Caldesmon Inhibits Active Crossbridges in Unstimulated Vascular Smooth Muscle

An Antisense Oligodeoxynucleotide Approach

From the Department of Physiology, MCP/Hahnemann School of Medicine, Allegheny University of the Health Sciences, Philadelphia, Pa.

Correspondence to Robert S. Moreland, PhD, Department of Physiology, Allegheny University of the Health Sciences, 415 S 19th St, Philadelphia, PA 19146. E-mail morelandrs{at}aol.com

	Abstract

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Abstract—Caldesmon is a thin-filament–associated protein believed to be important in the regulation of smooth muscle contraction, although the precise mechanism is unknown. We used antisense oligodeoxynucleotides to produce intact swine carotid smooth muscle tissue deficient in h-caldesmon. Caldesmon content was decreased by 78% after 7 days in culture with antisense oligodeoxynucleotides but was unchanged in tissues in the presence of sense oligodeoxynucleotides or vehicle. Antisense oligodeoxynucleotides produced a significant decrease in the caldesmon/actin ratio, but no change was measured in the calponin/actin ratio, suggesting that the effect was specific to caldesmon and not other thin-filament–associated proteins. Basal and KCl-stimulated levels of myosin light chain phosphorylation were not different among tissues from all 3 groups. In contrast, h-caldesmon–deficient tissues produced 62% less KCl-induced force than controls. Unstimulated h-caldesmon–deficient smooth muscle tissues stretched and then released, redeveloped force, demonstrating active crossbridge cycling; strips containing normal h-caldesmon content did not redevelop force on release. We suggest that in resting vascular smooth muscle, active crossbridges are inhibited by caldesmon. Therefore, regulation of smooth muscle includes a thin-filament–based disinhibition component.

Key Words: organ culture • crossbridge cycling • myosin • phosphorylation • thin filament

	Introduction

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Stimulation of vascular smooth muscle produces an increase in [Ca²⁺]_i, which in turn initiates a cascade of events resulting in an increase in myosin light chain (MLC) phosphorylation, crossbridge cycling, and force development.^{1 2} However, with continued stimulation, force remains elevated while [Ca²⁺]_i, crossbridge cycling rates, and, in most cases, MLC phosphorylation decrease to suprabasal levels. This phenomenon of force maintenance supported by slowly cycling crossbridges was termed the latch state.³

Since this hallmark publication, numerous studies have clearly demonstrated dramatic dissociations among the levels of force, MLC phosphorylation, crossbridge cycling, and [Ca²⁺]_i.^{4 5 6 7} Therefore, on the basis of a large body of literature, we⁸ proposed the hypothesis that smooth muscle is regulated by 2 independent regulatory systems acting in parallel. One required MLC phosphorylation and was responsible for the rapid development of force, and the other was independent of MLC phosphorylation and was responsible for the maintenance or slow development of force.^{8 9} Although the precise mechanisms involved in this second regulatory system have not been identified yet, one likely candidate for mediating this regulation is the thin-filament–associated protein caldesmon.

Since its discovery by Sobue et al,¹⁰ h-caldesmon (high-molecular-weight isoform of caldesmon) has attracted significant attention as a potential thin-filament regulatory protein in smooth muscle. Immunohistochemical studies have shown that caldesmon colocalizes with tropomyosin on thin filaments in proximity to myosin thick filaments.¹¹ Numerous studies using in vitro reconstituted tropomyosin/actin/myosin complexes have shown that caldesmon binds actin at low- and high-affinity sites and that occupancy of the high-affinity site of actin is associated with a significant inhibition of actin-activated myosin ATPase activity.¹² Because caldesmon contains both actin (C-terminal) and myosin (N-terminal) binding domains, one intriguing hypothesis suggests that caldesmon may act by cross-linking actin and myosin, forming a force-bearing noncycling crossbridge.^{13 14} In support of this hypothesis was the finding that the binding of myosin subfragments to actin is enhanced in the presence of caldesmon.¹² These in vitro studies support a role for caldesmon in the regulation of smooth muscle contraction.

