Forced expression of essential myosin light chain isoforms demonstrates their role in smooth muscle force production.

The molecular determinants of the contractile properties of smooth muscle are poorly understood, and have been suggested to be controlled by splice variant expression of the myosin heavy chain near the 25/50-kDa junction (Kelley, C. A., Takahashi, M., Yu, J. H., and Adelstein, R. S. (1993) J. Biol. Chem. 268, 12848-12854) as well as by differences in the expression of an acidic (MLC(17a)) and a basic (MLC(17b)) isoform of the 17-kDa essential myosin light chain (Nabeshima, Y., Nonomura, Y., and Fujii-Kuriyama, Y. (1987) J. Biol. Chem. 262, 106508-10612). To investigate the molecular mechanism that regulates the mechanical properties of smooth muscle, we determined the effect of forced expression of MLC(17a) and MLC(17b) on the rate of force activation during agonist-stimulated contractions of single cultured chicken embryonic aortic and gizzard smooth muscle cells. Forced expression of MLC(17a) in aortic smooth muscle cells increased (p < 0.05) the rate of force activation, forced expression of MLC(17b) in gizzard smooth muscle cells decreased (p < 0.05) the rate of force activation, while forced expression of the endogenous MLC(17) isoform had no effect on the rate of force activation. These results demonstrate that MLC(17) is a molecular determinant of the contractile properties of smooth muscle. MLC(17) could affect the contractile properties of smooth muscle by either changing the stiffness of the myosin lever arm or modulating the rate of a load-dependent step and/or transition in the actomyosin ATPase cycle.

To investigate the molecular mechanism that regulates the mechanical properties of smooth muscle, we determined the effect of forced expression of MLC 17a and MLC 17b on the rate of force activation during agonist-stimulated contractions of single cultured chicken embryonic aortic and gizzard smooth muscle cells. Forced expression of MLC 17a in aortic smooth muscle cells increased (p < 0.05) the rate of force activation, forced expression of MLC 17b in gizzard smooth muscle cells decreased (p < 0.05) the rate of force activation, while forced expression of the endogenous MLC 17 isoform had no effect on the rate of force activation. These results demonstrate that MLC 17 is a molecular determinant of the contractile properties of smooth muscle. MLC 17 could affect the contractile properties of smooth muscle by either changing the stiffness of the myosin lever arm or modulating the rate of a load-dependent step and/or transition in the actomyosin ATPase cycle.
The mechanical properties of smooth muscle are broadly classified as tonic and phasic (3)(4)(5). Tonic smooth muscle has slow rates of force activation, force relaxation, maximum velocity of muscle shortening (V max ), and actomyosin ATPase, while phasic smooth muscle has rapid rates of force activation, force relaxation, V max , and actomyosin ATPase (3)(4)(5). The molecular basis for tonic and phasic contractile properties is unknown, but has been suggested to be regulated by a variety of factors including splice variant isoforms of the myosin heavy chain (MHC) 1 at the 25/50-kDa junction (1) and/or splice variant isoforms of the essential myosin light chain (2) producing an acidic (MLC 17a ) and basic (MLC 17b ) isoform. The rate of force activation (6,7), V max (6,7), the velocity of actin movement in the in vitro motility assay (1,8), and ATPase activity (1,9) have been correlated with the expression of splice variant isoforms both of the MHC and MLC 17 . In this study, we tested the hypothesis that the mechanical properties of smooth muscle are regulated by splice variant expression of MLC 17 . We determined the effect of forced expression of MLC 17a and MLC 17b on the rate of force activation for agonist stimulated contractions of single cultured chicken embryonic aortic and gizzard smooth muscle cells (SMC).

EXPERIMENTAL PROCEDURES
Aorta and gizzard cells were isolated from primary cultures of ED 13-15 chicken embryos, as described previously (10). Briefly, after dissecting these tissues and removing the surrounding connective and epithelial tissues, gizzard or aorta were minced into fine pieces and resuspended in growth media (Dulbecco's modified Eagle's medium/ Ham's F-12, 1:1 mixture supplemented with 10% fetal calf serum and 50 units/ml penicillin, 50 g/ml streptomycin, Life Technologies, Inc.). The fine pieces in suspension medium were pipetted into culture dishes, and sedimented larger fragments were re-minced. This procedure was repeated for 2-4 cycles and all tissue fragments were incubated at 37°C with 5% CO 2. The culture media was changed the next day to remove the unattached fragments. Cells grew out from tissue explants in 2-3 days and they were passed and expanded after 0.05% trypsin digestion. Cells between the first and third passages were used for transfection and mechanical studies.
