Familial Hypertrophic Cardiomyopathy-linked Alterations in Ca2+ Binding of Human Cardiac Myosin Regulatory Light Chain Affect Cardiac Muscle Contraction
2004; Elsevier BV; Volume: 279; Issue: 5 Linguagem: Inglês
10.1074/jbc.m307092200
ISSN1083-351X
AutoresDanuta Szczesna‐Cordary, Georgianna Guzman, Shuk-Shin Ng, Jiaju Zhao,
Tópico(s)Viral Infections and Immunology Research
ResumoThe ventricular isoform of human cardiac regulatory light chain (HCRLC) has been shown to be one of the sarcomeric proteins associated with familial hypertrophic cardiomyopathy (FHC), an autosomal dominant disease characterized by left ventricular and/or septal hypertrophy, myofibrillar disarray, and sudden cardiac death. Our recent studies have demonstrated that the properties of isolated HCRLC could be significantly altered by the FHC mutations and that their detrimental effects depend upon the specific position of the missense mutation. This report reveals that the Ca2+ sensitivity of myofibrillar ATPase activity and steady-state force development are also likely to change with the location of the specific FHC HCRLC mutation. The largest effect was seen for the two FHC mutations, N47K and R58Q, located directly in or near the single Ca2+-Mg2+ binding site of HCRLC, which demonstrated no Ca2+ binding compared with wild-type and other FHC mutants (A13T, F18L, E22K, P95A). These two mutants when reconstituted in porcine cardiac muscle preparations increased Ca2+ sensitivity of myofibrillar ATPase activity and force development. These results suggest the importance of the intact Ca2+ binding site of HCRLC in the regulation of cardiac muscle contraction and imply its possible role in the regulatory light chain-linked pathogenesis of FHC. The ventricular isoform of human cardiac regulatory light chain (HCRLC) has been shown to be one of the sarcomeric proteins associated with familial hypertrophic cardiomyopathy (FHC), an autosomal dominant disease characterized by left ventricular and/or septal hypertrophy, myofibrillar disarray, and sudden cardiac death. Our recent studies have demonstrated that the properties of isolated HCRLC could be significantly altered by the FHC mutations and that their detrimental effects depend upon the specific position of the missense mutation. This report reveals that the Ca2+ sensitivity of myofibrillar ATPase activity and steady-state force development are also likely to change with the location of the specific FHC HCRLC mutation. The largest effect was seen for the two FHC mutations, N47K and R58Q, located directly in or near the single Ca2+-Mg2+ binding site of HCRLC, which demonstrated no Ca2+ binding compared with wild-type and other FHC mutants (A13T, F18L, E22K, P95A). These two mutants when reconstituted in porcine cardiac muscle preparations increased Ca2+ sensitivity of myofibrillar ATPase activity and force development. These results suggest the importance of the intact Ca2+ binding site of HCRLC in the regulation of cardiac muscle contraction and imply its possible role in the regulatory light chain-linked pathogenesis of FHC. The regulatory light chain (RLC) 1The abbreviations used are: RLCregulatory light chainHCRLChuman cardiac regulatory light chainTntroponinCTnCcardiac troponin CFHCfamilial hypertrophic cardiomyopathyWTwild typeELCessential light chainMREmean residue ellipticityCaMcalmodulinCDTA1,2-cyclohexylenedinitrilotetraacetic acid.1The abbreviations used are: RLCregulatory light chainHCRLChuman cardiac regulatory light chainTntroponinCTnCcardiac troponin CFHCfamilial hypertrophic cardiomyopathyWTwild typeELCessential light chainMREmean residue ellipticityCaMcalmodulinCDTA1,2-cyclohexylenedinitrilotetraacetic acid. of myosin is a major regulatory subunit of smooth-muscle and non-muscle myosins and a modulator of the troponin (Tn) -controlled regulation of the striated muscle contraction. The crystal structures of chicken skeletal S1 (1Rayment 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: 50-58Google Scholar) and the regulatory domain of scallop myosin, consisting of one RLC, one essential light chain (ELC), and a part of the myosin heavy chain (2Xie X. Harrison D.H. Schlichting I. Sweet R.M. Kalabokis V.N. Szent-Gyorgyi A.G. Cohen C. Nature. 