Evidence that caldesmon is involved in the regulation of contraction at the level of smooth muscle tissue is provided from several approaches. One approach has used a peptide analog to the C-terminal sequence of caldesmon, which contains essential residues for calmodulin and actin binding sites but does not inhibit myosin ATPase activity.¹⁵ Added to a permeabilized smooth muscle fiber, this peptide produced a sustained contraction believed to be due to competition with and displacement of endogenous caldesmon, resulting in disinhibition of myosin ATPase activity.¹⁶ Extraction of caldesmon from a permeabilized smooth muscle preparation increased Ca²⁺ sensitivity of force development.¹⁷ Conversely, addition of caldesmon to chicken gizzard permeabilized fibers inhibited contraction, and addition to contracted fibers induced relaxation.^{18 19 20}

As discussed above, to investigate the role of caldesmon in the regulation of contraction, investigators have used caldesmon fragments to compete with endogenous caldesmon in permeabilized smooth muscle cells,¹⁶ added excess exogenous caldesmon, and determined the effect on force and crossbridge cycling rates^{18 19} or extracted caldesmon from permeabilized preparations.¹⁷ It is often difficult to ascertain if a peptide did indeed displace the endogenous protein or if any exogenously added protein bound to normal physiological sites and if the extraction was specific for the protein of interest. Another approach to this question is to inhibit the synthesis of endogenous caldesmon by antisense oligodeoxynucleotides. This technique has been used to deplete caldesmon levels in nonmuscle cells²¹ but has not been attempted in muscle cells.

The goal of this study therefore was to develop a caldesmon-deficient strip of vascular smooth muscle by using organ culture of swine carotid artery and antisense oligodeoxynucleotide technology. Specifically, we were interested in determining if removal of endogenous caldesmon (1) abolished the maintenance of force, suggesting that caldesmon is an integral part of high force maintenance during conditions of low Ca²⁺ and MLC phosphorylation levels or (2) produced an active shortening muscle, suggesting that caldesmon inhibits active crossbridges in vascular smooth muscle.

	Materials and Methods

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Arterial Preparation
Swine carotid arteries were obtained at a local slaughterhouse and transported to the laboratory in ice-cold PSS of the following composition (mmol/L): NaCl 140, KCl 4.7, CaCl₂ 1.6, MgSO₄ 1.2, Na₂HPO₄ 1.2, Na₂EDTA 0.020, MOPS 2.0 (pH 7.4), and D-glucose 5.0. The arteries were cleaned of fat and connective tissue and dissected to produce thin (<100 µm) medial circumferential strips, as previously described.²²

Determination of Oligodeoxynucleotide Sequence
Oligodeoxynucleotides were synthesized and HPLC-purified commercially (CyberSyn). The oligodeoxynucleotides were phosphorothiolated,²³ and C-5 propyne-modified nucleotides (Glen Research) were used for thymidine [5-(1-propynyl)-2'-deoxyuridine] and cytidine [5-(1-propynyl)-2'-deoxycytidine]. These modified bases have been reported to increase the affinity of the oligodeoxynucleotides to mRNA and increase RNase H activity for the oligodeoxynucleotides/RNA heteroduplex.²⁴ The antisense oligodeoxynucleotide was targeted to a region initially identified in human caldesmon mRNA (GenBank accession No. m83216), with a preponderance of guanine and adenine bases and no secondary structure, as predicted by the computer program RNA Fold (Wisconsin Package v9.1, Genetics Computer Group). The homologous region was confirmed for porcine caldesmon mRNA by using reverse transcriptase–polymerase chain reaction (Promega) and primers F0530 and R1259 to amplify and sequence a 729-bp fragment from swine carotid artery RNA. The sequences of the oligodeoxynucleotide used in this study were antisense oligodeoxynucleotides=5'-UCCCUUGGUUUGGCUUCUC-3' and sense oligodeoxynucleotides=5'-GAGAAGCCAAACCAAGGGA-3', where underscored bases were C-5 propyne–modified nucleotides. For reverse transcriptase–polymerase chain reaction analysis and DNA sequencing, the following 2 primers were used: F0530=5'-GAATGATTGGAGAGATGC-3' and R1259=5'-TCATTTACCCATTTCCCTTC-3'.

