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Forced Expression of Essential Myosin Light Chain Isoforms Demonstrates Their Role in Smooth Muscle Force Production

1999; Elsevier BV; Volume: 274; Issue: 49 Linguagem: Inglês

10.1074/jbc.274.49.35095

1083-351X

Qi-Quan Huang, Steven A. Fisher, Frank V. Brozovich,

Muscle Physiology and Disorders

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 (MLC17a) and a basic (MLC17b) 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 MLC17a and MLC17b on the rate of force activation during agonist-stimulated contractions of single cultured chicken embryonic aortic and gizzard smooth muscle cells. Forced expression of MLC17a in aortic smooth muscle cells increased (p < 0.05) the rate of force activation, forced expression of MLC17b in gizzard smooth muscle cells decreased (p < 0.05) the rate of force activation, while forced expression of the endogenous MLC17 isoform had no effect on the rate of force activation. These results demonstrate that MLC17 is a molecular determinant of the contractile properties of smooth muscle. MLC17 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 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 (MLC17a) and a basic (MLC17b) 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 MLC17a and MLC17b on the rate of force activation during agonist-stimulated contractions of single cultured chicken embryonic aortic and gizzard smooth muscle cells. Forced expression of MLC17a in aortic smooth muscle cells increased (p < 0.05) the rate of force activation, forced expression of MLC17b in gizzard smooth muscle cells decreased (p < 0.05) the rate of force activation, while forced expression of the endogenous MLC17 isoform had no effect on the rate of force activation. These results demonstrate that MLC17 is a molecular determinant of the contractile properties of smooth muscle. MLC17 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. myosin heavy chain smooth muscle cells myosin light chain embryonic day reverse transcriptase-polymerase chain reaction green fluorescent protein The mechanical properties of smooth muscle are broadly classified as tonic and phasic (3Hartshorne D.J. Johnson L.R. Physiology of the Gastrointestinal Tract. Raven Press, New York1987: 432-482Google Scholar, 4Somlyo A.P. J. Muscle Res. Cell Motil. 1993; 14: 557-563Crossref PubMed Scopus (64) Google Scholar, 5Somlyo A.P. Somlyo A.V. Nature. 1994; 372: 231-236Crossref PubMed Scopus (1733) Google Scholar). 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 (3Hartshorne D.J. Johnson L.R. Physiology of the Gastrointestinal Tract. Raven Press, New York1987: 432-482Google Scholar, 4Somlyo A.P. J. Muscle Res. Cell Motil. 1993; 14: 557-563Crossref PubMed Scopus (64) Google Scholar, 5Somlyo A.P. Somlyo A.V. Nature. 1994; 372: 231-236Crossref PubMed Scopus (1733) Google Scholar). 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 (1Kelley C.A. Takahashi M. Yu J.H. Adelstein R.S. J. Biol. Chem. 1993; 268: 12848-12854Abstract Full Text PDF PubMed Google Scholar) and/or splice variant isoforms of the essential myosin light chain (2Nabeshima Y. Nonomura Y. Fujii-Kuriyama Y. J. Biol. Chem. 1987; 262: 10608-10612Abstract Full Text PDF PubMed Google Scholar) producing an acidic (MLC17a) and basic (MLC17b) isoform. The rate of force activation (6Malmqvist U. Arner A. Pflugers Arch. Eur. J. Physiol. 1992; 418: 523Crossref Scopus (109) Google Scholar, 7Mathew J.D. Khromov A.S. Trybus K.M. Somlyo A.V. Somlyo A.P. J. Biol. Chem. 1998; 273: 31289-31296Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar),V max (6Malmqvist U. Arner A. Pflugers Arch. Eur. J. Physiol. 1992; 418: 523Crossref Scopus (109) Google Scholar, 7Mathew J.D. Khromov A.S. Trybus K.M. Somlyo A.V. Somlyo A.P. J. Biol. Chem. 1998; 273: 31289-31296Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar), the velocity of actin movement in the in vitro motility assay (1Kelley C.A. Takahashi M. Yu J.H. Adelstein R.S. J. Biol. Chem. 1993; 268: 12848-12854Abstract Full Text PDF PubMed Google Scholar, 8Rovner A.S. Freyzon Y. Trybus K.M. J. Muscle Res. Cell Motil. 1997; 18: 103-110Crossref PubMed Scopus (115) Google Scholar), and ATPase activity (1Kelley C.A. Takahashi M. Yu J.H. Adelstein R.S. J. Biol. Chem. 1993; 268: 12848-12854Abstract Full Text PDF PubMed Google Scholar,9Hasegawa Y. Ueda Y. Watanabe M. Morita F. J. Biochem. ( Tokyo ). 