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• W1990421818 abstract "The functional significance of the actin-binding region at the N terminus of the cardiac myosin essential light chain (ELC) remains elusive. In a previous experiment, the endogenous ventricular ELC was replaced with a protein containing a 10-amino acid deletion at positions 5–14 (ELC1vΔ5–14, referred to as 1vΔ5–14), a region that interacts with actin (Sanbe, A., Gulick, J., Fewell, J., and Robbins, J. (2001) J. Biol. Chem. 276, 32682–32686). 1vΔ5–14 mice showed no discernable mutant phenotype in skinned ventricular strips. However, because the myofilament lattice swells upon skinning, the mutant phenotype may have been concealed by the inability of the ELC to reach the actin-binding site. Using the same mouse model, we repeated earlier measurements and performed additional experiments on skinned strips osmotically compressed to the intact lattice spacing as determined by x-ray diffraction. 1vΔ5–14 mice exhibited decreased maximum isometric tension without a change in calcium sensitivity. The decreased force was most evident in 5–6-month-old mice compared with 13–15-month-old mice and may account for the greater ventricular wall thickness in young 1vΔ5–14 mice compared with age-matched controls. No differences were observed in unloaded shortening velocity at maximum calcium activation. However, 1vΔ5–14 mice exhibited a significant difference in the frequency at which minimum complex modulus amplitude occurred, indicating a change in cross-bridge kinetics. We hypothesize that the ELC N-terminal extension interaction with actin inhibits the reversal of the power stroke, thereby increasing isometric force. Our results strongly suggest that an interaction between residues 5–14 of the ELC N terminus and the C-terminal residues of actin enhances cardiac performance. The functional significance of the actin-binding region at the N terminus of the cardiac myosin essential light chain (ELC) remains elusive. In a previous experiment, the endogenous ventricular ELC was replaced with a protein containing a 10-amino acid deletion at positions 5–14 (ELC1vΔ5–14, referred to as 1vΔ5–14), a region that interacts with actin (Sanbe, A., Gulick, J., Fewell, J., and Robbins, J. (2001) J. Biol. Chem. 276, 32682–32686). 1vΔ5–14 mice showed no discernable mutant phenotype in skinned ventricular strips. However, because the myofilament lattice swells upon skinning, the mutant phenotype may have been concealed by the inability of the ELC to reach the actin-binding site. Using the same mouse model, we repeated earlier measurements and performed additional experiments on skinned strips osmotically compressed to the intact lattice spacing as determined by x-ray diffraction. 1vΔ5–14 mice exhibited decreased maximum isometric tension without a change in calcium sensitivity. The decreased force was most evident in 5–6-month-old mice compared with 13–15-month-old mice and may account for the greater ventricular wall thickness in young 1vΔ5–14 mice compared with age-matched controls. No differences were observed in unloaded shortening velocity at maximum calcium activation. However, 1vΔ5–14 mice exhibited a significant difference in the frequency at which minimum complex modulus amplitude occurred, indicating a change in cross-bridge kinetics. We hypothesize that the ELC N-terminal extension interaction with actin inhibits the reversal of the power stroke, thereby increasing isometric force. Our results strongly suggest that an interaction between residues 5–14 of the ELC N terminus and the C-terminal residues of actin enhances cardiac performance. Muscle myosin (myosin II) is a hexamer that contains two heavy chains (MHCs) 3The abbreviations used are: MHCs, myosin heavy chains; ELC, essential light chain; NTG, non-transgenic; wt, wild-type; BES, N,N-bis(2-hydroexyethyl)-2-aminoethanesulfonic acid; ANOVA, analysis of variance. 