Introduction of Oligodeoxynucleotides and Organ Culture
The carotid arterial muscle strips were reversibly permeabilized to initially introduce the oligodeoxynucleotides into the muscle cells. The protocol used was similar to that previously described by Morgan and Morgan²⁵ for introduction of aequorin and by Lesh et al²⁶ for introduction of oligodeoxynucleotides into smooth muscle cells. Several muscle strips (24 maximum) were incubated on ice with agitation for 90 minutes in 10 mL of a solution of the following composition: (mmol/L): EGTA 10, KCl 120, ATP 5, MgCl₂ 2, and HEPES 20 (pH 6.8 at 4°C). The strips were transferred to 10 mL of a solution composed of (mmol/L) KCl 120, ATP 5, MgCl₂ 2, and HEPES 20 (pH 6.8 at 4°C) and agitated on ice for an additional 30 minutes. The strips were divided into 3 groups of 6 to 8 each and incubated on ice with agitation for 90 minutes in 900 µL of the same solution as above, but in addition, the solution contained 10 µmol/L sense oligodeoxynucleotides, antisense oligodeoxynucleotides, or vehicle. At the end of the 90-minute incubation, the total [MgCl₂] of the solution was increased to 10 mmol/L and allowed to shake on ice for 30 minutes. The muscle strips were washed twice in 900 µL of a solution composed of (mmol/L) NaCl 140, KCl 5, MgCl₂ 10, D-glucose 5.6, and HEPES 2 (pH 6.8 at 22°C). The muscle strips were transferred to 900 µL of the same solution containing oligodeoxynucleotides or vehicle and allowed to shake at room temperature for 30 minutes. At the end of this incubation, the total [CaCl₂] was increased in a stepwise manner. The [CaCl₂] was increased at 15-minute intervals to 0.001, 0.01, 0.1, and finally to 1.6 mmol/L.

The muscle strips were mounted for isometric force recording as described below to verify viability and subjected to organ culture. Oligodeoxynucleotide-containing strips were mounted using 0.2-mm stainless-steel minuten pins in polymerized clear silicon elastomer (Dow Corning Corp) in 15-cm culture dishes. The pins were positioned so that the strips were stretched slightly when raised 2 to 3 mm above the surface of the elastomer. Each culture dish held up to 6 muscle strips in a volume of 3.5 mL of serum-free medium composed of DMEM/F-12 (1:1 ratio, BRL/Life Technologies), 100 U/mL penicillin, 100 µg/mL streptomycin, 250 ng/mL amphotericin B, 35 µg/mL L-ascorbic acid, 5 µg/mL transferrin, 3.25 ng/mL selenium, 2.85 µg/mL insulin, and 3.0 µmol/L sense or antisense oligodeoxynucleotides. The muscle strips were maintained at 37°C, 5% CO₂, and 100% relative humidity on an orbital shaker at 70 to 90 rpm. The medium was changed daily for an average of 7 days.

Quantitation of Caldesmon, Calponin, and Actin Content
The content of h-caldesmon was quantified in all cultured arterial strips. The strips were homogenized using glass-glass homogenizers in a solution containing 1.0% SDS, 10% glycerol, and 20 mmol/L DTT and subjected to 1-dimensional SDS-PAGE using a 7.5% acrylamide concentration. Electrophoresis was performed at 6 mA/gel overnight then increased to 40 mA/gel until the tracking dye reached the bottom of the electrophoretic gel. At least 3 concentrations of purified caldesmon (kindly provided by Dr Samuel K. Chacko, Dept of Pathobiology, University of Pennsylvania, School of Veterinary Medicine, Philadelphia, Pa, and Dr Joseph M. Chalovich, Dept of Biochemistry, East Carolina University, School of Medicine, Greenville, NC) were electrophoresed simultaneously on each gel containing tissue samples. After electrophoresis, the proteins were transferred to nitrocellulose membranes at 0.8 A for 4 hours at 4°C in a buffer composed of 25 mmol/L Tris (pH 8.3), 192 mmol/L glycine, and 20% methanol. The membranes were washed in a Tris-saline solution composed of 20 mmol/L Tris (pH 7.5) and 0.5 mol/L NaCl, which also contained 3% Carnation-brand nonfat dry milk. The membranes were incubated overnight in Tris-saline plus 1% nonfat dry milk containing anti-caldesmon antibody (1:150 000, clone No. hHCD, Sigma). The next morning, the membranes were washed twice in Tris-saline containing 0.05% Tween 20, once in Tris-saline, followed by 2 hours in a Tris-saline solution containing 1% nonfat dry milk plus anti-mouse IgG conjugated with horseradish peroxidase (1:5000, Amersham). Caldesmon, both purified and from homogenized tissues, was visualized by enhanced chemiluminescence (ECL, Amersham). Quantitation of caldesmon in terms of density of spot on film was performed with the use of a personal laser densitometer (Molecular Dynamics). A standard curve of densitometric values versus micrograms of purified caldesmon was obtained for each ECL-exposed membrane and used to convert the densitometric values obtained for tissue homogenate caldesmon levels from the same film to micrograms of caldesmon.