1994; 368: 306-312Google Scholar), have revealed that the RLC is localized at the head-rod junction of the myosin heavy chain and, together with the ELC, stabilizes the α-helical neck of the myosin head. The N terminus of RLC is noncovalently bound to the myosin heavy chain between Asn-825 and Leu-842, whereas its C terminus wraps around the region located between Glu-808 and Val-826 of the myosin heavy chain (1Rayment 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: 50-58Google Scholar). The N-terminal domain of the RLC contains a divalent cation-binding site, located in the first helix-loop-helix motif, which binds both Ca2+ and Mg2+ (Fig. 1A). The N-terminal region of RLC also contains the myosin light chain kinase-specific phosphorylation site (Ser-15), which is located in the proximity of the cation-binding site (3Szczesna D. Curr. Drug Targets. 2003; 3: 187-197Google Scholar). regulatory light chain human cardiac regulatory light chain troponin cardiac troponin C familial hypertrophic cardiomyopathy wild type essential light chain mean residue ellipticity calmodulin 1,2-cyclohexylenedinitrilotetraacetic acid. regulatory light chain human cardiac regulatory light chain troponin cardiac troponin C familial hypertrophic cardiomyopathy wild type essential light chain mean residue ellipticity calmodulin 1,2-cyclohexylenedinitrilotetraacetic acid. Recent studies have revealed that the ventricular RLC is one of the sarcomeric proteins associated with familial hypertrophic cardiomyopathy (FHC) (4Poetter K. Jiang H. Hassanzadeh S. Master S.R. Chang A. Dalakas M.C. Rayment I. Sellers J.R. Fananapazir L. Epstein N.D. Nat. Genet. 1996; 13: 63-69Google Scholar, 5Flavigny J. Richard P. Isnard R. Carrier L. Charron P. Bonne G. Forissier J.F. Desnos M. Dubourg O. Komajda M. Schwartz K. Hainque B. J. Mol. Med. 1998; 76: 208-214Google Scholar, 6Andersen P.S. Havndrup O. Bundgaard H. Moolman-Smook J.C. Larsen L.A. Mogensen J. Brink P.A. Borglum A.D. Corfield V.A. Kjeldsen K. Vuust J. Christiansen M. J. Med. Genet. 2001; 38: E43Google Scholar). FHC is an autosomal dominant disease characterized by left ventricular hypertrophy, myofibrillar disarray, and sudden cardiac death. It is caused by missense mutations in various genes that encode for β-myosin heavy chain (7Geisterfer-Lowrance A.A. Kass S. Tanigawa G. Vosberg H.P. McKenna W. Seidman C.E. Seidman J.G. Cell. 1990; 62: 999-1006Google Scholar), myosin-binding protein C (8Watkins H. Conner D. Thierfelder L. Jarcho J.A. MacRae C. McKenna W.J. Maron B.J. Seidman J.G. Seidman C.E. Nat. Genet. 1995; 11: 434-437Google Scholar), ventricular RLC and ELC (4Poetter K. Jiang H. Hassanzadeh S. Master S.R. Chang A. Dalakas M.C. Rayment I. Sellers J.R. Fananapazir L. Epstein N.D. Nat. Genet. 1996; 13: 63-69Google Scholar, 5Flavigny J. Richard P. Isnard R. Carrier L. Charron P. Bonne G. Forissier J.F. Desnos M. Dubourg O. Komajda M. Schwartz K. Hainque B. J. Mol. Med. 1998; 76: 208-214Google Scholar, 6Andersen P.S. Havndrup O. Bundgaard H. Moolman-Smook J.C. Larsen L.A. Mogensen J. Brink P.A. Borglum A.D. Corfield V.A. Kjeldsen K. Vuust J. Christiansen M. J. Med. Genet. 