that the changes in velocity and force in mesenteric resistance vessels that lack SM-B require an adaptive response in the rest of the vascular system to preserve cardiovascular integrity (3). The combination of changes in caldesmon and calponin in aorta suggests that the adaptive response may involve conversion of aortic smooth muscle to an even more toniclike muscle (11, 13, 14, 33) to match the changes in performance that have occurred more distally. One approach we are now using to examine this issue further is to determine the consequence of knocking out those regulatory proteins singly and in combination with SM-B knockout. In any case, our findings suggest that the altered expression of key regulatory proteins, such as calponin and caldesmon, and the activation of ERK signaling pathway are important physiological adjustments that must be made to compensate for the loss of SM-B myosin.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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2. Arafat HA, Kim GS, DiSanto ME, Wein AJ, and Chacko S. Heterogeneity of bladder myocytes in vitro: modulation of myosin expression. Tissue Cell 33: 219232, 2001.[CrossRef][ISI][Medline]
3. Babu GJ, Loukianov E, Loukianova T, Pyne GJ, Huke S, Osol G, Low RB, Paul RJ, and Periasamy M. Loss of SM-B myosin affects muscle shortening velocity and maximal force development. Nat Cell Biol 3: 10251029, 2001.[CrossRef][ISI][Medline]
4. Babu GJ, Warshaw DM, and Periasamy M. Smooth muscle myosin heavy chain isoforms and their role in muscle physiology. Microsc Res Tech 50: 532540, 2000.[CrossRef][ISI][Medline]
5. Brayden JE, Halpern W, and Brann LR. Biochemical and mechanical properties of resistance arteries from normotensive and hypertensive rats. Hypertension 5: 1725, 1983.[Abstract]
6. Burton AC. Relation of structure to function of the tissues of the wall of blood vessels. Physiol Rev 34: 619642, 1954.
7. D'Angelo G, Graceffa P, Wang CA, Wrangle J, and Adam LP. Mammal-specific, ERK-dependent, caldesmon phosphorylation in smooth muscle. Quantitation using novel anti-phosphopeptide antibodies. J Biol Chem 274: 3011530121, 1999.
8. DiSanto ME, Cox RH, Wang Z, and Chacko S. NH2-terminal-inserted myosin II heavy chain is expressed in smooth muscle of small muscular arteries. Am J Physiol Cell Physiol 272: C1532C1542, 1997.
9. DiSanto ME, Stein R, Chang S, Hypolite JA, Zheng Y, Zderic S, Wein AJ, and Chacko S. Alteration in expression of myosin isoforms in detrusor smooth muscle following bladder outlet obstruction. Am J Physiol Cell Physiol 285: C1397C1410, 2003.
10. Eddinger TJ and Meer DP. Single rabbit stomach smooth muscle cell myosin heavy chain SMB expression and shortening velocity. Am J Physiol Cell Physiol 280: C309C316, 2001.
11. Fujishige A, Takahashi K, and Tsuchiya T. Altered mechanical properties in smooth muscle of mice with a mutated calponin locus. Zoolog Sci 19: 167174, 2002.[ISI][Medline]
12. Gerthoffer WT, Yamboliev IA, Shearer M, Pohl J, Haynes R, Dang S, Sato K, and Sellers JR. Activation of MAP kinases and phosphorylation of caldesmon in canine colonic smooth muscle. J Physiol 495: 597609, 1996.[Abstract]
13. Haeberle JR. Calponin decreases the rate of cross-bridge cycling and increases maximum force production by smooth muscle myosin in an in vitro motility assay. J Biol Chem 269: 1242412431, 1994.
14. Haeberle JR, Hathaway DR, and Smith CL. Caldesmon content of mammalian smooth muscles. J Muscle Res Cell Motil 13: 8189, 1992.[ISI][Medline]
15. Hedges JC, Oxhorn BC, Carty M, Adam LP, Yamboliev IA, and Gerthoffer WT. Phosphorylation of caldesmon by ERK MAP kinases in smooth muscle. Am J Physiol Cell Physiol 278: C718C726, 2000.
16. Huang QQ, Fisher SA, and Brozovich FV. Forced expression of essential myosin light chain isoforms demonstrates their role in smooth muscle force production. J Biol Chem 274: 3509535098, 1999.
17. Hypolite JA, DiSanto ME, Zheng Y, Chang S, Wein AJ, and Chacko S. Regional variation in myosin isoforms and phosphorylation at the resting tone in urinary bladder smooth muscle. Am J Physiol Cell Physiol 280: C254C264, 2001.
18. Je HD, Gangopadhyay SS, Ashworth TD, and Morgan KG. Calponin is required for agonist-induced signal transductionevidence from an antisense approach in ferret smooth muscle. J Physiol 537: 567577, 2001.