1992; 111: 798-803Crossref PubMed Scopus (21) Google Scholar) have been correlated with the expression of splice variant isoforms both of the MHC and MLC17. In this study, we tested the hypothesis that the mechanical properties of smooth muscle are regulated by splice variant expression of MLC17. We determined the effect of forced expression of MLC17a and MLC17b on the rate of force activation for agonist stimulated contractions of single cultured chicken embryonic aortic and gizzard smooth muscle cells (SMC). Aorta and gizzard cells were isolated from primary cultures of ED 13–15 chicken embryos, as described previously (10Fisher S.A. Ikebe M. Brozovich F. Circ. Res. 1997; 80: 885-893Crossref PubMed Scopus (34) Google Scholar). 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 μg/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% CO2. 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 (MLC17) 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 MLC17 cDNA sequence from position +44 to 66 and +528 to 552, this includes 5′-noncoding, all of the translated product and 3′-noncoding regions (2Nabeshima Y. Nonomura Y. Fujii-Kuriyama Y. J. Biol. Chem. 1987; 262: 10608-10612Abstract Full Text PDF PubMed Google Scholar). Briefly, 2.5 μg of total RNA was reverse-transcribed with the 3′-MLC17 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 MLC17 using 5′- and 3′-MLC17primers 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 MLC17a and MLC17b cDNAs produced were isolated by 2% agrose gel electrophoresis in TBE buffer, purified by QIAquick gel extraction kit (QIAGEN), and ligated to the pTracer-SV40" mammalian expression vector (Invitrogen) individually using standard molecular cloning techniques (11Huang Q.Q. Chen A.C. Jin J.P. Gene ( Amst. ). 1999; 229: 1-10Crossref PubMed Scopus (46) Google Scholar). The expression constructs encoding MLC17a or MLC17b were screened by PCR using 5′- and 3′-MLC17 primers and confirmed by DNA sequencing. The MLC17a or MLC17b 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 × 104-2 × 105 cells were resuspended in 100 μl of the electroporation buffer and mixed with 2 μg of either MLC17a or MLC17bexpression 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 and an interval of 1 s in a cuvette with 0.2-cm gap. The cells were placed on the MatrigelTM (Becton-Dickinson) coated culture dishes, or for mechanical studies, microcoverslips (10Fisher S.A. Ikebe M. Brozovich F. Circ. Res. 1997; 80: 885-893Crossref PubMed Scopus (34) Google Scholar) 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 MLC17expression constructs after transfection were identified by the expression of GFP. The vector expressed MLC17, driven by an SV40 promoter and GFP as a separate transcript driven by the cytomegalovirus promoter (pTracer-SV40, InvitrogenTM). GFP expression was visualized using a fluorescence microscope (Nikon) with a fluorescein isothiocyanate filter. Transcription of the exogenous MLC17a and MLC17b 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 5′- and 3′-MLC17 primers specific for MLC17 (10Fisher S.A. Ikebe M. Brozovich F. Circ. Res. 1997; 80: 885-893Crossref PubMed Scopus (34) Google Scholar), one applied the primers specific for MHC (10Fisher S.A. Ikebe M. Brozovich F. Circ. Res. 1997; 80: 885-893Crossref PubMed Scopus (34) Google Scholar) and the last used 5′-MLC17 and a 3′-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 3′ end of the transgene insert and will specifically anneal with the transcripts of exogenous MLC17 transcript. The experiments using the 3′-bovine growth hormone gene primer demonstrated that only cells transfected with the recombinant plasmid show a specific RNA transcript, indicating the expression of exogenous MLC17 (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 (12Shue G. Brozovich F.V. Biophys. J. 1999; 76: 2361-2369Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). SMCs were grown on MatrigelTM-coated coverslips (13Bowers C.W. Dahm L.M. Am. J. Physiol. 