3The abbreviations used are: MHCs, myosin heavy chains; ELC, essential light chain; NTG, non-transgenic; wt, wild-type; BES, N,N-bis(2-hydroexyethyl)-2-aminoethanesulfonic acid; ANOVA, analysis of variance., with each heavy chain having two types of light chains: the essential light chain (ELC) and the regulatory light chain. The MHCs separate into two globular heads at their N terminus, with the remainder being a coiled coil that, with the other MHC tail regions, forms the backbone of the thick filament. Muscle contraction is generated by the cyclic interaction of the myosin heads with actin in the thin filaments, drawing the filaments past one another. Both myosin light chains are involved in stabilizing the lever arm, the long α-helix extending from the catalytic motor part of the myosin head to the coiled-coil backbone. Most regulatory light chains modulate the interaction of the myosin head with actin through phosphorylation of residues near the N terminus (1Sweeney H.L. Bowman B.F. Stull J.T. Am. J. Physiol. 1993; 264: C1085-C1095Crossref PubMed Google Scholar, 2Tohtong R. Yamashita H. Graham M. Haeberle J. Simcox A. Maughan D. Nature. 1995; 374: 650-653Crossref PubMed Scopus (110) Google Scholar, 3Sweeney H.L. Am. J. Respir. Crit. Care Med. 1998; 158: S95-S99Crossref PubMed Scopus (21) Google Scholar, 4Bresnick A.R. Curr. Opin. Cell Biol. 1999; 11: 26-33Crossref PubMed Scopus (311) Google Scholar, 5Sanbe 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, 6Somlyo A.P. Somlyo A.V. J. Physiol. (Lond.). 2000; 522: 177-185Crossref Scopus (1069) Google Scholar, 7Davis J.S. Hassanzadeh S. Winitsky S. Lin H. Satorius C. Vemuri R. Aletras A.H. Wen H. Epstein N.D. Cell. 2001; 107: 631-641Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar). In contrast, most ELCs are believed to modulate myosin head-actin interaction through direct binding of an N-terminal extension to actin (8Milligan R.A. Whittaker M. Safer D. Nature. 1990; 348: 217-221Crossref PubMed Scopus (318) Google Scholar, 9Stepkowski D. FEBS Lett. 1995; 374: 6-11Crossref PubMed Scopus (22) Google Scholar, 10Sweeney H.L. Biophys. J. 1995; 68: 112S-1118PubMed Google Scholar, 11Schaub M.C. Hefti M.A. Zuellig R.A. Morano I. Cardiovasc. Res. 1998; 37: 381-404Crossref PubMed Scopus (104) Google Scholar, 12Morano I. J. Mol. Med. 1999; 77: 544-555Crossref PubMed Scopus (139) Google Scholar, 13Timson D.J. Trayer H.R. Smith K.J. Trayer I.P. J. Biol. Chem. 1999; 274: 18271-18277Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 14Timson D.J. Biochimie (Paris). 2003; 85: 639-645Crossref PubMed Scopus (54) Google Scholar). In human skeletal muscle, the A1-type fast (ELC1f) and slow (ELC1s) ELC isoforms contain 40–45 additional amino acids compared with the A2-type fast isoform (ELC3f) (14Timson D.J. Biochimie (Paris). 2003; 85: 639-645Crossref PubMed Scopus (54) Google Scholar). The longer skeletal muscle ELC isoforms cross-link to the C-terminal region of actin through their N-terminal α-amino group and four lysines within the first 10 residues (10Sweeney H.L. Biophys. J. 1995; 68: 112S-1118PubMed Google Scholar, 15Sutoh K. Biochemistry. 1982; 21: 3654-3661Crossref PubMed Scopus (257) Google Scholar, 16Hayashibara T. Miyanishi T. Biochemistry. 1994; 33: 12821-12827Crossref PubMed Scopus (45) Google Scholar, 17Andreev O.A. Saraswat L.D. Lowey S. Slaughter C. Borejdo J. Biochemistry. 1999; 38: 2480-2485Crossref PubMed Scopus (32) Google Scholar). In human cardiac muscle, both ventricular (ELC1v) and atrial (ELC1a) ELC isoforms have N-terminal extensions of approximately the same length as their A1-type counterparts in skeletal muscle. The atrial isoform N-terminal extension has been shown to interact with actin (13Timson D.J. Trayer H.R. Smith K.J. Trayer I.P. J. Biol. Chem. 1999; 274: 18271-18277Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar), and the ventricular isoform extension is generally assumed to do so as well. A previous study examined the role of the actin-binding region at the N terminus of the ventricular ELC in a transgenic mouse model (18Sanbe A. Gulick J. Fewell J. Robbins J. J. Biol. Chem. 2001; 276: 32682-32686Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar). The N-terminal extension of the ventricular ELC in the mouse is similar to that in the human (14Timson D.J. Biochimie (Paris). 