Examining the in Vivo Role of the Amino Terminus of the Essential Myosin Light Chain

The functional significance of the actin binding region at the amino terminus of the cardiac essential myosin light chain (ELC) remains obscure. Previous experiments carried out in vitro indicated that modulation of residues 5–14 could induce an inotropic effect, increasing maximal ATPase activity at submaximal Ca2+ concentrations (Rarick, H. M., Opgenorth, T. J., von Geldern, T. W., Wu-Wong, J. R., and Solaro, R. J. (1996) J. Biol. Chem. 271, 27039–27043). Using transgenesis, we effected a cardiac-specific replacement of ELC with a protein containing a 10-amino acid deletion at positions 5–14. Both the ventricular (ELC1vΔ5–14) and atrial (ELC1aΔ5–14) isoforms lacking this peptide were stably incorporated into the sarcomere at high efficiencies. Surprisingly when the kinetics of skinned fibers isolated from the ELC1vΔ5–14 or ELC1aΔ5–14 mice were examined, no alterations in either unloaded shortening or maximum shortening velocities were apparent. Myofibrillar Mg2+-ATPase activity was also unchanged in these preparations. No significant changes in the fiber kinetics in the cognate compartments were observed when either deletion-containing protein replaced endogenous ELC1v or ELC1a. The data indicate that the previously postulated importance of this region in mediating critical protein interactions between the cardiac ELCs and the carboxyl-terminal residues of actin in vivo should be reassessed. The functional significance of the actin binding region at the amino terminus of the cardiac essential myosin light chain (ELC) remains obscure. Previous experiments carried out in vitro indicated that modulation of residues 5–14 could induce an inotropic effect, increasing maximal ATPase activity at submaximal Ca2+ concentrations (Rarick, H. M., Opgenorth, T. J., von Geldern, T. W., Wu-Wong, J. R., and Solaro, R. J. (1996) J. Biol. Chem. 271, 27039–27043). Using transgenesis, we effected a cardiac-specific replacement of ELC with a protein containing a 10-amino acid deletion at positions 5–14. Both the ventricular (ELC1vΔ5–14) and atrial (ELC1aΔ5–14) isoforms lacking this peptide were stably incorporated into the sarcomere at high efficiencies. Surprisingly when the kinetics of skinned fibers isolated from the ELC1vΔ5–14 or ELC1aΔ5–14 mice were examined, no alterations in either unloaded shortening or maximum shortening velocities were apparent. Myofibrillar Mg2+-ATPase activity was also unchanged in these preparations. No significant changes in the fiber kinetics in the cognate compartments were observed when either deletion-containing protein replaced endogenous ELC1v or ELC1a. The data indicate that the previously postulated importance of this region in mediating critical protein interactions between the cardiac ELCs and the carboxyl-terminal residues of actin in vivo should be reassessed. myosin heavy chain myosin light chain essential myosin light chain transgenic nontransgenic atrial isoform of ELC1 ventricular isoform of ELC1 N,N-bis(2-hydroxyethyl)-2-aminoethane sulfonic acid Myosin is the major motor protein in the contractile apparatus of all muscle types and cyclically interacts with the thin filament to generate force (1Robbins J. Annu. Rev. Physiol. 2000; 62: 261-287Crossref PubMed Scopus (37) Google Scholar). The different “conventional” myosins, or myosin IIs, are thought to underlie the contractile properties of the different muscle types. Myosin II is a hexameric protein made up of two heavy chains (MyHCs)1 (M r∼229,000) and four light chains (MLCs) (M r ∼18,000–27,000). The MyHCs consist of two separate domains: a globular head region and a rod region that assumes an α-helical coiled coil. The ATPase activity underlying muscle contraction is localized at the amino-terminal end that corresponds to the globular head and neck of the molecule. Also associated with this heavy chain domain are the MLCs (2Rayment I. Holden H.M. Whittaker M. Yohn C.B. Lorenz M. Holmes K.C. Milligan R.A. Science. 