Aliquots of the homogenized tissue samples that were used for determination of micrograms of caldesmon were processed for total protein content by the method of Lowry et al,²⁷ using bovine serum albumin as a standard. Blank tubes and all standards were mixed in a solution of identical content as the tissue homogenates. The determination of total protein in each individual muscle strip allowed quantitation of caldesmon content in micrograms of caldesmon per milligram of tissue protein.

All cultured tissues were also processed for the determination of caldesmon/actin and actin/calponin ratios. Aliquots of the homogenized tissues were subjected to SDS-PAGE. The separating gel was composed of a 5-cm 12% acrylamide concentration portion and a 7-cm 7.5% acrylamide concentration portion. A standard stacking gel was used in this system. The use of 2 distinct acrylamide concentrations provided better resolution for caldesmon, actin, and calponin compared with either a single acrylamide concentration or a gradient gel. Three different quantities of each tissue homogenate were loaded onto the PAGE to ensure linearity of response for quantitation. Electrophoresis followed by transfer to nitrocellulose membranes was performed as described above for caldesmon alone. After protein transfer, the membranes were washed as described above and incubated overnight in a solution composed of Tris-saline containing 1% nonfat dry milk, anti–h-caldesmon antibody (1:150 000, clone number hHCD, Sigma), anti–smooth muscle actin antibody (1:1 500 000, clone No. 1A4, Sigma), and anti-calponin antibody (1:1 500 000, clone No. hCP, Sigma). The next morning the membranes were washed as described above, incubated for 2 hours in a Tris-saline solution containing 1% nonfat dry milk plus anti-mouse IgG conjugated with horseradish peroxidase (1:5000, Amersham), washed as described above, and visualized by ECL.

Quantitation of the ECL-exposed films was performed using the personal laser densitometer. Because 3 different quantities of tissue homogenate were loaded for each tissue sample, we could ensure that all values obtained were within the linear range of the technique. Densitometric data in arbitrary units was used to calculate the ratio of caldesmon to actin and the ratio of actin to calponin.

Quantitation of MLC Phosphorylation Levels
Basal and stimulated MLC phosphorylation levels were quantified as described previously by this laboratory.^{28 29} Briefly, cultured tissues were mounted for isometric force recording as described below and frozen at rest or after 3 minutes of exposure to 110 mmol/L KCl-PSS (equimolar substitution of KCl for NaCl) by immersion in an acetone–dry ice slurry containing 6% trichloroacetic acid. The frozen tissues were slowly thawed to room temperature, and the dry weight was obtained. The tissues were homogenized in glass-glass homogenizers and subjected to 2-dimensional gel electrophoresis and transferred to nitrocellulose membranes by methods previously described.²⁹ Proteins were visualized using AuroDye forte colloidal gold protein stain (Amersham) and quantified by laser scanning densitometry. The density of the spot corresponding to the phosphorylated MLC as a percentage of the total density of the spots corresponding to phosphorylated and unphosphorylated MLC was used as the value of MLC phosphorylation, expressed in mol P_i/mol MLC.