The cDNAs encoding splice variants of the chicken 17-kDa smooth muscle essential myosin light chain (MLC 17 ) were obtained by reverse transcription-polymerase chain reaction (RT-PCR) from the adult chicken bladder total RNA extracted by Trizol reagent (Life Technologies, Inc.). The primers were synthesized according to the MLC 17 cDNA sequence from position ϩ44 to 66 and ϩ528 to 552, this includes 5Ј-noncoding, all of the translated product and 3Ј-noncoding regions (2). Briefly, 2.5 g of total RNA was reverse-transcribed with the 3Ј-MLC 17 primer by Superscript II reverse transcriptase (Life Technologies, Inc.) at 42°C for 2 h. 1/10 of the RT product was subject to PCR to amplify both isoforms of MLC 17 using 5Ј-and 3Ј-MLC 17 primers with PCR programmed at 95°C denaturing for 1 min, 55°C annealing for 1 min, and 72°C elongation for 1 min for a total of 35 cycles. The MLC 17a and MLC 17b cDNAs produced were isolated by 2% agrose gel electrophoresis in TBE buffer, purified by QIAquick gel extraction kit (QIAGEN), and ligated to the pTracer-SV40Љ mammalian expression vector (Invitrogen) individually using standard molecular cloning techniques (11). The expression constructs encoding MLC 17a or MLC 17b were screened by PCR using 5Ј-and 3Ј-MLC 17 primers and confirmed by DNA sequencing.
The MLC 17a or MLC 17b expression plasmids were prepared by an endotoxin-removal column (QIAGEN) according to the manufacturer's protocol. Transfections were performed by electroporation with the GenePulser II RF Module electroporator (Bio-Rad). The protocol was optimized for transfection efficiency. Primary cultured embryonic chicken aorta or gizzard SMC were collected and washed once in an electroporation buffer (27 mM sodium phosphate, 150 mM sucrose, pH 7.5). After centrifugation (RTH-750 rotor (Sorvall), 1,000 rpm at 4°C for 4 min), approximately 5 ϫ 10 4 -2 ϫ 10 5 cells were resuspended in 100 l of the electroporation buffer and mixed with 2 g of either MLC 17a or MLC 17b expression plasmid DNA, or the buffer as a control. Electroporation was performed at the following conditions: total voltage, 180; 100% modulation; frequency, 40 kHz; bursts, 10 at a duration of 3 ms * This work was supported by a grant-in-aid from the American Heart Association (to F. V. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
and an interval of 1 s in a cuvette with 0.2-cm gap. The cells were placed on the Matrigel TM (Becton-Dickinson) coated culture dishes, or for mechanical studies, microcoverslips (10) in low serum (0.5% fetal calf serum) medium after electroporation.
For analysis of steady state levels of mRNA, the population of transfection positive SMC was sorted through a flow cytometer (Elite ESP, Coulter, Miami, FL) and SMC with green fluorescent protein (GFP) fluorescence were collected. Transfection efficiency ranged from 15 to 60%.
The individual cultured cells that carried the MLC 17 expression constructs after transfection were identified by the expression of GFP. The vector expressed MLC 17 , driven by an SV40 promoter and GFP as a separate transcript driven by the cytomegalovirus promoter (pTracer-SV40, Invitrogen TM ). GFP expression was visualized using a fluorescence microscope (Nikon) with a fluorescein isothiocyanate filter.
Transcription of the exogenous MLC 17a and MLC 17b were confirmed by RT-PCR. Total RNA extracted from the cultured cells was subjected to three RT-PCR reactions at the same time. One of them applied the 5Јand 3Ј-MLC 17 primers specific for MLC 17 (10), one applied the primers specific for MHC (10) and the last used 5Ј-MLC 17 and a 3Ј-bovine growth hormone gene primer which was synthesized according to the sequence of bovine growth hormone gene polyadenylation signal in the vector DNA. This primer flanks the 3Ј end of the transgene insert and will specifically anneal with the transcripts of exogenous MLC 17 transcript. The experiments using the 3Ј-bovine growth hormone gene primer demonstrated that only cells transfected with the recombinant plasmid show a specific RNA transcript, indicating the expression of exogenous MLC 17 (data not shown).
For mechanical measurements, single SMC were attached to a force transducer (series 400A, Aurora Scientific Inc., Aurora, Ontario, Canada) with glue (Polycel, Maklanberg-Duncan, Oklahoma City, OK) as described previously (12). SMCs were grown on Matrigel TM -coated coverslips (13) in low serum culture dishes for 24 -48 h after transfection and were then transferred to a chamber on the movable stage of a Nikon inverted florescent microscope. The SMC were incubated in physiological saline solution, and healthy single SMC either with GFP or without GFP expression were selected for mechanical studies. SMC were activated with 10 M phenylephrine. The rate of force activation was computed from the t1 ⁄2 of the force response (time to reach 50% of the maximum force), where rate ϭ (t1 ⁄2 ) Ϫ1 .