1993; 264: C229-C236Crossref PubMed Google Scholar) 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 μmphenylephrine. The rate of force activation was computed from thet 12 of the force response (time to reach 50% of the maximum force), where rate = (t 12)−1. Cultured aortic and gizzard embryonic SMC were transfected with GFP and MLC17. A bicistronic expression vector was used in which a cytomegalovirus promoter drove GFP expression and an SV40 promoter drove MLC17 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 MLC17 isoform on the ratio of MLC17a/MLC17b was determined (Fig.1). Culturing both aortic and gizzard SMC on MatrigelTM maintains the ratio of MLC17a/MLC17b observed in tissue (TableI and Ref. 10Fisher S.A. Ikebe M. Brozovich F. Circ. Res. 1997; 80: 885-893Crossref PubMed Scopus (34) Google Scholar). Transfection of aortic and gizzard SMC with the endogenous isoform of MLC17increased the expression of MLC17b in cultured aortic cells and MLC17a in cultured gizzard SMCs (Table I). Transfection of aortic cells with MLC17a increased the level of message for MLC17a from 15 to 80% of all transcripts (Table I). Similarly, transfection of gizzard cells with MLC17bincreased the level of message for MLC17b from 60% of all transcripts to almost exclusively MLC17b (Table I). These results suggest that transfection of cultured SMC with MLC17 results in the overexpression of MLC17band/or MLC17a. In addition, overexpression of MLC17 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 MLC17 did not effect the expression of the MHC.Table IMLC17b and MLC17a transcript ratios for cultured chicken embryonic SMCCell typeTransfected MLC17MLC17b expression1-aMLC17a and MLC17b are expressed as the percentage of total transcripts (mean ± S.E., n = 5–12 for each experiment). The ratio of MLC17a and MLC17b 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."MLC17aexpression1-aMLC17a and MLC17b are expressed as the percentage of total transcripts (mean ± S.E., n = 5–12 for each experiment). The ratio of MLC17a and MLC17b 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."%AortaNone84.86 ± 1.7115.05 ± 1.73AortaMLC17a20.67 ± 3.6679.33 ± 3.66AortaMLC17b96.50 ± 1.433.50 ± 1.43GizzardNone59.52 ± 1.4640.48 ± 1.46GizzardMLC17a24.60 ± 3.8075.45 ± 3.80GizzardMLC17b97.69 ± 1.192.31 ± 1.191-a MLC17a and MLC17b are expressed as the percentage of total transcripts (mean ± S.E., n = 5–12 for each experiment). The ratio of MLC17a and MLC17b bands was determined using NIH image 159 software as previously described (10Fisher S.A. Ikebe M. Brozovich F. Circ. Res. 1997; 80: 885-893Crossref PubMed Scopus (34) Google Scholar). SMC were grown on Matrigel™ and the transfected cells separated as described under "Experimental Procedures." Open table in a new tab The rate of force activation of GFP positive SMC was compared with non-transfected control SMC. For the nontransfected controls, phenylephrine (10 μm) 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, TableII). These results are in agreement with others (10Fisher S.A. Ikebe M. Brozovich F. Circ. Res. 1997; 80: 885-893Crossref PubMed Scopus (34) Google Scholar) and suggest that when cultured on MatrigelTM, embryonic aortic and gizzard SMC retain their native tonic and phasic contractile properties, respectively. For the aortic SMC, forced expression of MLC17a resulted in an increase in the rate of force activation (p < 0.05) to that observed in the untransfected gizzard cells while overexpression of MLC17bdid not change (p > 0.05) the rate of force activation (Fig. 2, Table II). Similarly for gizzard SMC, forced expression of MLC17a did not change the rate of force activation (p > 0.05), while overexpression of MLC17bslowed the rate of force activation (p < 0.05) to that observed in the untransfected aortic SMC (Fig. 2, Table II). Transfection did not influence the maximal steady state force (TableII), 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 (14Hai C.M. Murphy R.A. Am. J. Physiol. 