2003; 85: 639-645Crossref PubMed Scopus (54) Google Scholar, 18Sanbe A. Gulick J. Fewell J. Robbins J. J. Biol. Chem. 2001; 276: 32682-32686Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar), suggesting that their physiological roles are similar. In skinned atrial and ventricular strips from transgenic mice in which ELC residues 5–14 (ELC1vΔ5–14, referred to as 1vΔ5–14) were deleted, some of which interact with actin (10Sweeney H.L. Biophys. J. 1995; 68: 112S-1118PubMed Google Scholar, 15Sutoh K. Biochemistry. 1982; 21: 3654-3661Crossref PubMed Scopus (257) Google Scholar, 16Hayashibara T. Miyanishi T. Biochemistry. 1994; 33: 12821-12827Crossref PubMed Scopus (45) Google Scholar, 17Andreev O.A. Saraswat L.D. Lowey S. Slaughter C. Borejdo J. Biochemistry. 1999; 38: 2480-2485Crossref PubMed Scopus (32) Google Scholar), there was a surprising lack of morphological and functional differences between transgenic (1vΔ5–14) and non-transgenic (NTG) controls. The calcium sensitivity of isometric tension of 1vΔ5–14 was similar to that of controls (absolute tensions were not reported), and there were no observed differences in MgATPase activity and shortening velocity at maximum calcium activation (pCa 5) (18Sanbe A. Gulick J. Fewell J. Robbins J. J. Biol. Chem. 2001; 276: 32682-32686Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar). The lack of morphological and functional differences was unexpected because other experiments involving the addition of 10-residue peptides consisting of the deleted region or portions thereof (ELC residues 1–10 (19Nieznanska H. Nieznanski K. Stepkowski D. Acta Biochim. Pol. 2002; 49: 709-719Crossref PubMed Scopus (7) Google Scholar) and 5–14 (20Morano I. Ritter O. Bonz A. Timek T. Vahl C.F. Michel G. Circ. Res. 1995; 76: 720-725Crossref PubMed Scopus (72) Google Scholar, 21Rarick 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)) showed effects on cross-bridge kinetics. For example, adding ELC residues 5–14 to rat ventricle myofibrils resulted in an ∼2-fold increase in MgATPase at submaximum calcium concentrations (pCa 6.5–5.875) (21Rarick 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). Likewise, a peptide corresponding to A1-type fast ELC residues 1–10 added to myofibrillar preparations from rabbit fast muscles enhanced MgATPase, but over a higher range of calcium concentrations (pCa 5.75–4.59) (19Nieznanska H. Nieznanski K. Stepkowski D. Acta Biochim. Pol. 2002; 49: 709-719Crossref PubMed Scopus (7) Google Scholar) compared with the other peptide. In contrast, a peptide corresponding to slow ELC residues 1–10 added to myofibrils from slow muscles decreased MgATPase over the entire range of calcium concentrations examined. Incubating human heart strips in solutions containing peptides corresponding to human ELC extension residues 5–14, 5–10, and 5–8 resulted in increased isometric tension (20Morano I. Ritter O. Bonz A. Timek T. Vahl C.F. Michel G. Circ. Res. 1995; 76: 720-725Crossref PubMed Scopus (72) Google Scholar). Treatment with peptide 5–14 (which enhanced isometric tension more than the other peptides) also produced an increased maximum rate of tension rise, an increased maximum rate of relaxation, and increased shortening velocity compared with untreated strips. Thus, given the contrasting experimental results, the functional significance of the actin-binding residues in the N terminus of the cardiac ELC remains elusive. Lattice spacing (the distance between thick and thin filaments) increases upon skinning of intact muscle fibers (22Irving T.C. Konhilas J. Perry D. Fischetti R. de Tombe P.P. Am. J. Physiol. 2000; 279: H2568-H2573Crossref PubMed Google Scholar, 23Irving T. Bhattacharya S. Tesic I. Moore J. Farman G. Simcox A. Vigoreaux J. Maughan D. J. Muscle Res. Cell Motil. 2001; 22: 675-683Crossref PubMed Scopus (18) Google Scholar). Thus, the increased distance between filaments may have compromised the ability of the ELC N-terminal extension to reach actin in previous skinned strip experiments using the 1vΔ5–14 mouse (18Sanbe A. Gulick J. Fewell J. Robbins J. J. Biol. Chem. 2001; 276: 32682-32686Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar), thereby masking the effect of the 10-amino acid deletion. A similar concern was expressed with regard to the effect of lattice spacing on the function of the regulatory light chain extension in Drosophila flight muscle, which bears a striking resemblance to the vertebrate ELC extension (23Irving T. Bhattacharya S. Tesic I. Moore J. Farman G. Simcox A. Vigoreaux J. Maughan D. J. Muscle Res. Cell Motil. 