2001; 38: E43Google Scholar, 9Epstein N.D. Adv. Exp. Med. Biol. 1998; 453: 105-114Google Scholar), troponin T (10Watkins H. McKenna W.J. Thierfelder L. Suk H.J. Anan R. O'Donoghue A. Spirito P. Matsumori A. Moravec C.S. Seidman J.G. Seidman C.E. N. Engl. J. Med. 1995; 332: 1058-1064Google Scholar), troponin I (11Kimura A. Harada H. Park J.E. Nishi H. Satoh M. Takahashi M. Hiroi S. Sasaoka T. Ohbuchi N. Nakamura T. Koyanagi T. Hwang T.H. Choo J.A. Chung K.S. Hasegawa A. Nagai R. Okazaki O. Nakamura H. Matsuzaki M. Sakamoto T. Toshima H. Koga Y. Imaizumi T. Sasazuki T. Nat. Genet. 1997; 16: 379-382Google Scholar), troponin C (TnC) (12Hoffmann B. Schmidt-Traub H. Perrot A. Osterziel K.J. Gessner R. Hum. Mutat. 2001; 17: 524Google Scholar), α-tropomyosin (13Thierfelder L. Watkins H. MacRae C. Lamas R. McKenna W. Vosberg H.P. Seidman J.G. Seidman C.E. Cell. 1994; 77: 701-712Google Scholar), actin (14Mogensen J. Klausen I.C. Pedersen A.K. Egeblad H. Bross P. Kruse T.A. Gregersen N. Hansen P.S. Baandrup U. Borglum A.D. J. Clin. Investig. 1999; 103: R39-R43Google Scholar), and titin (15Satoh M. Takahashi M. Sakamoto T. Hiroe M. Marumo F. Kimura A. Biochem. Biophys. Res. Commun. 1999; 262: 411-417Google Scholar). Depending on the affected gene and the site of the mutation, FHC has a variable presentation with regard to its degree, severity, and extent of myocardial disarray. The clinical manifestations of FHC range from benign to severe heart failure and to sudden cardiac death. The best characterized clinical cases include patients with β-myosin heavy chain mutations, who show a high level of cardiac hypertrophy, and those with troponin T mutations, who have less hypertrophy but a higher incidence of sudden cardiac death in young adults. To date 10 RLC FHC mutations have been identified, eight of which are single point mutations and two are intronic splice site mutations (Fig. 1B). The first three mutations, identified in an American population, A13T, E22K, and P95A, were shown to have a rare cardiac phenotype that involved massive hypertrophy of the cardiac papillary muscles and adjacent ventricular tissue causing a mid-cavity obstruction (4Poetter K. Jiang H. Hassanzadeh S. Master S.R. Chang A. Dalakas M.C. Rayment I. Sellers J.R. Fananapazir L. Epstein N.D. Nat. Genet. 1996; 13: 63-69Google Scholar). Two other RLC mutations, F18L and R58Q, identified in a French population (5Flavigny J. Richard P. Isnard R. Carrier L. Charron P. Bonne G. Forissier J.F. Desnos M. Dubourg O. Komajda M. Schwartz K. Hainque B. J. Mol. Med. 1998; 76: 208-214Google Scholar), were associated with a classic form of hypertrophic cardiomyopathy that causes increased left ventricular wall thickness and abnormal electrocardiogram findings with no mid-cavity obliteration. A new report by Richard et al. (16Richard P. Charron P. Carrier L. Ledeuil C. Cheav T. Pichereau C. Benaiche A. Isnard R. Dubourg O. Burban M. Gueffet J.-P. Millaire A. Desnos M. Schwartz K. Hainque B. Komajda M. for the EUROGENE Heart Failure Project Circulation. 2003; 107: 2227-2232Google Scholar) identified two additional mutations of French origin, D166L and a splice site mutation, intervening sequences 5-2 A → G of intron 5. Three subsequent RLC FHC mutations were found in the Danish cohort, N47K, K104E, and another splice site mutation, intervening sequences 6-1 G → C of intron 6 (6Andersen P.S. Havndrup O. Bundgaard H. Moolman-Smook J.C. Larsen L.A. Mogensen J. Brink P.A. Borglum A.D. Corfield V.A. Kjeldsen K. Vuust J. Christiansen M. J. Med. Genet. 2001; 38: E43Google Scholar) (Fig. 1). A C → A transversion in exon 3 of the RLC gene, resulting in the N47K substitution (Fig. 1B), was identified in the 60-year-old proband of a Danish family (6Andersen P.S. Havndrup O. Bundgaard H. Moolman-Smook J.C. Larsen L.A. Mogensen J. Brink P.A. Borglum A.D. Corfield V.A. Kjeldsen K. Vuust J. Christiansen M. J. Med. Genet. 