19. Jones R, Steudel W, White S, Jacobson M, and Low R. Microvessel precursor smooth muscle cells express head-inserted smooth muscle myosin heavy chain (SM-B) isoform in hyperoxic pulmonary hypertension. Cell Tissue Res 295: 453465, 1999.[CrossRef][ISI][Medline]
20. Kelley CA, Takahashi M, Yu JH, and Adelstein RS. An insert of seven amino acids confers functional differences between smooth muscle myosins from the intestines and vasculature. J Biol Chem 268: 1284812854, 1993.
21. Lauzon AM, Tyska MJ, Rovner AS, Freyzon Y, Warshaw DM, and Trybus KM. A 7-amino-acid insert in the heavy chain nucleotide binding loop alters the kinetics of smooth muscle myosin in the laser trap. J Muscle Res Cell Motil 19: 825837, 1998.[CrossRef][ISI][Medline]
22. Leinweber BD, Leavis PC, Grabarek Z, Wang CL, and Morgan KG. Extracellular regulated kinase (ERK) interaction with actin and the calponin homology (CH) domain of actin-binding proteins. Biochem J 344: 117123, 1999.[CrossRef][ISI][Medline]
23. Low RB, Mitchell J, Woodcock-Mitchell J, Rovner AS, and White SL. Smooth-muscle myosin heavy-chain SM-B isoform expression in developing and adult rat lung. Am J Respir Cell Mol Biol 20: 651657, 1999.
24. Matthew JD, Khromov AS, Trybus KM, Somlyo AP, and Somlyo AV. Myosin essential light chain isoforms modulate the velocity of shortening propelled by nonphosphorylated cross-bridges. J Biol Chem 273: 3128931296, 1998.
25. Menice CB, Hulvershorn J, Adam LP, Wang CA, and Morgan KG. Calponin and mitogen-activated protein kinase signaling in differentiated vascular smooth muscle. J Biol Chem 272: 2515725161, 1997.
26. Morgan KG and Gangopadhyay SS. Cross-bridge regulation by thin filament-associated proteins. J Appl Physiol 91: 953962, 2001.
27. Obara K, Szymanski PT, Tao T, and Paul RJ. Effects of calponin on isometric force and shortening velocity in permeabilized taenia coli smooth muscle. Am J Physiol Cell Physiol 270: C481C487, 1996.
28. Paul RJ, Bowman PS, and Kolodney MS. Effects of microtubule disruption on force, velocity, stiffness and [Ca2+]i in porcine coronary arteries. Am J Physiol Heart Circ Physiol 279: H2493H2501, 2000.
29. Rovner AS, Fagnant PM, and Trybus KM. The two heads of smooth muscle myosin are enzymatically independent but mechanically interactive. J Biol Chem 278: 2693826945, 2003.
30. Rovner AS, Freyzon Y, and Trybus KM. An insert in the motor domain determines the functional properties of expressed smooth muscle myosin isoforms. J Muscle Res Cell Motil 18: 103110, 1997.[CrossRef][ISI][Medline]
31. Sjuve R, Haase H, Morano I, Uvelius B, and Arner A. Contraction kinetics and myosin isoform composition in smooth muscle from hypertrophied rat urinary bladder. J Cell Biochem 63: 8693, 1996.[CrossRef][ISI][Medline]
32. Somlyo AP. Myosin isoforms in smooth muscle: how may they affect function and structure? J Muscle Res Cell Motil 14: 557563, 1993.[ISI][Medline]
33. Takahashi K, Yoshimoto R, Fuchibe K, Fujishige A, Mitsui-Saito M, Hori M, Ozaki H, Yamamura H, Awata N, Taniguchi S, Katsuki M, Tsuchiya T, and Karaki H. Regulation of shortening velocity by calponin in intact contracting smooth muscles. Biochem Biophys Res Commun 279: 150157, 2000.[CrossRef][ISI][Medline]
34. Uyama Y, Imaizumi Y, Watanabe M, and Walsh MP. Inhibition by calponin of isometric force in demembranated vascular smooth muscle strips: the critical role of serine-175. Biochem J 319: 551558, 1996.[ISI][Medline]
35. Wang Z, Jiang H, Yang ZQ, and Chacko S. Both N-terminal myosin-binding and C-terminal actin-binding sites on smooth muscle caldesmon are required for caldesmon-mediated inhibition of actin filament velocity. Proc Natl Acad Sci USA 94: 1189911904, 1997.
36. Wetzel U, Lutsch G, Haase H, Ganten U, and Morano I. Expression of smooth muscle myosin heavy chain B in cardiac vessels of normotensive and hypertensive rats. Circ Res 83: 204209, 1998.
37. White SL, Zhou MY, Low RB, and Periasamy M. Myosin heavy chain isoform expression in rat smooth muscle development. Am J Physiol Cell Physiol 275: C581C589, 1998.