RESULTS AND DISCUSSION
Cultured aortic and gizzard embryonic SMC were transfected with GFP and MLC 17 . A bicistronic expression vector was used in which a cytomegalovirus promoter drove GFP expression and an SV40 promoter drove MLC 17 isoform expression. SMC carrying the transgene were detected by fluorescence. GFP positive and negative cells are easily identified, and since cells for the mechanical studies were selected based on the presence of a marker protein, GFP, transfection efficiency was not a factor.
The effect of forced expression of one MLC 17 isoform on the ratio of MLC 17a /MLC 17b was determined (Fig. 1). Culturing both aortic and gizzard SMC on Matrigel TM maintains the ratio of MLC 17a /MLC 17b observed in tissue (Table I and (Table  I). Transfection of aortic cells with MLC 17a increased the level of message for MLC 17a from 15 to 80% of all transcripts (Table  I). Similarly, transfection of gizzard cells with MLC 17b increased the level of message for MLC 17b from 60% of all transcripts to almost exclusively MLC 17b (Table I). These results suggest that transfection of cultured SMC with MLC 17 results in the overexpression of MLC 17b and/or MLC 17a . In addition, overexpression of MLC 17 did not effect MHC splicing (Fig. 1). Both ED 14 cultured aortic and gizzard SMC express exclusively the splice-out isoform of MHC and the forced expression of MLC 17 did not effect the expression of the MHC.
The rate of force activation of GFP positive SMC was compared with non-transfected control SMC. For the nontransfected controls, phenylephrine (10 M) produced contractions of both single cultured ED 13-15 aortic and gizzard SMC. Aortic SMC displayed a slow rate of force activation compared with single cultured ED 13-15 gizzard SMC (Fig. 2, Table II). These results are in agreement with others (10) and suggest that when cultured on Matrigel TM , embryonic aortic and gizzard SMC retain their native tonic and phasic contractile properties, respectively. For the aortic SMC, forced expression of MLC 17a resulted in an increase in the rate of force activation (p Ͻ 0.05) to that observed in the untransfected gizzard cells while overexpression of MLC 17b did not change (p Ͼ 0.05) the rate of force activation (Fig. 2, Table II). Similarly for gizzard SMC, forced expression of MLC 17a did not change the rate of force activation (p Ͼ 0.05), while overexpression of MLC 17b slowed the rate of force activation (p Ͻ 0.05) to that observed in the untransfected aortic SMC (Fig. 2, Table II). Transfection did not influence the maximal steady state force (Table II), and transfection of both embryonic aortic and gizzard SMC with the vector alone, expressing GFP, neither effected the rate of force activation nor the maximal steady state force (data not shown). Similar to contractions of intact tissues (14), the agonist stimulated contractions of both cultured embryonic aortic and gizzard SMC were accompanied by transient increases in MLC 20 phosphorylation (data not shown).
The mechanical properties of smooth muscle are thought to arise from differences in the expression of the contractile proteins (3)(4)(5). For smooth muscle, the distribution of both MHC  a MLC 17a and MLC 17b are expressed as the percentage of total transcripts (mean Ϯ S.E., n ϭ 5-12 for each experiment). The ratio of MLC 17a and MLC 17b bands was determined using NIH image 159 software as previously described (10). SMC were grown on Matrigel™ and the transfected cells separated as described under "Experimental Procedures." and MLC 17 varies for both the same tissues across species and among tissues within a species (6). In general, tonic tissues express predominantly the splice-out isoform of MHC and MLC 17b , while phasic tissues express predominantly the splice-in isoform of MHC and MLC 17a . During development, the mechanical properties of the aorta and gizzard correlate with the changes in contractile protein expression. Early during embryogenesis both aorta and gizzard predominantly exclude exons for expression of MHC and MLC 17b (10) and both tissues display tonic contractile properties (15). The gizzard develops phasic contractile properties at ED 12-14, coincident with the change in contractile protein expression to predominantly splice-in MHC and MLC 17b (10,15). However, a correlation between changes in protein expression and changes in mechanical properties could arise from a variety of factors unrelated to differences in the expression of MHC and/or MLC 17 .
In the present study, neither expression of an irrelevant cytosolic protein (GFP) nor the endogenous isoform of MLC 17 in SMC effected the rate of force activation. Overexpression of MLC 17 isoforms also did not change the maximum force of contraction. Thus, the differences in the rate of force activation were due to neither transfection alone nor an unphysiologic level of protein expression. The vector expresses two independent proteins, GFP and MLC 17 , which rules out the possibility that the results were due only to an affect of a fusion protein.