2001; 22: 675-683Crossref PubMed Scopus (18) Google Scholar, 24Moore J.R. Dickinson M.H. Vigoreaux J.O. Maughan D.W. Biophys. J. 2000; 78: 1431-1440Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). In this study, we re-examined the phenotype of the 1vΔ5–14 transgenic mouse both in vivo and in isolated skinned strips that were osmotically compressed to intact lattice spacing. We also examined the effect of age because the phenotype may be age-dependent. Our results strongly suggest that an interaction between residues 5–14 of the ELC N terminus and the C-terminal residues of actin enhances cardiac performance. Materials—All reagents were purchased from Sigma except where noted. Mouse Model—We used the same 1vΔ5–14 transgenic and control (NTG) lines as in previous studies (5Sanbe 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, 18Sanbe A. Gulick J. Fewell J. Robbins J. J. Biol. Chem. 2001; 276: 32682-32686Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar). In mouse ventricular muscle, the extended ELC N-terminal region rich in alanine and proline residues and capped by a region rich in lysine residues is MAPKKPEPKKDDAKAAAPKAAPAPAAAPAAAPAAAPEPERPKEAEFDASKIKIE (boldface underlined residues are those deleted in the 1vΔ5–14 line). Female mice were used in all experiments. Echocardiography—1vΔ5–14 and NTG female mice were examined by echocardiography at two ages: young (20 weeks) and old (53 weeks). Mice were lightly anesthetized with halothane and placed in the left lateral decubitus position. Parasternal short-axis images of the heart at the mid-papillary level were obtained using a clinical echocardiograph machine (ACUSON Sequoia, Siemens Medical Solutions USA, Inc., Mountain View, CA) equipped with a 13-MHz probe. Using the two-dimensional short-axis image to correctly position the probe, M-mode echo data were recorded. Left ventricular volumes and wall thickness values were quantified from the digitized M-mode images using standard software (ACUSON Sequoia). Three representative cardiac cycles, each obtained from separate traces, were analyzed during each study to minimize selection bias. Transcript and Myosin Heavy Chain Analyses—Transcript analysis with sequence-specific oligonucleotides was performed as described previously (5Sanbe 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, 25Sanchez A. Jones W.K. Gulick J. Doetschman T. Robbins J. J. Biol. Chem. 1991; 266: 22419-22426Abstract Full Text PDF PubMed Google Scholar). Oligonucleotides corresponding to atrial natriuretic factor, phospholamban, sarcoplasmic reticulum ATPase 2A, skeletal α-actin, and glyceraldehyde-3-phosphate dehydrogenase (used as a loading control) were used to examine gene expression related to cardiac hypertrophy and sarcoplasmic reticulum calcium handling (26Fewell J.G. Osinska H. Klevitsky R. Ng W. Sfyris G. Bahrehmand F. Robbins J. Am. J. Physiol. 1997; 273: H1595-H1605PubMed Google Scholar). Ventricular protein was loaded onto a 5% glycerol gel and electrophoresed to separate the α- and β-MHC proteins. A mouse in which 70% of the α-MHC was replaced with β-MHC through genetic manipulation (27Krenz M. Sanbe A. Bouyer-Dalloz F. Gulick J. Klevitsky R. Hewett T.E. Osinska H.E. Lorenz J.N. Brosseau C. Federico A. Alpert N.R. Warshaw D.M. Perryman M.B. Helmke S.M. Robbins J. J. Biol. Chem. 2003; 278: 17466-17474Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar) was used as a marker. Both transcript and MHC analyses were performed on mice at 15 weeks, and an additional line (ELC1v-wt), defined in previous studies (5Sanbe 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, 18Sanbe A. Gulick J. Fewell J. Robbins J. J. Biol. Chem. 2001; 276: 32682-32686Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar), was used as a second control. Solutions—Relaxing solution (pCa 8) contained 20 mm BES, 5 mm EGTA, 40 mm disodium creatine phosphate, 240 units/ml creatine phosphokinase, 5.38 mm N2ATP, 7.71 mm MgCl2, 0.25 mm KH2PO4, 0.12 mm CaCl2, and 1 mm 1,4-dithio-dl-threitol titrated with 1.78 mm KOH to pH 7.0 and adjusted with 17.48 mm sodium methanesulfonate to an ionic strength of 190 milliequivalents, yielding 5 mm MgATP, 1 mm Mg+2, and 0.25 mm free phosphate according to a computer program that solves the ionic equilibria (28Godt R.E. Lindley B.D. J. Gen. Physiol. 