2001; 38: E43Google Scholar). The patient had severe septal and ventricular hypertrophy, abnormal electrocardiogram findings, and a high, relatively fixed mid-ventricular flow gradient, as well as diastolic filling abnormalities. The mid-ventricular flow gradient was caused not only by the pronounced septal hypertrophy but also by a significant increase in the size of the papillary muscle apparatus. Interestingly, a marked progression in the septal hypertrophy, from 31 to 45 mm, was seen over a 2-year span from age 60 to 62 years. The N47K mutation was not identified in the 150 healthy controls or in the other 197 probands. No other mutations in the additional seven FHC genes that were screened in parallel were identified in this patient (6Andersen P.S. Havndrup O. Bundgaard H. Moolman-Smook J.C. Larsen L.A. Mogensen J. Brink P.A. Borglum A.D. Corfield V.A. Kjeldsen K. Vuust J. Christiansen M. J. Med. Genet. 2001; 38: E43Google Scholar). The amino acid sequence analysis of cardiac RLC from different organisms reveals a high sequence homology among RLC from different species and demonstrates that the FHC-mutated residues of HCRLC are highly conserved in all of the presented RLC sequences (Fig. 2A). The structure of the N terminus of RLC is similar to other EF-hand Ca2+-binding proteins such as calmodulin (CaM) or TnC (Fig. 2B), whereas the C terminus is considerably less similar (1Rayment 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: 50-58Google Scholar). Interestingly, sequence comparison of HCRLC and other EF-hand Ca2+-binding proteins reveals that amino acids of both Ca2+ binding site mutations, N47K and R58Q of HCRLC, appear as Lys and Gln in the equivalent positions of the CaM and TnC sequences (Fig. 2B). This study presents a physiological characterization of the FHC HCRLC mutants when reconstituted in porcine cardiac muscle preparations and also reports new solution data for the N47K-HCRLC mutation. We demonstrate that two Ca2+ binding site mutants, N47K and R58Q, in which the Ca2+ binding ability was lost due to the FHC mutation, increased Ca2+ sensitivity of myofibrillar ATPase activity and steady-state force. Our results suggest that the FHC-associated perturbations of the HCRLC Ca2+ binding site that lead to its inactivation and produce alterations in the Ca2+-dependent ATPase/force could be a key mechanism of the RLC-linked pathogenesis of FHC. We also propose the importance of the intact Ca2+ binding site of HCRLC in the regulation of cardiac muscle contraction in the normal and diseased state of the heart. Mutation, Expression, and Purification of Wild-type HCRLC and the FHC Mutants—The cDNA for wild-type (WT) human cardiac RLC (HCRLC) was cloned by reverse transcription PCR using primers based on the published cDNA sequence (GenBank™ accession number AF020768) and standard methods (17Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. John Wiley & Sons, New York1995: 8.0.1-8.5.9Google Scholar). The FHC RLC mutants, A13T, F18L, E22K, N47K, R58Q, and P95A, were generated using overlapping sequential PCR (17Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. John Wiley & Sons, New York1995: 8.0.1-8.5.9Google Scholar). Wild-type and mutant cDNAs were constructed with an NcoI site at the N-terminal ATG and a BamHI site following the stop codon, to facilitate ligation into the NcoI-BamHI cloning site of the pET-3d (Novagen) plasmid vector and transformation into DH5α cloning host bacteria for expression of the cDNAs of WT-HCRLC and the FHC mutants. The CDNAs of the proteins were transformed into BL21 expression host cells, and proteins were expressed in large (16 