Quantitation of Isometric Force Development
Cultured tissues were mounted for measurement of isometric force between 2 clips, one of which was fixed to a micrometer for control of length, and the other was attached to a Grass FT.03 force transducer and Grass model 7D polygraph. Mounted strips were placed into water-jacketed baths containing PSS (composition described above) at 37°C and aerated with 100% O₂. The tissues were stretched to a length that produced approximately 8 grams of force and allowed to equilibrate for at least 90 minutes. After equilibration, the tissues were released to a length that produced approximately 1.5 grams of force. Based on previous studies using the swine carotid artery, this level of passive force produces maximal active force development.^{7 22 28}

Statistics
All values are presented as the mean±SEM for n determinations. N values denote individual tissues from different arteries. Statistical significance between means was determined using the Student t test for unpaired values. A value of P<0.05 was considered to be significant.

Materials
Amphotericin B, L-ascorbic acid, ATP, DTT, HEPES, insulin, penicillin, selenium, streptomycin, and transferrin were obtained from Sigma Chemical Co. Antibodies, ECL reagents, molecular biological supplies, and culture medium were obtained as listed in the text. All electrophoretic and blotting chemicals and materials were obtained from Bio-Rad. All other reagents were analytical grade or better and were obtained from Thomas Scientific.

	Results

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Caldesmon Content in Cultured Arterial Tissues
Thin medial strips of swine carotid artery were cultured as described in Materials and Methods in the presence of 3.0 µmol/L antisense or sense oligodeoxynucleotides to a region 5' to exon 3b in h-caldesmon. The strips were cultured for 7 days with fresh medium containing 3.0 µmol/L antisense or sense oligodeoxynucleotides daily. At the end of the 7 days, caldesmon content in the tissues was determined. Figure 1 shows the results of these experiments. Control values represent strips cultured in the presence of vehicle. Exposure of the strips to sense oligodeoxynucleotides had no significant effect on caldesmon content quantified as micrograms of caldesmon per milligram of tissue protein compared with control. The strips cultured in the presence of antisense oligodeoxynucleotides had significantly decreased levels of caldesmon. The caldesmon content decreased by 78% from control values of 0.159±0.034 to 0.035±0.005 µg caldesmon/mg tissue protein.

View larger version (27K): [in this window] [in a new window]   Figure 1. Caldesmon content in strips of swine carotid artery cultured for 7 days in the presence of vehicle, sense, or antisense oligodeoxynucleotides. Caldesmon content was quantified in the 3 groups of muscle strips as described in Materials and Methods. Caldesmon content of strips cultured in the presence of sense oligodeoxynucleotides was not significantly different than that measured in strips cultured in vehicle. In contrast, arterial strips cultured for 7 days in the presence of antisense oligodeoxynucleotides had significantly reduced levels of caldesmon. Values are mean±SE for 8 to 11 determinations. *P<0.05 vs control and sense oligodeoxynucleotides.

Caldesmon content with respect to the content of 2 other thin-filament proteins, actin and calponin, was also determined in the cultured strips. As shown in Figure 2, no difference in the ratio of caldesmon to actin was determined in tissues cultured in the presence of sense oligodeoxynucleotides compared with control tissues cultured for 7 days. Consistent with the decrease in actual caldesmon content shown in Figure 1, tissues cultured in the presence of antisense oligodeoxynucleotides exhibited a significant decrease in caldesmon-to-actin ratio. No culture condition, control, sense oligodeoxynucleotides, or antisense oligodeoxynucleotides altered calponin-to-actin ratios.

View larger version (22K): [in this window] [in a new window]   Figure 2. Ratio of caldesmon to actin and actin to calponin in strips of swine carotid artery cultured for 7 days in the presence of vehicle, sense, or antisense oligodeoxynucleotides. Strips were processed as described in Materials and Methods for quantitation of caldesmon-to-actin and actin-to-calponin ratios. There was no difference between caldesmon-to-actin ratio in strips cultured in the presence of sense oligodeoxynucleotides compared with control. The caldesmon-to-actin ratio in strips cultured in the presence of antisense oligodeoxynucleotides was significantly lower than either of the other 2 groups. There were no differences among the 3 groups of strips in terms of actin-to-calponin ratios. Values are mean±SE for 4 to 8 determinations. *P<0.05 vs control and sense oligodeoxynucleotides.

