Identification of a Functionally Critical Protein Kinase C Phosphorylation Residue of Cardiac Troponin T
2003; Elsevier BV; Volume: 278; Issue: 37 Linguagem: Inglês
10.1074/jbc.m306325200
ISSN1083-351X
AutoresMarius P. Sumandea, W. Glen Pyle, Tomoyoshi Kobayashi, Pieter P. de Tombe, R. John Solaro,
Tópico(s)Viral Infections and Immunology Research
ResumoCardiac Troponin T (cTnT) is one prominent substrate through which protein kinase C (PKC) exerts its effect on cardiomyocyte function. To determine the specific functional effects of the cTnT PKC-dependent phosphorylation sites (Thr197, Ser201, Thr206, and Thr287) we first mutated these residues to glutamate (E) or alanine (A). cTnT was selectively mutated to generate single, double, triple, and quadruple mutants. Bacterially expressed mutants were evaluated in detergent-treated mouse left ventricular papillary muscle fiber bundles where the endogenous troponin was replaced with a recombinant troponin complex containing either cTnT phosphorylated by PKC-α or a mutant cTnT. We simultaneously determined isometric tension development and actomyosin Mg-ATPase activity of the exchanged fiber bundles as a function of Ca2+ concentration. Our systematic analysis of the functional role of the multiple PKC phosphorylation sites on cTnT identified a localized region that controls maximum tension, ATPase activity, and Ca2+ sensitivity of the myofilaments. An important and novel finding of our study was that Thr206 is a functionally critical cTnT PKC phosphorylation residue. Its exclusive phosphorylation by PKC-α or replacement by Glu (mimicking phosphorylation) significantly decreased maximum tension, actomyosin Mg-ATPase activity, myofilament Ca2+ sensitivity, and cooperativity. On the other hand the charge modification of the other three residues together (T197/S201/T287-E) had no functional effect. Fibers bundles containing phosphorylated cTnT-wt (but not the T197/S201/T206/T287-E) exhibited a significant decrease of tension cost as compared with cTnT-wt. Cardiac Troponin T (cTnT) is one prominent substrate through which protein kinase C (PKC) exerts its effect on cardiomyocyte function. To determine the specific functional effects of the cTnT PKC-dependent phosphorylation sites (Thr197, Ser201, Thr206, and Thr287) we first mutated these residues to glutamate (E) or alanine (A). cTnT was selectively mutated to generate single, double, triple, and quadruple mutants. Bacterially expressed mutants were evaluated in detergent-treated mouse left ventricular papillary muscle fiber bundles where the endogenous troponin was replaced with a recombinant troponin complex containing either cTnT phosphorylated by PKC-α or a mutant cTnT. We simultaneously determined isometric tension development and actomyosin Mg-ATPase activity of the exchanged fiber bundles as a function of Ca2+ concentration. Our systematic analysis of the functional role of the multiple PKC phosphorylation sites on cTnT identified a localized region that controls maximum tension, ATPase activity, and Ca2+ sensitivity of the myofilaments. An important and novel finding of our study was that Thr206 is a functionally critical cTnT PKC phosphorylation residue. Its exclusive phosphorylation by PKC-α or replacement by Glu (mimicking phosphorylation) significantly decreased maximum tension, actomyosin Mg-ATPase activity, myofilament Ca2+ sensitivity, and cooperativity. On the other hand the charge modification of the other three residues together (T197/S201/T287-E) had no functional effect. Fibers bundles containing phosphorylated cTnT-wt (but not the T197/S201/T206/T287-E) exhibited a significant decrease of tension cost as compared with cTnT-wt. Contraction and relaxation of the sarcomere of cardiac muscle represents the integrated activity of highly synchronized and fine-tuned protein-protein interactions. Post-translational modifications and mutations leading to alterations in the charges of sarcomeric proteins are critical to these protein-protein interactions. Charge-charge interactions are involved in the reaction of the molecular motor myosin with actin and in the control of the actin-myosin interaction by Tm and the troponin complex (Tn). 