The differences in the rate of force activation of cultured embryonic SMC are also unlikely to be the result of differences in splice variant expression of MHC near the 25/50-kDa junction (1) since MHC splicing was not effected by the forced expression of MLC 17 in our study. The results of the present study of single cultured embryonic SMC demonstrate that the expression of splice-variant isoforms of MLC 17 is a determinant of the rate of force activation and MLC 17 is a molecular marker for the contractile phenotype of smooth muscle.
Our results are in general agreement with those of others (9) who have demonstrated that an increase in both the V max and K m for the actin-activated ATPase of smooth muscle myosin correlates with an increase in the ratio of MLC 17a /MLC 17b . However, a correlation of the ATPase and the ratio of MLC 17a / MLC 17b is not universal (1,8), and changes in the rate of force activation may not be related to changes in the V max and K m of the ATPase. Our results in single cultured embryonic SMC are similar to those reported for endothelin-1-treated cultured SMC (10) and permeabilized trifluoperazine-treated smooth muscle strips (7). Chronic endothelin-1 treatment of cultured gizzard smooth muscle cells slowed the rate of force activation and increased the expression of MLC 17b (10). However, for the endothelin-1-treated cultured SMC, a change in the rate of force activation could have been due to a variety of factors other than a change in MLC 17 expression. For trifluoperazinetreated permeabilized strips of smooth muscle (7), the contractions were unaccompanied by either a change in Ca 2ϩ or MLC 20 phosphorylation, and occurred after the exchange of MLC 17 isoforms, upon transfer of the preparation from a solution containing trifluoperazine to a relaxing solution. This result raises the possibility that the differences in force activation and muscle V max could be due to the trifluoperazine treatment alone and unrelated to the regulation of the mechanical properties of intact smooth muscle.
An insert at the 25/50-kDa junction of the MHC (1) has also been suggested to determine the functional properties of smooth muscle (1,8,16). These investigators (1,8) demonstrated that both the ATPase activity and the velocity of actin translation in the in vitro motility assay was higher for the splice-in isoform of MHC with either MLC 17a or MLC 17b than the splice-in isoform of MHC with either isoform of MLC 17 . This difference in motility could be due to an increase, by a factor of 2, in the attachment time for actin and myosin for the splice-out MHC compared with the splice-in MHC (16). Similarly, the velocity of actin translation by skeletal muscle MHC from in vitro motility assays was primarily determined by the MHC isoform and the essential MLC isoform played a minor, modulatory role (17  of actin movement in the in vitro motility assay measure the properties of unloaded myosin, and these parameters are thought to correlate with muscle V max . However, the effect of elevating ADP on the velocity of actin movement by myosin in the in vitro motility assay (18) and skinned fiber V max (19) are opposite. The results of these studies (18,19) suggest that a change in the ATPase activity and velocity of actin translation during in vitro motility assays do not always correlate with a change in the mechanical properties of muscle. It is also possible that the mechanical properties of smooth muscle could be determined by a number of factors; i.e. splicing at the 25/50-kDa junction of MHC could regulate the ability of the unloaded cross-bridge to hydrolyze ATP and thus could control V max , while MLC 17 could regulate the properties of loaded crossbridges, and thus the rate of force activation.
The results of the present study of cultured embryonic SMC clearly demonstrate that, in the absence of any other confounding variables, a change in the ratio of MLC 17a /MLC 17b expression alone changes the contractile properties of the SMC. Our data suggests that MLC 17a may have a dominant effect; above a threshold level of MLC 17a , the rate of force activation could be fast while below the threshold, force activation might be slow. Force activation was rapid for control gizzard cells and RT-PCR of gizzard cells showed 40% MLC 17a and 60% MLC 17b , and increasing MLC 17a to 75% did not influence the rate of force activation while decreasing the expression of MLC 17b to 2% slowed the rate of force activation. Similarly in the aorta, increasing the expression of MLC 17a from 15 to 80% increased the rate of force activation. Alternatively above a threshold level of MLC 17a expression, there may be a dose-response relationship between the relative content of MLC 17a and a further increase in the speed of the tissue. Our experiments were not designed to investigate this question, but rather only whether changing the relative expression of MLC 17a /MLC 17b influences the rate of force activation.
Our results clearly demonstrate that splice variant expression of MLC 17 is a determinant of the rate of force activation and that MLC 17 is a molecular marker for the tonic and phasic contractile phenotype. The ability of MLC 17 to modulate the rate of force activation could be due to a change either in the stiffness of the myosin lever arm (7) or a change in the rate of a load-dependent transition within the actomyosin ATPase.