liters) cultures. Expressed proteins were purified as described previously (18Szczesna D. Ghosh D. Li Q. Gomes A.V. Guzman G. Arana C. Zhi G. Stull J.T. Potter J.D. J. Biol. Chem. 2001; 276: 7086-7092Google Scholar). Flow Dialysis—Flow dialysis was performed in a solution of 100 mm KCl, 20 mm imidazole buffer, pH 7.0 (22 °C). All proteins were equilibrated in this buffer prior to the measurements. The flow dialysis experiments were performed according to Colowick and Womack (19Colowick S.P. Womack F.C. J. Biol. Chem. 1969; 244: 774-777Google Scholar) with slight modifications described in detail previously (18Szczesna D. Ghosh D. Li Q. Gomes A.V. Guzman G. Arana C. Zhi G. Stull J.T. Potter J.D. J. Biol. Chem. 2001; 276: 7086-7092Google Scholar). Data were calculated using Scatchard analysis (20Scatchard G. Ann. N. Y. Acad. Sci. 1949; 51: 660-672Google Scholar) as described (18Szczesna D. Ghosh D. Li Q. Gomes A.V. Guzman G. Arana C. Zhi G. Stull J.T. Potter J.D. J. Biol. Chem. 2001; 276: 7086-7092Google Scholar). CD Measurements—Far-UV circular dichroism (CD) spectra were obtained using a 1-mm-path quartz cell in a Jasco J-720 spectropolarimeter. Spectra were recorded at 195-250 nm with a bandwidth of 1 nm at a speed of 50 nm/min and a resolution of 0.2 nm. Analysis and processing of data were done using the Jasco system software (Windows standard analysis, version 1.20). Ten scans were averaged, base lines subtracted, and no numerical smoothing applied. Values of mean residue ellipticity ([θ]MRE, in degree·cm2/dmol) for the spectra were calculated (utilizing the same Jasco system software) using the following equation (21Greenfield N. Fasman G.D. Biochemistry. 1969; 8: 4108-4116Google Scholar, 22Holt J.C. Lowey S. Biochemistry. 1975; 14: 4600-4609Google Scholar, 23Huang W. Wilson G.J. Brown L.J. Lam H. Hambly B.D. Eur. J. Biochem. 1998; 257: 457-465Google Scholar),[θ]MRE=[θ]/(10·Cr·1)(Eq. 1) where [θ] is the measured ellipticity in millidegrees, Cr is the mean residue molar concentration, and l is the path length in cm. The optical activity of the buffer was subtracted from relevant protein spectra. The α-helical content for each protein was calculated using the standard equation for [θ] at 222 nm (24Chen Y.H. Yang J.T. Biochem. Biophys. Res. Commun. 1971; 44: 1285-1291Google Scholar),[θ]222=-30,300fH-2,340(Eq. 2) where fH is the fraction of α-helical content (fH × 100, expressed in %). The measurements were performed at 5 and 22 °C in 30 mm NaCl, 0.3 mm EGTA, 0.7 mm MgCl2, and 3 mm Tris-HCl buffer at pH 7.4. Spectra were presented as mean residue ellipticity as shown previously (18Szczesna D. Ghosh D. Li Q. Gomes A.V. Guzman G. Arana C. Zhi G. Stull J.T. Potter J.D. J. Biol. Chem. 2001; 276: 7086-7092Google Scholar). Depletion of Endogenous RLC from Porcine Cardiac Myofibrils (CMF) Followed by Reconstitution with CTnC and FHC HCRLC Mutants—CMF were prepared from the left ventricular walls and papillary muscles of porcine hearts from healthy young adult animals according to Solaro et al. (25Solaro R.J. Pang D.C. Briggs F.N. Biochim. Biophys. Acta. 1971; 245: 259-262Google Scholar). They were stored at a concentration of about 20 mg/ml in SB buffer containing 30 mm imidazole, 60 mm KCl, 2 mm MgCl2, pH 7, and 50% glycerol at -20 °C. Before experiments were performed, CMF were removed from 50% glycerol stock, diluted with an equal volume of SB solution containing 1 mm dithiothreitol (SB1), and pelleted at 800 × g. They were re-suspended, tested for protein concentration (determined by a Coomassie protein assay), and diluted to 2.5 mg/ml in the same buffer. The RLC extraction was initiated with a wash of CMF with a solution containing 5 mm CDTA, 50 mm KCl, 1% Triton X-100, 40 mm Tris-HCl, pH 8.4, 