Force and MLC Phosphorylation Levels in Cultured Arterial Tissues
Arterial strips cultured for 7 days in the presence of vehicle, sense oligodeoxynucleotides, or antisense oligodeoxynucleotides were mounted for measurement of isometric force recording. Figure 3 shows the results of experiments to quantify the magnitude of stress (force/cross-sectional area) of these strips in response to membrane depolarization. Maximal steady-state stress in response to 110 mmol/L KCl-PSS was not significantly different between tissues cultured in the presence of vehicle or sense oligodeoxynucleotides. The values of developed stress in these 2 groups were also not significantly different from normal strips of the swine carotid artery not subjected to culture conditions.^{7 22 30 31} Moreover, isometric force measured in grams before culture in the presence of vehicle or sense oligodeoxynucleotides was within 5% of the grams of isometric force measured after culture (n=17, data not shown). In contrast, tissues cultured in the presence of antisense oligodeoxynucleotides exhibited a 62% decrease in KCl-PSS–induced stress. In all strips tested, any stress that developed was maintained for at least 20 minutes.

View larger version (28K): [in this window] [in a new window]   Figure 3. Stress development in strips of swine carotid artery cultured for 7 days in the presence of vehicle, sense, or antisense oligodeoxynucleotides. Strips were mounted for isometric force recording and stimulated with 110 mmol/L KCl-PSS. There were no significant differences in stress development between strips cultured in vehicle or sense oligodeoxynucleotides. Strips cultured in the presence of antisense oligodeoxynucleotides produced significantly less stress than either of the 2 other groups of strips. Values are mean±SE for 4 to 10 determinations. *P<0.05 vs control and sense oligodeoxynucleotides.

MLC phosphorylation levels were determined in resting and stimulated arterial strips after culture. The strips were mounted for isometric force recording and then frozen during either basal, resting conditions, or after 3 minutes of KCl-PSS stimulation. The results are shown in Figure 4. Basal levels of MLC phosphorylation were similar in tissues cultured in the presence of vehicle, sense, or antisense oligodeoxynucleotides. Stimulated levels of MLC phosphorylation were also similar in tissues from the 3 culturing conditions. This demonstrates that subjecting the arterial strips to culture neither increased basal MLC phosphorylation levels nor decreased stimulation-dependent cellular metabolic activity.

View larger version (27K): [in this window] [in a new window]   Figure 4. MLC phosphorylation levels in strips of swine carotid artery cultured for 7 days in the presence of vehicle, sense, or antisense oligodeoxynucleotides. Strips were mounted for isometric force recording and frozen for quantitation of MLC phosphorylation levels at rest and after 3 minutes of stimulation with 110 mmol/L KCl-PSS. There were no significant differences in levels of MLC phosphorylation among the 3 groups at rest or after membrane depolarization. Values are mean±SE for 3 to 5 determinations.

Force Redevelopment in Unstimulated Caldesmon-Deficient Arterial Tissues
We hypothesized that the decrease in KCl-stimulated stress development without a concomitant change in MLC phosphorylation in caldesmon-deficient tissues was due to either a decrease in stress-generating capacity or disinhibition of active crossbridges. We performed an experiment to test the latter possibility, and the results are shown in Figure 5. It is well known that if active muscle, striated or smooth, is released to a shorter length it will redevelop force. Conversely, if inactive muscle is released to a shorter length, it will not redevelop any force. There may be a small elastic recoil but no true force redevelopment. Arterial muscle strips that were cultured in vehicle, sense, and antisense oligodeoxynucleotide conditions were mounted for isometric force recording and allowed to equilibrate for at least 90 minutes. The tissues were then passively stretched to a length that produced approximately 8 grams of force and allowed to stress-relax until a stable force recording was obtained. The muscles were stretched to allow them to be shortened without stimulation or activation of any kind. The tissues were then rapidly released (shortened) by means of a micrometer attached to the rod and a clip holding one end of the muscle strip. As shown by the original recordings in Figure 5, the muscle strips cultured in antisense oligodeoxynucleotides and therefore caldesmon-deficient redeveloped force with a time course consistent with normal cycling crossbridges. The muscle strips that were cultured in vehicle or sense oligodeoxynucleotides and therefore had normal caldesmon content did not redevelop any force on release. This result supports our hypothesis that caldesmon inhibits active crossbridges in arterial smooth muscle.