1982; 80: 279-297Crossref PubMed Scopus (330) Google Scholar). Activating solution (pCa 4, pH 7, 190 milliequivalents) contained the same concentrations of MgATP, Mg+2, and free phosphate and other constituents as in relaxing solution, except for an increased calcium concentration (5.28 mm CaCl2) and slight adjustments in the concentrations of other salts (5.53 mm Na2ATP, 7.55 mm MgCl2, 11.3 mm KOH, and 7.01 mm sodium methanesulfonate). ATP-free rigor solution (pCa 4.5, pH 7, 190 milliequivalents) contained 4.97 mm CaCl2, 1.0 mm MgCl2, 9.8 mm KOH, and 154.8 mm sodium methanesulfonate. Storage solution had the same constituents as relaxing solution plus 10 μg/ml leupeptin and 50% (w/v) glycerol. Skinning solution had the same constituents as storage solution plus 1% (w/v) Triton X-100 (a nonionic detergent). For the x-ray diffraction experiments, 0–10% (w/v) dextran T-500 (an osmotic compression agent) was added to relaxing solution to establish the concentration required to compress the skinned strip lattice to its intact spacing. For the mechanical experiments, 5.4% (w/v) dextran T-500 (the concentration required to compress the skinned strip lattice to its intact spacing) was added to relaxing, activating, and rigor solutions. Myocardial Strip Preparation—Mice were killed by cervical dislocation, and the hearts were rapidly excised and placed in O2 (95%)/CO2 (5%)-bubbled Krebs solution containing 30 mm 2,3-butanedione monoxime at 22 °C (29Mulieri L.A. Hasenfuss G. Ittleman F. Blanchard E.M. Alpert N.R. Circ. Res. 1989; 65: 1441-1449Crossref PubMed Google Scholar). The right ventricle was trimmed, and the papillary muscles from the left ventricle were removed. Papillary muscles were dissected to yield at least three thin strips (∼100–140 μm in diameter and ∼600 μm in length) consisting of longitudinally oriented bundles of myocytes as described previously (29Mulieri L.A. Hasenfuss G. Ittleman F. Blanchard E.M. Alpert N.R. Circ. Res. 1989; 65: 1441-1449Crossref PubMed Google Scholar, 30Blanchard E. Seidman C. Seidman J.G. LeWinter M. Maughan D. Circ. Res. 1999; 84: 475-483Crossref PubMed Scopus (80) Google Scholar). Strips were skinned for 2 h at 22 °C and stored at –20 °C for no more than 1 week. (Generally, myocardial strip experiments were performed the day of dissection or the day after.) Shortly before each experiment, an aluminum T-clip was attached to each end, producing an undisturbed central segment of ∼200 μm. X-ray Diffraction—Papillary muscle strips from four 1vΔ5–14 and four NTG mice at 13 weeks of age were prepared as described above, secured between adjustable hooks in a perfusion chamber that contained relaxing solution, and stretched to 2.2-μm sarcomere length. The perfusion chamber had thin Mylar windows that allowed the x-ray beam to pass through the strip. X-ray diffraction patterns were obtained using the small angle instrument on the BioCAT beam line (31Irving T.C. Fischetti R. Rosenbaum G. Bunker G.B. Nucl. Instr. and Meth. A. 2000; 448: 250-254Crossref Scopus (21) Google Scholar) and a CCD detector at the Advanced Photon Source (Argonne, IL) as described previously (22Irving T.C. Konhilas J. Perry D. Fischetti R. de Tombe P.P. Am. J. Physiol. 2000; 279: H2568-H2573Crossref PubMed Google Scholar). The separation of the 1,0 equatorial reflections in diffraction patterns is inversely proportional to the distance between the crystallographic lattice planes of hexagonally packed thick filaments (d1,0). Lattice spacing (d1,0) was converted to interfilament spacing (d1,0× 2/√3) as a measure of the center-to-center spacing between thick filaments (22Irving T.C. Konhilas J. Perry D. Fischetti R. de Tombe P.P. Am. J. Physiol. 