1The abbreviations used are: Tn, troponin; cTn, cardiac troponin; cTnT, cardiac troponin T; cTnI, cardiac troponin I; cTnC, cardiac troponin C; Tm, tropomyosin; PKC, protein kinase C; PKA, cAMP-dependent protein kinase; FHC, familial hypertrophic cardiomyopathy; DCM, dilated cardiomyopathy; MOPS, 3-[N-morpholino]propane-sulfonic acid; BES, N,N-bis[2-hydroxyethyl]-2-aminoethanesulfonic acid; DTT, dithiothreitol; wt, wild type; ATPγS, adenosine 5′-O-(thiotriphosphate). Troponin is a heterotrimeric protein consisting of a Ca2+-binding protein, TnC; an actomyosin Mg-ATPase-inhibiting protein, TnI, and a Tm-binding protein, TnT (for reviews see Refs. 1Tobacman L.S. Annu. Rev. Physiol. 1996; 58: 447-481Crossref PubMed Scopus (461) Google Scholar, 2Gordon A.M. Homsher E. Regnier M. Physiol. Rev. 2000; 80: 853-924Crossref PubMed Scopus (1342) Google Scholar, 3Solaro R.J. Berne R.M. Handbook of Physiology Section 2: The Cardiovascular System. Vol. I. Oxford University Press, UK2001: 264-300Google Scholar, 4Solaro R.J. Rarick H.M. Circ. Res. 1998; 83: 471-480Crossref PubMed Scopus (292) Google Scholar). Excellent examples of the major significance of the charged amino acids in these proteins are the functional effects of missense mutations and deletions that are causal in familial hypertrophic cardiomyopathies (FHC) (5Watkins 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: 1058-1064Crossref PubMed Scopus (782) Google Scholar, 6Watkins H. Seidman C.E. Seidman J.G. Feng H.S. Sweeney H.L. J. Clin. Invest. 1996; 98: 2456-2461Crossref PubMed Scopus (112) Google Scholar) or dilated cardiomyopathies (DCM) (7Kamisago M. Sharma S.D. DePalma S.R. Solomon S. Sharma P. McDonough B. Smoot L. Mullen M.P. Woolf P.K. Wigle E.D. Seidman J.G. Seidman C.E. N. Engl. J. Med. 2000; 343: 1688-1696Crossref PubMed Scopus (589) Google Scholar). Moreover, charge modifications induced by phosphorylation of cTnI at tissue-specific PKA sites and at PKC sites modifies the Ca2+ sensitivity, tension, and velocity of shortening of the myofilaments (8Burkart E.M. Sumandea M.P. Kobayashi T. Nili M. Martin A.F. Homsher E. Solaro R.J. J. Biol. Chem. 2003; 278: 11265-11272Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 9van der Velden J. de Jong J.W. Owen V.J. Burton P.B. Stienen G.J. Cardiovasc. Res. 2000; 46: 487-495Crossref PubMed Scopus (81) Google Scholar, 10Kajiwara H. Morimoto S. Fukuda N. Ohtsuki I. Kurihara S. Biochem. Biophys. Res. Commun. 2000; 272: 104-110Crossref PubMed Scopus (27) Google Scholar). In experiments reported here we have focused on the functional significance of PKC phosphorylation and specific charge modifications, mimicking phosphorylation, of cardiac cTnT. cTnT is the "lever" that transmits the signal generated by Ca2+-induced conformational changes in cTnC-cTnI structures to the filamentous protein, Tm. Upon Ca2+-activation Tm undergoes both a movement and a rotation on the actin filament to facilitate the binding of myosin heads to actin and to promote the process of contraction (11Bacchiocchi C. Lehrer S.S. Biophys. J. 2002; 82: 1524-1536Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 12Squire J.M. Morris E.P. FASEB J. 1998; 12: 761-771Crossref PubMed Scopus (168) Google Scholar, 13Lorenz M. Poole K.J. Popp D. Rosenbaum G. Holmes K.C. J. Mol. Biol. 1995; 246: 108-119Crossref PubMed Scopus (192) Google Scholar). As a critically positioned molecule in the thin filament, it is clear that modifications of cTnT have the potential for versatile interactions with adjacent proteins and for producing significant functional effects (14Communal C. Sumandea M. de Tombe P. Narula J. Solaro R.J. Hajjar R.