1 μg/ml pepstatin A, 0.6 mm NaN3, and 0.2 mm phenylmethylsulfonyl fluoride for 5 min at room temperature. Then the CMF were pelleted, resuspended to 2.5 mg/ml, and incubated in the same solution for another 30 min at room temperature. They were washed three times with SB1 buffer, assayed for protein concentration, and diluted to 2.5 mg/ml. The RLC-depleted CMF were then reconstituted with a 40 μm solution of HCRLC-WT and/or FHC mutants and 8 μm porcine cardiac TnC (CTnC) (for which partial extraction could take place during the RLC depletion process) for 1.5 h at room temperature (Fig. 4). The HCRLC- and CTnC-reconstituted CMF were washed twice with SB1 buffer, assayed for protein concentration, and diluted to 2.5 mg/ml in SB1 buffer. 100-μl aliquots of CMF were then used for myofibrillar ATPase assays. Myofibrillar ATPase Assays—Myofibrillar ATPase activity assays of the control (not extracted), RLC-depleted, and HCRLC/CTnC-reconstituted CMF were performed in a solution of 30 mm imidazole, pH 7, 50 mm KCl, 2 mm MgCl2, and different concentrations of Ca2+ from pCa 9 to 4.5. After a 5-min incubation at 30 °C, the reaction was initiated with 2.5 mm MgATP and terminated after 5 min with 5% trichloroacetic acid. Released inorganic phosphate was measured according to Fiske and Subbarow (26Fiske C. Subbarow Y. J. Biol. Chem. 1925; 66: 375-400Google Scholar). Skinned Cardiac Muscle Fibers—Freshly isolated porcine hearts were placed in oxygenated physiological salt solution of 140 mm NaCl, 4 mm KCl, 1.8 mm CaCl2, 1.0 mm MgCl2, 1.8 mm NaH2PO4, 5.5 mm glucose, and 50 mm Hepes buffer, pH 7.4. The papillary muscles of the left ventricles were isolated, dissected into muscle bundles of about 20 × 3 mm, and chemically skinned in a 50% glycerol, 50% pCa 8 solution (10-8m [Ca2+], 1mm [Mg2+], 7mm EGTA, 5 mm [MgATP2+], 20 mm imidazole, pH 7.0, 15 mm creatinine phosphate; ionic strength = 150 mm adjusted with potassium propionate) containing 1% Triton X-100 for 24 h at 4 °C. Then the fibers were transferred to the same solution without Triton X-100 and stored at -20 °C for about 2 months. CDTA Extraction of Cardiac Muscle Fibers—Endogenous RLC depletion from porcine cardiac muscle fiber preparations was achieved in the same solution utilized for CMF extraction, containing 5 mm CDTA, 40 mm Tris, 50 mm KCl, 1 μg/ml pepstatin A, 0.6 mm NaN3, 0.2 mm phenylmethylsulfonyl fluoride, and 1% Triton X-100, pH 8.4. The fibers were incubated in this solution for 5 min at room temperature and then transferred to the fresh solution of the same composition for another 30 min. The extent of RLC extraction was tested by SDS-PAGE (Fig. 6A). Depletion of the endogenous RLC may result in partial extraction of the endogenous TnC, and therefore, both of these proteins were added back into the CDTA-treated fibers (Fig. 6). Reconstitution of the CDTA-depleted Fibers with CTnC and FHC HCRLC Mutants—Reconstitution of the RLC-depleted fibers with porcine CTnC and HCRLC-WT or A13T, F18L, E22K, N47K, R58Q, and P95A mutants was performed in pCa 8 solution containing 40 μm HCRLC and 15 μm CTnC. The solution of CTnC was included in the reconstitution protein mixture during the first 30 min of fiber incubation followed by a 30-min incubation with fresh HCRLC solution at room temperature. The addition of CTnC was to assure that the fibers were not deficient in CTnC, because its partial extraction could affect the Ca2+ sensitivity of force development. Reconstituted fibers were then washed in pCa 8 solution and subjected to force measurements. The SDS-PAGE of the control, CDTA-depleted, and CTnC- and HCRLC-WT- or FHC mutant-reconstituted fibers is presented in Fig. 6. Steady-state Force Development—A bundle of 3-5 single fibers isolated