View larger version (8K): [in this window] [in a new window]   Figure 5. Force redevelopment after a decrease in tissue length in strips of swine carotid artery cultured for 7 days in the presence of vehicle, sense, or antisense oligodeoxynucleotides. Unstimulated strips of carotid artery were stretched to a length that produced approximately 8 grams of force, allowed to stress-relax, and then released to a shorter length. Only strips cultured in the presence of caldesmon antisense oligodeoxynucleotides redeveloped force. Strips cultured in the presence of vehicle or sense oligodeoxynucleotides produced a small elastic recoil but no active redevelopment of force. Tracings of the original recordings representative of 5 experiments are shown.

	Discussion

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It is now well accepted that the regulation of a smooth muscle contraction cannot be completely accounted for by MLC phosphorylation acting as a simple Ca²⁺-dependent switch. Therefore, additional regulatory elements must be present in smooth muscle. Although numerous "second" regulatory systems have been postulated, none have been clearly demonstrated to play an important role in the generation or maintenance of a contraction. We believe the results of the present study may represent the first direct evidence to support an integral role for caldesmon in the regulation of smooth muscle contraction. Classically, smooth muscle has been believed to be a pure activation system in which unphosphorylated myosin is inactive and must be activated by phosphorylation of the 20-kDa MLC to allow actin and myosin interactions. Our results suggest that smooth muscle regulation, at least in part, involves disinhibition of active crossbridges. Removal of caldesmon in the arterial strips produced active crossbridge cycling as shown by force redevelopment in an unstimulated tissue; thus disinhibition of caldesmon allowed the expression of active crossbridges.

In addition, our results provide strong support for the utilization of antisense oligodeoxynucleotide and organ culture techniques in experiments designed to understand the mechanisms of contraction in multicellular preparations of smooth muscle. The caldesmon antisense oligodeoxynucleotide was apparently specific for caldesmon with little or no nonspecific actions of the phosphorothiolated compounds. We found no change in the calponin-to-actin ratio after 7 days in organ culture in the presence of the antisense oligodeoxynucleotide, although the ratio of caldesmon to actin and the absolute content of caldesmon were significantly depressed. Consistent with the lack of nonspecific effects was the finding that neither 7 days of culture alone (control) nor in the presence of a phosphorothiolated compound (sense oligodeoxynucleotide) had significant effects on any parameter measured. Lindqvist et al³² provided similar results in that serum-free organ culture of rat tail arterial segments did not alter contractility.

From our data suggesting the antisense oligodeoxynucleotides were specific for inhibition of caldesmon expression, we conclude that any change in the physiological function of the muscle tissue can be attributed to loss of caldesmon. Two fundamental changes in contractile function were found in the arterial strips deficient in caldesmon: stimulation-induced levels of stress were significantly depressed, and unstimulated tissues redeveloped force after a release to shorter lengths. The decrease in maximal stress development in the caldesmon-deficient tissues could be explained by at least 3 possible mechanisms. First, the loss of caldesmon may alter thin-filament structure such that actin and myosin interactions are reduced. Our results do not directly address this possibility. However, the finding that force redeveloped after a release suggests that any alteration in the interaction between actin and myosin was most likely not significant. Second, a decrease in stimulation induced MLC phosphorylation levels or an uncoupling of MLC phosphorylation levels from force. We could find no significant changes in either basal or stimulated levels of MLC phosphorylation. The basal values measured in our study are somewhat elevated compared with those reported previously by our group.^{7 22} However, the lower values for MLC phosphorylation were quantified using Coomassie blue–stained spots of 2-dimensional gels. If MLC phosphorylation levels are quantified by Auroforte dye staining of Western blots, basal levels are consistently higher and similar to those reported in the present study.^{29 33 34} We do not understand the reason for this difference in quantitation on the basis of staining method. We have no data concerning uncoupling of force from MLC phosphorylation, and therefore this remains a possibility. The salient point to be made is that there were no differences in basal or stimulated levels of MLC phosphorylation as a function of cell culture alone or in the presence of either sense or antisense oligodeoxynucleotide.