2000; 279: H2568-H2573Crossref PubMed Google Scholar). Interfilament spacing was measured in relaxing solution before skinning (intact spacing) and after skinning in the presence of 0–10% (w/v) dextran T-500. Skinned Myocardial Strip Mechanics—We conducted sinusoidal analysis on skinned myocardial strips obtained from mice that had echocardiograms. Papillary muscle strips were isolated from 1vΔ5–14 and NTG mice at 24 (young) and 58 (old) weeks, skinned, and T-clipped as described above. A strip was mounted between a piezoelectric motor (Physik Instrumente, Auburn, MA) and a strain gauge (SensorNor, Horten, Norway), lowered into a 30-μl droplet of relaxing solution maintained at 27 °C, and incrementally stretched to (and maintained at) 2.2-μm sarcomere length as determined using a filar micrometer (30Blanchard E. Seidman C. Seidman J.G. LeWinter M. Maughan D. Circ. Res. 1999; 84: 475-483Crossref PubMed Scopus (80) Google Scholar). Papillary muscle strips were calcium-activated by exchanging equal volumes of bathing solution for activating solution, thereby incrementally increasing the free calcium concentration from pCa 8.0 to pCa 4.25. Individual recordings of normalized isometric tension were fit to the Hill equation: [Ca2+]n/([Ca2+]50n + [Ca2+]n), where [Ca2+]50 is the calcium concentration at half-activation, pCa50 is –log [Ca2+]50, and n is the Hill coefficient. At each calcium concentration, sinusoidal perturbations of amplitude 0.125% strip length were applied at 42 discrete frequencies (0.125–100 Hz). Complex stiffness (expressed as the amplitude ratio and the phase shift between the force response and length perturbation) was calculated at each frequency (30Blanchard E. Seidman C. Seidman J.G. LeWinter M. Maughan D. Circ. Res. 1999; 84: 475-483Crossref PubMed Scopus (80) Google Scholar, 32Kawai M. Saeki Y. Zhao Y. Circ. Res. 1993; 73: 35-50Crossref PubMed Scopus (114) Google Scholar, 33Kawai M. Brandt P.W. J. Muscle Res. Cell Motil. 1980; 1: 279-303Crossref PubMed Scopus (240) Google Scholar). Complex modulus (i.e. normalized complex stiffness) was calculated by multiplying the complex stiffness by the length of the muscle preparation and then dividing by the cross-sectional area. Thus, the amplitude of the complex modulus is the ratio of the stress response to the strain perturbation, whereas the phase of the complex modulus is the shift of the stress with respect to the strain. Because the strain perturbation was held constant in these experiments, the amplitude and phase of the complex modulus provide insight into changes in stress. At maximum calcium activation (pCa 4.25), the velocity of unloaded shortening was determined using the Edman slack test (34Edman K.A. J. Physiol. (Lond.). 1979; 291: 143-159Crossref Scopus (553) Google Scholar). Experimental runs were concluded by conducting sinusoidal analysis on strips bathed in rigor solution, followed by chemical fixation (10% (v/v) glutaraldehyde added to rigor solution) that allowed a measure of end compliance (i.e. the compliance of the T-clipped portion in series with the fixed central muscle segment). Data Analysis—Data are presented as means ± S.E. All statistical analyses were performed using SPSS Version 11.0. Statistical tests were considered significant at the p < 0.05 level. A one-way analysis of variance (ANOVA) was performed to determine the strain effects (1vΔ5–14 versus NTG) on the interfilament spacing data. A two-way ANOVA was performed to determine the effects of strain (1vΔ5–14 versus NTG) and age (young versus old) on the echocardiogram, morphology, velocity, isometric tension, power, and frequency of maximum power data (data independent of strip oscillation frequency). A one-way ANOVA was also performed on the same data to determine additional relationships between the various means. If the one-way ANOVA differences were significant, the least significant difference post hoc test was used to determine which means differed. A repeated measures ANOVA with strip oscillation frequency as the repeated measure was performed on the elastic and viscous moduli to examine whether