1993; 261: 58-65Crossref PubMed Scopus (1441) Google Scholar). Myosin-actin cross-bridge interactions play an important role in determining cardiac systolic and diastolic function. Cardiac MyHC is encoded by two genes, α- and β-MyHC, and each heavy chain is associated with two MLC types, the regulatory MLC and essential MLCs (ELCs). X-ray crystallographic analyses have demonstrated that both the ELC and regulatory MLC are associated with the neck region of the MyHC (2Rayment I. Holden H.M. Whittaker M. Yohn C.B. Lorenz M. Holmes K.C. Milligan R.A. Science. 1993; 261: 58-65Crossref PubMed Scopus (1441) Google Scholar, 3Rayment I. Rypniewski W.R. Schmidt-Base K. Smith R. Tomchick D.R. Benning M.M. Winkelmann D.A. Wesenberg G. Holden H.M. Science. 1993; 261: 50-58Crossref PubMed Scopus (1850) Google Scholar). Striated muscle can contain two ELC isoforms, ELC1f and ELC3f. The sequences of the two are quite similar except that the ELC1f isoform contains an amino-terminal extension of 40 amino acids. The cross-bridge kinetics of ELC3f fibers are significantly faster than ELC1f fibers (4Sweeney H.L. Biophys. J. 1995; 68: 112S-119SPubMed Google Scholar), and the functional significance of the amino-terminal extension has been the subject of multiple investigations (5Bottinelli R. Betto R. Schiaffino S. Reggiani C. J. Muscle Res. Cell. Motil. 1994; 15: 413-419Crossref PubMed Scopus (115) Google Scholar, 6Lowey S. Waller G.S. Trybus K.M. J. Biol. Chem. 1993; 268: 20414-20418Abstract Full Text PDF PubMed Google Scholar, 7Morano I. Ritter O. Bonz A. Timek T. Vahl C.F. Michel G. Circ. Res. 1995; 76: 720-725Crossref PubMed Scopus (72) Google Scholar, 8Rarick H.M. Opgenorth T.J. von Geldern T.W. Wu-Wong J.R. Solaro R.J. J. Biol. Chem. 1996; 271: 27039-27043Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 9Sweeney H.L. Kushmerick M.J. Mabuchi K. Sreter F.A. Gergely J. J. Biol. Chem. 1988; 263: 9034-9039Abstract Full Text PDF PubMed Google Scholar). We have shown previously that transgenic (TG) substitution of the atrial ELC1 isoform (ELC1a) for the ventricular isoform results in faster myocardial fiber kinetics as well as increased contractile function at the whole organ level (10Sanbe A. Gulick J. Hayes E. Warshaw D. Osinska H. Chan C.B. Klevitsky R. Robbins J. Am. J. Physiol. Heart Circ. Physiol. 2000; 279: H1355-H1364Crossref PubMed Google Scholar). Because the amino-terminal alanine- and proline-rich region of the ELC1a is shorter than that of the ELC1v, the amount of interaction between the ELC and actin may be reduced, effectively attenuating the tether action and leading to increased function (11Morano I. Haase H. FEBS Lett. 1997; 408: 71-74Crossref PubMed Scopus (36) Google Scholar, 12Schaub M.C. Hefti M.A. Zuellig R.A. Morano I. Cardiovasc. Res. 1998; 37: 381-404Crossref PubMed Scopus (104) Google Scholar). Thus, different ELC isoforms can regulate aspects of contractility in cardiac muscle. In vitro and NMR data showed that the amino-terminal region of ELC, encompassed within the first 11 residues of the ELC1f, ELC1a, and ELC1v isoforms, can bind to the carboxyl-terminal residues of actin where it presumably affects myosin-actin cross-bridge cycling (4Sweeney H.L. Biophys. J. 1995; 68: 112S-119SPubMed Google Scholar,12Schaub M.C. Hefti M.A. Zuellig R.A. Morano I. Cardiovasc. Res. 1998; 37: 381-404Crossref PubMed Scopus (104) Google Scholar, 13Andreev O.A. Saraswat L.D. Lowey S. Slaughter C. Borejdo J. Biochemistry. 1999; 38: 2480-2485Crossref PubMed Scopus (32) Google Scholar, 14Timson D.J. Trayer I.P. FEBS Lett. 1997; 400: 31-36Crossref PubMed Scopus (24) Google Scholar, 15Timson D.J. Trayer H.R. Trayer I.P. Eur. J. Biochem. 