The third possibility to account for the decrease in stimulation-induced stress in the caldesmon-deficient tissues is that the tissues were partially activated at rest, before stimulation. Because smooth muscles have a finite level of maximal force that can be generated, the magnitude of stimulation-induced force would be limited by any initial activation. In support of this suggestion is the fact that unstimulated, caldesmon-deficient tissues redeveloped force after a release to a shorter length. Redevelopment of force after a release is indicative of active cycling crossbridges.³⁵ Therefore, as we have stated above, the tissues deficient in caldesmon allow active crossbridges to develop force in the absence of tissue activation. Tissues that were cultured in the presence of vehicle or sense oligodeoxynucleotides and contained normal caldesmon content did not redevelop force after a release, indicating that no active cycling crossbridges were expressed. We believe this represents the first demonstration of the presence of active crossbridges in unstimulated vascular smooth muscle tissue.

The finding that force did not redevelop in unstimulated tissues with a normal caldesmon content suggests that all of the resting force was passive. In contrast, because force redeveloped in the caldesmon-deficient tissues, this would suggest that most, if not all, of the resting force was active. Our protocol did not allow for a precise determination of the magnitude or even presence of passive force in the mounted and stretched caldesmon-deficient tissues. Therefore, it is possible that the caldesmon-deficient tissues were maintained at a shorter length relative to the length for optimal force development than the control tissues. This should not, however, alter our interpretations, because it has been previously demonstrated that the relationship among [Ca²⁺], force, and MLC phosphorylation is not altered at lengths shorter than optimal.^{36 37} We have no direct information concerning the effect of length on thin-filament regulation, but because the Ca²⁺-force relationship was not altered by decreasing length,³⁷ one may assume that both thick- and thin-filament regulatory mechanisms are also unchanged.

The development of force by active cycling crossbridges in unstimulated caldesmon-deficient tissue provides important insight into the regulation of vascular smooth muscle contraction. It can be concluded from our results that caldesmon inhibits crossbridge interactions in resting smooth muscle. What is not yet known is whether caldesmon inhibits a fraction of active unphosphorylated crossbridges and thus represents a novel regulatory pathway for contraction distinct from MLC phosphorylation, as we have previously suggested,^{7 8} or acts in concert with MLC phosphorylation and therefore inhibits active phosphorylated crossbridges.

Another thin-filament protein, calponin, was previously shown to inhibit slowly cycling unphosphorylated crossbridges in single cells of the toad stomach.³⁸ In that study, calponin was extracted chemically, and the physiologically relevant parameters measured were cell shortening and isometric force. Similar to the results of the present study, significant levels of cellular activation were measured in the absence of MLC phosphorylation and apparently the result of disinhibition of a thin-filament protein. Whether calponin and caldesmon function differently in mammalian compared with nonmammalian smooth muscle is not known, but in our opinion, it is not likely. It is possible that the 2 thin proteins function in concert to inhibit active crossbridges or that removal of either calponin or caldesmon alters thin-filament conformation in such a way as to allow active myosin heads to interact with actin and produce force.

	Acknowledgments

 
This study was supported, in part, by the National Heart, Lung, and Blood Institute grants HL 37956 and HL 46704 (Dr Moreland) and funds from the Emilie T. deHellebranth Endowment for Research. The authors would like to thank Mr William Jack for the timely and reliable delivery of the arteries used in this study. All swine carotid arteries were obtained from the Hatfield Meat Packing Plant, Hatfield, Pa.

	Footnotes

 
This manuscript was sent to Leslie A. Leinwand, Consulting Editor, for review by expert referees, editorial decision, and final disposition.

Received June 5, 1998; accepted July 28, 1998.

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