1988; 254: C99-C106Crossref PubMed Google Scholar), the agonist stimulated contractions of both cultured embryonic aortic and gizzard SMC were accompanied by transient increases in MLC20 phosphorylation (data not shown).Table IIContractile response for aortic and gizzard SMCsCell typeTransfected MLCsnt 1/2ForcesμNAortaNone816.66 ± 2.172.0 ± 0.2AortaMLC17b715.52 ± 1.371.7 ± 0.2AortaMLC17a69.01 ± 2.382.7 ± 0.7GizzardNone119.28 ± 0.631.7 ± 0.2GizzardMLC17b1014.05 ± 2.353.5 ± 0.9GizzardMLC17a68.18 ± 1.832.4 ± 0.3Values are mean ± S.E. The t 1/2 was the time to reach 50% maximum force, and the rate of force activation is (t 1/2)−1. Open table in a new tab Values are mean ± S.E. The t 1/2 was the time to reach 50% maximum force, and the rate of force activation is (t 1/2)−1. The mechanical properties of smooth muscle are thought to arise from differences in the expression of the contractile proteins (3Hartshorne D.J. Johnson L.R. Physiology of the Gastrointestinal Tract. Raven Press, New York1987: 432-482Google Scholar, 4Somlyo A.P. J. Muscle Res. Cell Motil. 1993; 14: 557-563Crossref PubMed Scopus (64) Google Scholar, 5Somlyo A.P. Somlyo A.V. Nature. 1994; 372: 231-236Crossref PubMed Scopus (1733) Google Scholar). For smooth muscle, the distribution of both MHC and MLC17varies for both the same tissues across species and among tissues within a species (6Malmqvist U. Arner A. Pflugers Arch. Eur. J. Physiol. 1992; 418: 523Crossref Scopus (109) Google Scholar). In general, tonic tissues express predominantly the splice-out isoform of MHC and MLC17b, while phasic tissues express predominantly the splice-in isoform of MHC and MLC17a. 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 MLC17b (10Fisher S.A. Ikebe M. Brozovich F. Circ. Res. 1997; 80: 885-893Crossref PubMed Scopus (34) Google Scholar) and both tissues display tonic contractile properties (15Ogut O. Brozovich F.V. Biophys. J. 1999; 76: A284Google Scholar). The gizzard develops phasic contractile properties at ED 12–14, coincident with the change in contractile protein expression to predominantly splice-in MHC and MLC17b (10Fisher S.A. Ikebe M. Brozovich F. Circ. Res. 1997; 80: 885-893Crossref PubMed Scopus (34) Google Scholar, 15Ogut O. Brozovich F.V. Biophys. J. 1999; 76: A284Google Scholar). 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 MLC17. In the present study, neither expression of an irrelevant cytosolic protein (GFP) nor the endogenous isoform of MLC17 in SMC effected the rate of force activation. Overexpression of MLC17 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 MLC17, 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 (1Kelley C.A. Takahashi M. Yu J.H. Adelstein R.S. J. Biol. Chem. 1993; 268: 12848-12854Abstract Full Text PDF PubMed Google Scholar) since MHC splicing was not effected by the forced expression of MLC17 in our study. The results of the present study of single cultured embryonic SMC demonstrate that the expression of splice-variant isoforms of MLC17 is a determinant of the rate of force activation and MLC17 is a molecular marker for the contractile phenotype of smooth muscle. Our results are in general agreement with those of others (9Hasegawa Y. Ueda Y. Watanabe M. Morita F. J. Biochem. ( Tokyo ). 1992; 111: 798-803Crossref PubMed Scopus (21) Google Scholar) who have demonstrated that an increase in both the V maxand K m for the actin-activated ATPase of smooth muscle myosin correlates with an increase in the ratio of MLC17a/MLC17b. However, a correlation of the ATPase and the ratio of MLC17a/MLC17b is not universal (1Kelley C.A. Takahashi M. Yu J.H. Adelstein R.S. J. Biol. Chem. 1993; 268: 12848-12854Abstract Full Text PDF PubMed Google Scholar, 8Rovner A.S. Freyzon Y. Trybus K.M. J. Muscle Res. Cell Motil. 1997; 18: 103-110Crossref PubMed Scopus (115) Google Scholar), and changes in the rate of force activation may not be related to changes in the V max andK m of the ATPase. Our results in single cultured embryonic SMC are similar to those reported for endothelin-1-treated cultured SMC (10Fisher S.A. Ikebe M. Brozovich F. Circ. Res. 1997; 80: 885-893Crossref PubMed Scopus (34) Google Scholar) and permeabilized trifluoperazine-treated smooth muscle strips (7Mathew J.D. Khromov A.S. Trybus K.M. Somlyo A.V. Somlyo A.P. J. Biol. Chem. 1998; 273: 31289-31296Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Chronic endothelin-1 treatment of cultured gizzard smooth muscle cells slowed the rate of force activation and increased the expression of MLC17b (10Fisher S.A. Ikebe M. Brozovich F. Circ. Res. 1997; 80: 885-893Crossref PubMed Scopus (34) Google Scholar). 