1998; 255: 654-662Crossref PubMed Scopus (62) Google Scholar). Using a skinned fiber system, this ELC-actin interaction can be ablated by incubation with a synthetic peptide corresponding to amino acid residues 5–14 with a concomitant increase in the rate of fiber shortening (7Morano I. Ritter O. Bonz A. Timek T. Vahl C.F. Michel G. Circ. Res. 1995; 76: 720-725Crossref PubMed Scopus (72) Google Scholar). Similar experiments carried out with isolated rat cardiac myofibrils showed that exogenous peptide led to a stimulation of Mg2+-ATPase activity at submaximal Ca2+levels (8Rarick H.M. Opgenorth T.J. von Geldern T.W. Wu-Wong J.R. Solaro R.J. J. Biol. Chem. 1996; 271: 27039-27043Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). The ELC-actin interaction depends on the regional charges; in particular, ELC lysines 3, 4, 8, and 9 appear to be important and interact with actin carboxyl-terminal residues Glu-360, Glu-361, Asp-363, and Glu-364 (4Sweeney H.L. Biophys. J. 1995; 68: 112S-119SPubMed Google Scholar). However, mutational analysis of actin in Dictyostelium revealed that Glu-360, Glu-361, Asp-363, and Glu-364 were not necessary for cross-bridge cycling (16Johara M. Toyoshima Y.Y. Ishijima A. Kojima H. Yanagida T. Sutoh K. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2127-2131Crossref PubMed Scopus (70) Google Scholar). Thus,in vivo the functional significance of the interaction between the ELC amino-terminal region and the actin carboxyl-terminal region is unclear. Using cardiac-specific promoters to drive expression in TG animals is an effective means of replacing endogenous sarcomeric components with transgenically encoded protein. The α-MyHC promoter can drive cardiac-specific TG expression such that ventricular and atrial sarcomeric isoforms can be efficiently exchanged with the TG-encoded species (17Fewell J.G. Hewett T.E. Sanbe A. Klevitsky R. Hayes E. Warshaw D. Maughan D. Robbins J. J. Clin. Invest. 1998; 101: 2630-2639Crossref PubMed Scopus (74) Google Scholar, 18Palermo J. Gulick J. Colbert M. Fewell J. Robbins J. Circ. Res. 1996; 78: 504-509Crossref PubMed Scopus (115) Google Scholar). Replacement of endogenous species with proteins containing single site mutations have also been reported (10Sanbe A. Gulick J. Hayes E. Warshaw D. Osinska H. Chan C.B. Klevitsky R. Robbins J. Am. J. Physiol. Heart Circ. Physiol. 2000; 279: H1355-H1364Crossref PubMed Google Scholar), allowing the structure-function relationships of single residues to be explored in vivo. To clarify the potential importance of the amino-terminal light chain-actin interactions within the whole organ and whole animal contexts, we created TG mice with cardiac-specific expression of mutated ELC1v and ELC1a isoforms. Lines of TG mice in which the endogenous ELC was replaced with a protein containing a 10-amino acid amino-terminal deletion between residues 5–14 were made. The data show that interactions mediated by this region of the protein are not essential for cross-bridge cycling in the heart. Previously isolated full-length murine ELC1a and ELC1v cDNAs were used as starting templates (17Fewell J.G. Hewett T.E. Sanbe A. Klevitsky R. Hayes E. Warshaw D. Maughan D. Robbins J. J. Clin. Invest. 1998; 101: 2630-2639Crossref PubMed Scopus (74) Google Scholar,19James J. Osinska H. Hewett T.E. Kimball T. Klevitsky R. Witt S. Hall D.G. Gulick J. Robbins J. Transgenic Res. 1999; 8: 9-22Crossref PubMed Scopus (30) Google Scholar). Primers with SalI sites engineered at the termini were made to the 3′- and 5′-untranslated regions of the cDNA. The 5′ primer also contained the mutated bases necessary to delete the amino-terminal amino acid residues KPEPKKETAK in ELC1a and KPEPKKDDAK in ELC1v (Fig. 1B). The polymerase chain reaction product was sequenced, and the fragment was linked to the mouse α-MyHC promoter. The final constructs