these data changed with oscillation frequency. If a significant frequency by strain or age interaction was found, then a two-way ANOVA was performed at each frequency to determine the effects of strain and age. Echocardiographic and Weight Measures—Although echocardiographic methods indicated a significant increase in posterior wall thickness for young 1vΔ5–14 mice (TABLE ONE), overall, the ventricular-to-body weight ratios were not significantly different between the cohorts (TABLE TWO). The end systolic and end diastolic left ventricular diameters of 1vΔ5–14 mice were similar to those of NTG mice (TABLE ONE), demonstrating that the internal chamber volumes and hence ejection fraction were comparable between the different strains and ages. Heart rates decreased significantly (18%) with age for 1vΔ5–14 mice, whereas the heart rates for NTG mice did not change with age (TABLE ONE), as reported in a previous study (35Yang B. Larson D.F. Watson R. Am. J. Physiol. 1999; 277: H1906-H1913Crossref PubMed Google Scholar).TABLE ONEEchocardiogram measurements EDD ESD PDWT PSWT FS HR mm mm mm mm % bpm Young mice NTG (n = 10) 2.87 ± 0.07 1.33 ± 0.05 1.14 ± 0.05 1.56 ± 0.08 53 ± 2 449 ± 19 1vΔ5-14 (n = 16) 2.88 ± 0.06 1.25 ± 0.05 1.41 ± 0.06aSignificantly different from young NTG mice (p < 0.05). 1.86 ± 0.08aSignificantly different from young NTG mice (p < 0.05). 57 ± 2 496 ± 17 Old mice NTG (n = 9) 2.96 ± 0.08 1.38 ± 0.10 1.28 ± 0.07 1.89 ± 0.08aSignificantly different from young NTG mice (p < 0.05). 54 ± 2 468 ± 4 1vΔ5-14 (n = 11) 3.04 ± 0.11 1.46 ± 0.08 1.30 ± 0.05 1.93 ± 0.04aSignificantly different from young NTG mice (p < 0.05). 52 ± 1 406 ± 24bSignificantly different from young 1vΔ5-14 mice (p < 0.05). Significant difference Strain, strain by age Strain by age Strain by agea Significantly different from young NTG mice (p < 0.05).b Significantly different from young 1vΔ5-14 mice (p < 0.05). Open table in a new tab TABLE TWOBody weight and ventricle/body weight ratios BW VENT/BW LV/BW RV/BW Young mice NTG (n = 3) 24.4 ± 1.2 4.87 ± 0.64 3.93 ± 0.41 0.94 ± 0.23 1vΔ5-14 (n = 4) 22.9 ± 2.1 5.80 ± 0.40 4.44 ± 0.20 1.36 ± 0.23 Old mice NTG (n = 3) 27.8 ± 0.4 5.05 ± 0.11 4.07 ± 0.15 0.98 ± 0.14 1vΔ5-14 (n = 3) 26.7 ± 1.8 5.06 ± 0.33 3.97 ± 0.23 1.09 ± 0.14 Open table in a new tab Transcript and Myosin Heavy Chain Analyses—To confirm the absence of a pathological hypertrophic response, we analyzed a series of transcripts that are normally altered during and whose up- or down-regulation can precede the development of a hypertrophic response or onset of heart failure (36Fewell J.G. Hewett T.E. Sanbe A. Klevitsky R. Hayes E. Warshaw D. Maughan D. Robbins J. J. Clin. Investig. 1998; 101: 2630-2639Crossref PubMed Scopus (74) Google Scholar, 37Jones W.K. Grupp I.L. Doetschman T. Grupp G. Osinska H. Hewett T.E. Boivin G. Gulick J. Ng W.A. Robbins J. J. Clin. Investig. 1996; 98: 1906-1917Crossref PubMed Scopus (162) Google Scholar). No overt changes could be detected in the transcript levels among the NTG and 1vΔ5–14 cohorts (Fig. 1A). α- and β-MHC protein levels were also similar among 1vΔ5–14, NTG, and ELC1v-wt mice (Fig. 1B). The similarity in gene expression and MHC protein levels indicates that no significant pathological hypertrophic response occurred at the molecular and protein levels. X-ray Diffraction—Prior to conducting the mechanical experiments on isolated skinned myocardial strips, we carried out small angle x-ray diffraction experiments to determine the osmotic pressure required to restore the myofilament lattice of the skinned preparation to its intact spacing. The thick-to-thick filament distance (44.9 nm) in the intact strip from 1vΔ5–14 mice was not significantly different compared with NTG mice (Fig. 2). The lattice of both strains expanded by ∼8% upon skinning, consistent with the loss of the osmotic constraint to swelling ordinarily imposed by the pla" @default.
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• W1990421818 title "The Essential Light Chain N-terminal Extension Alters Force and Fiber Kinetics in Mouse Cardiac Muscle" @default.
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