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 6252-6256Crossref PubMed Scopus (330) Google Scholar). Even so, the specific effects of cTnT phosphorylation on tension and ATP hydrolysis have not been well studied in the myofilament lattice. There is no evidence for phosphorylation of cTnT by PKA. However, in vitro studies demonstrated that there are four main sites for PKC-dependent phosphorylation on cTnT (15Jideama N.M. Noland Jr., T.A. Raynor R.L. Blobe G.C. Fabbro D. Kazanietz M.G. Blumberg P.M. Hannun Y.A. Kuo J.F. J. Biol. Chem. 1996; 271: 23277-23283Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar), which are at Thr197, Ser201, Thr206, and Thr287 (mouse sequence). These sites are located in the functionally significant C-terminal half of the molecule. It is this region of cTnT that is likely to interact with cTnI and cTnC and possibly with Tm, and is essential in the transmission of the Ca2+-binding signal to Tm-actin (16Perry S.V. J. Muscle Res. Cell Motil. 1998; 19: 575-602Crossref PubMed Scopus (258) Google Scholar). Noland and Kuo (17Noland Jr., T.A. Kuo J.F. J. Mol. Cell Cardiol. 1993; 25: 53-65Abstract Full Text PDF PubMed Scopus (74) Google Scholar) reported that exclusive phosphorylation of cTnT results in a ∼50% decrease in maximum actomyosin Mg-ATPase activity using in vitro fully reconstituted systems. When cTnI was exclusively phosphorylated under the same conditions, there was a much smaller, ∼20%, decrease. A recent study in our laboratory has indicated that pan-activation of PKC results in phosphorylation of cTnT and cTnI and induces a 30% reduction of maximum force of mouse cardiac myofilaments (18Montgomery D.E. Chandra M. Huang Q. Jin J. Solaro R.J. Am. J. Physiol. Heart Circ. Physiol. 2001; 280: H1011-H1018Crossref PubMed Google Scholar). When cTnT was partially replaced with a fast skeletal (fs) isoform, which is not phosphorylated by PKC, the effect of PKC activation was no longer evident. These results indicated that cTnT phosphorylation might be pivotal for the PKC-induced depression of tension in the myofilament. However given the large number of PKC isoforms, multiple phosphorylation sites, the lack of homogeneously phosphorylated samples and inability of performing site-directed phosphorylation, the role of each cTnT phosphorylation site remains unknown. In the present study we generated cTnT mutants in which Glu (mimic of phosphorylation) or Ala residues were placed at the PKC phosphorylation sites. cTnT was selectively mutated to generate single, double, triple, and quadruple mutants (see Fig. 3). The wild-type and mutated cTnTs were used to reconstitute a recombinant cTn complex, which was incorporated into detergent extracted left ventricular papillary muscle fiber bundles by replacing the endogenous troponin complex (19Brenner B. Kraft T. Yu L.C. Chalovich J.M. Biophys. J. 1999; 77: 2677-2691Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 20She M. Trimble D. Yu L.C. Chalovich J.M. J. Muscle Res. Cell Motil. 2000; 21: 737-745Crossref PubMed Scopus (21) Google Scholar). We simultaneously determined tension development and actomyosin Mg-ATPase activity in the reconstituted preparations as a function of Ca2+ concentration (21Janssen P.M. de Tombe P.P. Am. J. Physiol. 1997; 273: H2415-H2422PubMed Google Scholar). Our data provide the first evidence that Thr206 is the functionally critical phosphorylation residue. Its exclusive phosphorylation by PKC-α or replacement by Glu (mimicking phosphorylation) significantly decreased maximum tension, actomyosin Mg-ATPase activity, myofilament Ca2+-sensitivity and cooperativity. On the other hand the charge modification of the other 3 residues together (T197/S201/T287-E) had no functional effect. Fibers bundles containing cTnT-wt-P (but not T197/S201/T206/T287-E) exhibited a significant decrease of tension cost as compared with cTnT-wt implicating cTnT phosphorylation in regulation of cross-bridge detachment rate. Preparation of Site-directed cTnT Mutants—cDNA of adult mouse cardiac TnT (a generous gift of Dr. Jil Tardiff) was previously cloned into a pSBETa vector (22Chandra M. Montgomery D.E. Kim J.J. Solaro R.J. J. Mol. Cell Cardiol. 