containing a deleted ELC1v from position 5–14 (ELC1vΔ5–14) and a deleted ELC1a at the same region (ELC1aΔ5–14) were digested free of vector sequence with NotI, purified from agarose, and used to generate TG mice as described previously (20Sanbe A. Fewell J.G. Gulick J. Osinska H. Lorenz J. Hall D.G. Murray L.A. Kimball T.R. Witt S.A. Robbins J. J. Biol. Chem. 1999; 274: 21085-21094Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). Transcript analysis was performed by RNA blots with transcript-specific probes as described previously (20Sanbe A. Fewell J.G. Gulick J. Osinska H. Lorenz J. Hall D.G. Murray L.A. Kimball T.R. Witt S.A. Robbins J. J. Biol. Chem. 1999; 274: 21085-21094Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). For myofibrillar protein analysis, the left ventricular apex and atrial flaps were obtained from euthanized ELC1v, ELC1a, ELC1vΔ5–14 TG, ELC1aΔ5–14 TG, and nontransgenic (NTG) mice. Myofibrillar sample preparation, gel preparation, electrophoretic conditions, and gel staining have been described previously (20Sanbe A. Fewell J.G. Gulick J. Osinska H. Lorenz J. Hall D.G. Murray L.A. Kimball T.R. Witt S.A. Robbins J. J. Biol. Chem. 1999; 274: 21085-21094Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). For fiber isolation and analysis, the animals were heparinized (500 IU/kg intraperitoneally) and then euthanized with CO2. After a thoracotomy, the heart was removed and placed in relaxing solution (5.37 mmATP, 30 mm phosphocreatine, 5.0 mm EGTA, 20.0 mm BES, 7.33 mm MgCl2, 0.12 mm CaCl2, 10 mmdithiothreitol, 10 µg/ml leupeptin, 32 mmpotassium methanesulfonate, and 240 units/ml creatine phosphokinase (pCa 8.0, pH 7.0, ionic strength 175 mm)) at 4 °C. The solution contained 30 mm 2,3-butanedione monoxime to protect the myocardial tissue from cutting injury (10Sanbe A. Gulick J. Hayes E. Warshaw D. Osinska H. Chan C.B. Klevitsky R. Robbins J. Am. J. Physiol. Heart Circ. Physiol. 2000; 279: H1355-H1364Crossref PubMed Google Scholar, 21Sanbe A. Nelson D. Gulick J. Setser E. Osinska H. Wang X. Hewett T.E. Klevitsky R. Hayes E. Warshaw D.M. Robbins J. Circ. Res. 2000; 87: 296-302Crossref PubMed Scopus (40) Google Scholar,22Yang Q. Sanbe A. Osinska H. Hewett T.E. Klevitsky R. Robbins J. J. Clin. Invest. 1998; 102: 1292-1300Crossref PubMed Scopus (153) Google Scholar). Small sections of either left ventricular papillary muscles or left atrial trabeculae were dissected in the 2,3-butanedione monoxime relaxing solution to yield 0.5-mm-diameter and 2–3-mm-long strips. The strips were skinned by incubation in 5.5 mm ATP, 5.0 mm EGTA, 20.0 mm BES, 6.13 mmMgCl2, 0.11 mm CaCl2, 10 mm dithiothreitol, 10 µg/ml leupeptin, 121.8 mm potassium methanesulfonate (pCa 8.0, pH 7.0, ionic strength 175 mm), and 50% glycerol containing 0.5 w/v% Triton X-100 for 12 h at 4 °C. Strips were transferred into the same solution minus detergent and stored at −20 °C. The skinned fiber experiments were performed using a commercially available apparatus (Scientific Instruments, Heidelberg, Germany). Fibers were dissected into bundles of 125–175-µm diameter, 1.3–2.0-mm long under a dissecting microscope and mounted isometrically between a force transducer (KG3, Scientific Instruments) and a length-step generator in relaxing solution. Sarcomere length at resting tension was always 2.0 µm. Contracting solution had the same composition as the relaxing solution except that EGTA was substituted with 5 mmCa-EGTA. The free Ca2+ concentration was obtained by mixing the relaxing and contraction solutions in the appropriate proportions. Strip tension (millinewton/mm2) was calculated by dividing force by fiber cross-sectional area calculated from widths measured at the major axis. Activating solution had the same ionic composition as relaxing solution. The solutions were formulated by solving a set of simultaneous