from a batch of glycerinated fibers was attached by tweezer clips to a force transducer, placed in a 1-ml cuvette, and bathed in pCa 8 solution containing 1% Triton X-100. The fibers were then tested for steady-state force development in pCa 4 solution (composition is the same as pCa 8 buffer except [Ca2+] = 10-4m) and relaxed in the pCa 8 solution. Steady-state force development was monitored for control, CDTA-depleted, and then CTnC- and WT-, A13T-, F18L-, E22K-, N47K-, R58Q-, and P95A-reconstituted fibers. Ca2+ Dependence of Force Development—After the initial steady-state force was determined, the fibers were relaxed in the pCa 8 buffer and then exposed to solutions of increasing Ca2+ concentrations (from pCa 8 to 4). The maximal force was measured in each "pCa" solution followed by a short relaxation of the fibers in pCa 8 solution. Data were analyzed using the following equations,%forcerestored=100×(forcerestored-residualforce)/initialforce(Eq. 3) %changeinforce=100×[Ca2+]nH/([Ca2+]nH+[Ca502+]nH)(Eq. 4) where [Ca2+50] is the free Ca2+ concentration that produces 50% force and nH is the Hill coefficient. SDS Gel Electrophoresis—Control, CDTA-depleted and CTnC/WT-, A13T-, F18L-, E22K-, N47K-, R58Q-, and P95A-reconstituted porcine cardiac myofibrils and fibers were run on 15% SDS-PAGE according to Laemmli (27Laemmli U.K. Nature. 1970; 227: 680-685Google Scholar) (Figs. 4 and 6). The respective HCRLC protein bands were quantified utilizing densitometry with the Scion Image for Windows (version beta 4.0.2). The percent of RLC depletion and/or reconstitution was calculated from the net intensity of the RLC bands compared with the control untreated fibers (100%). Differences in gel loading were corrected by comparing the RLC bands with the ELC bands (not affected by the RLC extraction/reconstitution procedure) of the respective fibers. Statistical Analysis—The significant difference between the pCa50 values of the Ca2+ sensitivity of myofibrillar ATPase activity and force development among WT and respective FHC HCRLC mutants was determined utilizing an unpaired Student's t test (Sigma Plot 8.0), with significance defined as p < 0.05. Effect of N47K Mutation in HCRLC on Ca2+ Binding and the CD Spectrum—We have recently published the Ca2+ binding properties and the CD spectra of human cardiac RLC and five recombinant FHC HCRLC mutants (A13T, F18L, E22K, R58Q,and P95A) (18Szczesna D. Ghosh D. Li Q. Gomes A.V. Guzman G. Arana C. Zhi G. Stull J.T. Potter J.D. J. Biol. Chem. 2001; 276: 7086-7092Google Scholar). Three of the FHC mutations, A13T, F18L,and P95A, decreased the KCa ∼3-fold, whereas two other mutations, E22K and R58Q, changed the Ca2+ binding properties in a more drastic way. Compared with WT-HCRLC (KCa = 6.67 ± 0.21 × 105m-1), the E22K mutation decreased the KCa value by ∼17-fold, whereas the R58Q mutation eliminated Ca2+ binding to HCRLC (18Szczesna D. Ghosh D. Li Q. Gomes A.V. Guzman G. Arana C. Zhi G. Stull J.T. Potter J.D. J. Biol. Chem. 2001; 276: 7086-7092Google Scholar) (Table I). Flow dialysis experiments utilizing 45Ca2+ performed for the new N47K mutant showed no Ca2+ binding to the single Ca2+-Mg2+ site of HCRLC (Table I). Therefore, the Asn → Lys substitution in the second from last coordinating position of the Ca2+ binding loop of HCRLC abolished its Ca2+ binding.Table IEffect of FHC mutations in the HCRLC on Ca2+ binding to isolated HCRLC (data from Ref.18Szczesna D. Ghosh D. Li Q. Gomes A.V. Guzman G. Arana C. Zhi G. Stull J.T. Potter J.D. J. Biol. Chem. 2001; 276: 7086-7092Google Scholar) and on the apparent Ca2+ dissociation constants (Kapp) of HCRLC- and CTnC-reconstituted porcine cardiac myofibrils and fibersHCRLC proteinIsolated HCRLCaApparent Ca2+ dissociation constants (1/KCa) of isolated HCRLC-WT and FHC mutants (from Ref. 18). Flow dialysis was performed in a solution of 100 mm KCl, 20 mm imidazole buffer, pH 7.0 (22 °C) No Mg2+MyofibrilsbKapp of reconstituted cardiac myofibrils expressing the free Ca2+ concentration, which produces 50% of myofibrillar ATPase activity (pCa50 values ±S.E.) (n is specified in Fig. 5C)2 mm Mg2+FiberscKapp of reconstituted skinned fibers expressing the free Ca2+ concentration, which produces 50% of steady state force (pCa50 values ±S.E.) (n is specified in Fig. 7)1 mm Mg2+Force recovery compared with intact fibersμmμmμm% (n)WT1.50 ± 0.020.200 ± 0.0092.88 ± 0.1984.1 ± 4.5 (8)A13T4.85 ± 0.310.191 ± 0.0153.02 ± 0.0676.7 ± 3.8 (4)F18L4.20 ± 0.260.224 ± 0.0622.63 ± 0.1074.3 ± 2.1 (5)E22K25.64 ± 3.480.178 ± 0.0012.63 ± 0.1066.4 ± 2.8 (6)N47KNo binding0.141 ± 0.0092.51 ± 0.1074.0 ± 3.2 (8)R58QNo binding0.170 ± 0.0042.19 ± 0.1077.7 ± 2.0 (4)P95A4.74 ± 1.050.200 ± 0.0112.75 ± 0.2570.7 ± 4.7 (4)a Apparent Ca2+ dissociation constants (1/KCa) of isolated HCRLC-WT and FHC mutants (from Ref. 18Szczesna D. Ghosh D. Li Q. Gomes A.V. Guzman G. Arana C. Zhi G. Stull J.T. Potter J.D. J. Biol. Chem. 2001; 276: 7086-7092Google Scholar). Flow dialysis was performed in a solution of 100 mm KCl, 20 mm imidazole buffer, pH 7.0 (22 °C)b Kapp of reconstituted cardiac myofibrils expressing the free Ca2+ concentration, which produces 50% of myofibrillar ATPase activity (pCa50 values ±S.E.) (n is specified in Fig. 5C)c Kapp of reconstituted skinned fibers expressing the free Ca2+ concentration, which produces 50% of steady state force (pCa50 values ±S.E.) (n is specified in Fig. 7) Open table in a new tab Far-UV CD spectroscopy was used to analyze whether the N47K mutation altered the secondary structure of HCRLC. As shown in Fig. 3, the mutation did not introduce any significant changes to the CD spectrum of HCRLC, and the calculated α-helical content (at wavelength 222 nm) of N47K was 18.7 versus 18.5%, determined for WT-HCRLC (n = 10). These values of α-helical content were calculated from the mean residue ellipticity at 222 nm (at 22 °C), [θ]MRE ≈ -8020 or -7930 for N47K or HCRLC-WT, respectively. As demonstrated by others (23Huang W. Wilson G.J. Brown L.J. Lam H. Hambly B.D. Eur. J. Biochem. 1998; 257: 457-465Google Scholar, 28Wu C.S. Yang J.T. Biochemistry. 1976; 15: 3007-3014Google Scholar), they were similar to the values presented for rabbit skeletal RLC, although the percent of α-helical content calculated by these authors was shown to be higher (23Huang W. Wilson G.J. Brown L.J. Lam H. Hambly B.D. Eur. J. Biochem. 1998; 257: 457-465Google Scholar, 28Wu C.S. Yang J.T. Biochemistry. 1976; 15: 3007-3014Google Scholar). The effect of the N47K mutation on the CD spectrum of HCRLC was tested further at 5 °C. This was to assess the difference between these proteins at a low temperature-induced, possibly more folded, structure. Surprisingly, the CD spectra of these proteins did not significantly change, and their calculated α-helical content was about 19%. Interestingly, there was no change in the CD spectra of WT and N47K when monitored at 5 °C and then at 22 °C followed by the measurement at 5 °C. This suggests that the secondary structure of HCRLC is quite stable in this range of temperatures. Effect of FHC HCRLC Mutations on ATPase Activity in Reconstituted CMF—Extraction of endogenous RLC from porcine CMF was achieved with a 5-min wash followed by a 30-min incubation in the CDTA-containing solution (see "Materials and Methods"). Even though the CDTA solution contained 50 mm KCl to prevent extraction of TnC, the RLC- and possibly CTnC-depleted CMF were reconstituted
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