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 MLC17 expression. For trifluoperazine-treated permeabilized strips of smooth muscle (7Mathew J.D. Khromov A.S. Trybus K.M. Somlyo A.V. Somlyo A.P. J. Biol. Chem. 1998; 273: 31289-31296Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar), the contractions were unaccompanied by either a change in Ca2+ or MLC20 phosphorylation, and occurred after the exchange of MLC17 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 (1Kelley C.A. Takahashi M. Yu J.H. Adelstein R.S. J. Biol. Chem. 1993; 268: 12848-12854Abstract Full Text PDF PubMed Google Scholar) has also been suggested to determine the functional properties of smooth muscle (1Kelley C.A. Takahashi M. Yu J.H. Adelstein R.S. J. Biol. Chem. 1993; 268: 12848-12854Abstract Full Text PDF PubMed Google Scholar,8Rovner A.S. Freyzon Y. Trybus K.M. J. Muscle Res. Cell Motil. 1997; 18: 103-110Crossref PubMed Scopus (115) Google Scholar, 16Lauzon A.-M. Tyska M.J. Rovner A.S. Freyzon Y. Warshaw D.M. Trybus K.M.A.J. J. Muscle Res. Cell Motil. 1998; 19: 825-837Crossref PubMed Scopus (119) Google Scholar). These investigators (1Kelley C.A. Takahashi M. Yu J.H. Adelstein R.S. J. Biol. Chem. 1993; 268: 12848-12854Abstract Full Text PDF PubMed Google Scholar, 8Rovner A.S. Freyzon Y. Trybus K.M. J. Muscle Res. Cell Motil. 1997; 18: 103-110Crossref PubMed Scopus (115) Google Scholar) 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 MLC17a or MLC17b than the splice-in isoform of MHC with either isoform of MLC17. 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 (16Lauzon A.-M. Tyska M.J. Rovner A.S. Freyzon Y. Warshaw D.M. Trybus K.M.A.J. J. Muscle Res. Cell Motil. 1998; 19: 825-837Crossref PubMed Scopus (119) Google Scholar). 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 (17Lowey S. Waller G.S. Trybus K.M. J. Biol. Chem. 1993; 268: 20414-20418Abstract Full Text PDF PubMed Google Scholar). Both the ATPase activity and the velocity of actin movement in the in vitromotility assay measure the properties of unloaded myosin, and these parameters are thought to correlate with muscleV max. However, the effect of elevating ADP on the velocity of actin movement by myosin in the in vitromotility assay (18Homsher E. Wang F. Sellers J. Adv. Exp. Med. Biol. 1993; 332: 279-290Crossref PubMed Scopus (18) Google Scholar) and skinned fiber V max (19Fulbright R.M. Barsotti R.J. Biophys. J. 1998; 72: A278Google Scholar) are opposite. The results of these studies (18Homsher E. Wang F. Sellers J. Adv. Exp. Med. Biol. 1993; 332: 279-290Crossref PubMed Scopus (18) Google Scholar, 19Fulbright R.M. Barsotti R.J. Biophys. J. 1998; 72: A278Google Scholar) suggest that a change in the ATPase activity and velocity of actin translation duringin 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 MLC17 could regulate the properties of loaded cross-bridges, 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 MLC17a/MLC17b expression alone changes the contractile properties of the SMC. Our data suggests that MLC17a may have a dominant effect; above a threshold level of MLC17a, 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% MLC17a and 60% MLC17b, and increasing MLC17a to 75% did not influence the rate of force activation while decreasing the expression of MLC17bto 2% slowed the rate of force activation. Similarly in the aorta, increasing the expression of MLC17a from 15 to 80% increased the rate of force activation. Alternatively above a threshold level of MLC17a expression, there may be a dose-response relationship between the relative content of MLC17a 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 MLC17a/MLC17binfluences the rate of force activation. Our results clearly demonstrate that splice variant expression of MLC17 is a determinant of the rate of force activation and that MLC17 is a molecular marker for the tonic and phasic contractile phenotype. The ability of MLC17 to modulate the rate of force activation could be due to a change either in the stiffness of the myosin lever arm (7Mathew J.D. Khromov A.S. Trybus K.M. Somlyo A.V. Somlyo A.P. J. Biol. Chem. 1998; 273: 31289-31296Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar) or a change in the rate of a load-dependent transition within the actomyosin ATPase. We thank Dr. Richard Walsh for comments on the manuscript.