equations describing the multiple equilibrium of ions in the solutions. A total of four constructs were used in these studies (Fig.1 A). To rule out any phenotype due to overexpression of either the atrial- or ventricular-specific ELC isoforms, lines carrying the wild type cDNAs (18Palermo J. Gulick J. Colbert M. Fewell J. Robbins J. Circ. Res. 1996; 78: 504-509Crossref PubMed Scopus (115) Google Scholar, 19James J. Osinska H. Hewett T.E. Kimball T. Klevitsky R. Witt S. Hall D.G. Gulick J. Robbins J. Transgenic Res. 1999; 8: 9-22Crossref PubMed Scopus (30) Google Scholar) were used as controls along with NTG animals. Although previous in vitro studies focused exclusively on the amino terminus of the ventricular isoform, we extended these studies to include the atrial isoform as well. The amino-terminal extension is shorter in ELC1a but also contains the 5–14-residue region (KPEPKK(D/E)(D/T)AK) that has been implicated in actin binding (Fig. 1 B). Multiple lines were generated for each TG construct; for the purposes of this study we focused on lines that yielded high levels of replacement. For each of the constructs expressing the wild type proteins, lines were used in which replacement in the ectopic cardiac compartment was >90%: both the ELC1a and ELC1v “wild type” lines have been reported previously (18Palermo J. Gulick J. Colbert M. Fewell J. Robbins J. Circ. Res. 1996; 78: 504-509Crossref PubMed Scopus (115) Google Scholar, 19James J. Osinska H. Hewett T.E. Kimball T. Klevitsky R. Witt S. Hall D.G. Gulick J. Robbins J. Transgenic Res. 1999; 8: 9-22Crossref PubMed Scopus (30) Google Scholar). The ELC1vΔ5–14 TG line 188 showed significant levels of replacement (Fig. 1 C). Analyses of ventricular RNA levels showed that line 188 had a 15.6-fold increase as compared with the endogenous transcript (data not shown) resulting in ∼85% replacement (Fig. 1 C). The ELC1aΔ5–14 line used in the studies below (line 22) had 21.7-fold higher atrial transcript levels as compared with the endogenous ELC1a. This level of TG expression resulted in >95% replacement of ELC1a in the atria (Fig.1 C). The overall appearance, behavior, and of the lines were all analyses revealed the of cardiac and no (data not the were for cardiac using both animals at and as well as means such as (20Sanbe A. Fewell J.G. Gulick J. Osinska H. Lorenz J. Hall D.G. Murray L.A. Kimball T.R. Witt S.A. Robbins J. J. Biol. Chem. 1999; 274: 21085-21094Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). The was a increase in ventricular in the ELC1aΔ5–14 animals. all heart in ELC1vΔ5–14 TG mice were the ELC1aΔ5–14 TG mice show a significant increase in the left ventricular Previously we showed that an ELC1v ELC1a protein replacement has no on (17Fewell J.G. Hewett T.E. Sanbe A. Klevitsky R. Hayes E. Warshaw D. Maughan D. Robbins J. J. Clin. Invest. 1998; 101: 2630-2639Crossref PubMed Scopus (74) Google Scholar), that the may in be of the deletion at the amino However, it should be that both ELC1v ELC1vΔ5–14 and ELC1a ELC1aΔ5–14 no it as the deletion also be with an isoform to the and line line line line 22) from NTG are left total total from NTG in a are left total total the of the in vitro which were obtained using fiber in which of were to any we to changes in either the in the TG mice and in the ELC1aΔ5–14 animals as well. To this end we first the Mg2+-ATPase and relationships using skinned fiber from the left ventricular papillary muscles of the TG lines (Fig. In to the data obtained using the peptide no in either the Mg2+-ATPase activity or in maximum force as a function of the Ca2+ concentration (Fig. were in the ELC1vΔ5–14 TG mice. Previously we have shown that expression of ELC1a in the not to changes in these (10Sanbe A. Gulick J. Hayes E. Warshaw D. Osinska H. Chan C.B. Klevitsky R. Robbins J. Am. J. Physiol. Heart Circ. Physiol. 2000; 279: H1355-H1364Crossref PubMed Google Scholar). Similar results were obtained when the fibers containing replacement of the endogenous protein with ELC1aΔ5–14 were II and Mg2+-ATPase are Mg2+-ATPase activity of was by the activity at 5.0 from the activity at not in a are Mg2+-ATPase activity of was by the activity at 5.0 from the activity at not We the unloaded shortening maximum shortening and maximum in the TG Previous experiments have indicated that expression of ELC1v in the heart not to changes in these ventricular (10Sanbe A. Gulick J. Hayes E. Warshaw D. Osinska H. Chan C.B. Klevitsky R. Robbins J. Am. J. Physiol. Heart Circ. Physiol. 2000; 279: H1355-H1364Crossref PubMed Google Scholar). Surprisingly replacement of the endogenous protein with the ELC1vΔ5–14 species resulted in to the NTG controls (Fig. 3, Similar results were obtained when the fibers containing replacement of the endogenous protein with ELC1aΔ5–14 were (Fig. 3, and The amino-terminal residues that were deleted in both ELC1v and ELC1a contain an site (7Morano I. Ritter O. Bonz A. Timek T. Vahl C.F. Michel G. Circ. Res. 1995; 76: 720-725Crossref PubMed Scopus (72) Google Scholar, 15Timson D.J. Trayer H.R. Trayer I.P. Eur. J. Biochem. 1998; 255: 654-662Crossref PubMed Scopus (62) Google Scholar), and deletion of these residues ELC-actin attenuating the actin of the of skinned ventricular fibers with the 5–14 amino-terminal presumably the carboxyl-terminal binding site of resulting in an increase in tension at both submaximal and maximal Ca2+ (7Morano I. Ritter O. Bonz A. Timek T. Vahl C.F. Michel G. Circ. Res. 1995; 76: 720-725Crossref PubMed Scopus (72) Google Scholar). experiments carried out with similar showed that the ELC1v amino-terminal fragment a increase in fiber Mg2+-ATPase activity at submaximal Ca2+levels with no at and maximum Ca2+ levels (8Rarick H.M. Opgenorth T.J. von Geldern T.W. Wu-Wong J.R. Solaro R.J. J. Biol. Chem. 1996; 271: 27039-27043Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Although data not on the potential of an exogenous peptide to fiber all the data indicate that the protein interactions between the amino-terminal 5–14 ELC region and the terminus of actin are not essential for myosin motor function in vivo. results are with data that were obtained when mutations at the actin terminus residues and were these not significantly either velocities or force in actin analyses (16Johara M. Toyoshima Y.Y. Ishijima A. Kojima H. Yanagida T. Sutoh K. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2127-2131Crossref PubMed Scopus (70) Google Scholar). on the of peptide to actin or the thin filament that were used (8Rarick H.M. Opgenorth T.J. von Geldern T.W. Wu-Wong J.R. Solaro R.J. J. Biol. Chem. 1996; 271: 27039-27043Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar), the amount of peptide for the is high thin studies showed that the amino terminus of the ELC could the terminus of actin when MyHC to two actin O.A. Borejdo J. Circ. Res. 1997; PubMed Scopus Google Scholar). data the importance of the ELC-actin binding in cross-bridge kinetics or the of force with to Ca2+ vitro data that the ELC1v amino-terminal peptide Mg2+-ATPase activity an interaction with This in results in a to the thin filament (8Rarick H.M. Opgenorth T.J. von Geldern T.W. Wu-Wong J.R. Solaro R.J. J. Biol. Chem. 1996; 271: 27039-27043Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). in where fiber and is we were to any on or the of any effect, a similar made in the atrial isoform also resulted in fiber Because replacement was in the ELC1vΔ5–14 line 188 mice and in the ELC1aΔ5–14 it that residues 5–14 are not for of fiber or function in vivo. of function the of the animal is by the of increased or in the TG We it that the faster cycling that are observed in ELC1a and ELC3f fibers to fibers may in the neck region of We for