1999; 31: 867-880Abstract Full Text PDF PubMed Scopus (72) Google Scholar) and selectively mutated using the Quick-Change Site-directed mutagenesis kit (Stratagene) according to the manufacturer's instructions. Sense primers (from Operon) shown in Table I were used for the respective mutations. Mutated residues are underlined. The identity of constructs was verified by DNA sequencing.Table IOligonucleotide primerscTnT-T197A-15′-gaagcaggctcaggctgagcggaagagtg-3′cTnT-T197E-15′-gaagcaggctcaggaagagcggaagagtg-3′cTnT-T206A-15′-gtgggaagagacaggctgagagagagaagaag-3′cTnT-T206E-15′-gtgggaagagacaggaagagagagagaagaag-3′cTnT T287A-15′-gggaaggccaaagtcgctgggcgttggaaatagg-3′cTnT-T287E-15′-gggaaggccaaagtcgaagggcgttggaaatagg-3′cTnT-T197A/S201A5′-gaagcaggctcaggctgagcggaaggctg-3′cTnT-T197E/S201E5′-gaagcaggctcaggaagagcggaaggaag-3′cTnT-S201A/T206A5′-gctggggaagagacaggctgagagagagaagaag-3′cTnT-S201E/T206E5′-gaagggaagagacaggaagagagagagaagaag-3′cTnT-T197E/S201E/T206E5′-ggaagagcggaaggaagggaagagacaggaag-3′cTnT-T197A/S201A/T206A5′-ggctgagcggaaggctgggaagagacaggctg-3′ Open table in a new tab Contractile Protein Expression and Purification—Human cardiac TnC and mouse cardiac TnI were expressed and purified as previously described (23Guo X. Wattanapermpool J. Palmiter K.A. Murphy A.M. Solaro R.J. J. Biol. Chem. 1994; 269: 15210-15216Abstract Full Text PDF PubMed Google Scholar). Bovine cardiac Tm for the cosedimentation assay was purified as previously described by Tobacman and Adelstein (24Tobacman L.S. Adelstein R.S. Biochemistry. 1986; 25: 798-802Crossref PubMed Scopus (62) Google Scholar). Recombinant adult mouse cTnT-wt and mutants were expressed and purified by a modified method of Chandra et al. (25Chandra M. Kim J.J. Solaro R.J. Biochem. Biophys. Res. Commun. 1999; 263: 219-223Crossref PubMed Scopus (32) Google Scholar). cTnT was expressed in BL21(DE3) cells using the pSBETa expression plasmid. BL21(DE3) cells grown over night in Luria Broth supplemented with 30 μg/ml kanamycin, were collected by centrifugation at 6000 × g for 10 min. at 4 °C. The cell pellet was then resuspended well in a suitable volume (about 200 ml) of TnT-Buffer A (20 mm Tris, pH 8.0, 6 m urea, 5 mm EDTA, 0.2 mm phenylmethylsulfonyl fluoride, 1 mm benzamidine, 1 mm DTT) containing 0.5% Triton X-100. The cells were lysed by sonication on ice, followed by 60 min of centrifugation at 48,000 × g, at 4 °C. The supernatant fraction was subjected to ammonium sulfate fractionation as previously reported (25Chandra M. Kim J.J. Solaro R.J. Biochem. Biophys. Res. Commun. 1999; 263: 219-223Crossref PubMed Scopus (32) Google Scholar). The final ammonium sulfate pellet was solubilized in TnT-Buffer A, and then dialyzed at 4 °C against TnT-Buffer A. The dialyzed sample was applied on a DEAE-Fast Flow Sepharose column (Amersham Biosciences) connected to a FPLC System (Amersham Biosciences). cTnT was eluted with a 0.0–0.4 m KCl gradient in TnT-Buffer A. The fractions containing cTnT were analyzed by 12% SDS-PAGE and those containing >90% pure cTnT were pooled, extensively dialyzed against 0.1% formic acid, 1 mm DTT in H2O, lyophilized, and stored in powder form at –80 °C. PKC-α Expression and Purification—Recombinant human PKC-α (26Finkenzeller G. Marme D. Hug H. Nucleic Acids Res. 1990; 18: 2183Crossref PubMed Scopus (55) Google Scholar) was purchased from ATCC (American Type Culture Collection, ATCC 80045). The following sense primers (from Operon) were used for the creation of a 5′-BglII and 3′-EcoRI restriction sites by PCR, for the subsequent subcloning into pVL1392 vector (BD Pharmingen): PKCAB-glII, 5′-ata ata aga tct atg gct gac gtt ttc ccg ggc aac-3′; PKCAEcoRI 5′-att aat gaa ttc tca tac tgc act ctg taa gat ggg-3′. Mutated residues are underlined. The identity of constructs was verified by DNA sequencing. Sf-9 cells (Invitrogen) transfected with PKC-α using the BaculoGold Kit (BD Pharmingen) were used to produce high-titer baculovirus stock. For protein expression, Sf-9 cells were grown at 27 °C in suspension culture to 2 × 106 cells/ml and infected with a MOI of 10. After 3 days cells were harvested and resuspended in 40 ml of Lysis Buffer containing 25 mm Tris-HCl, pH 7.5, 10 mm EGTA, 2 mm EDTA, 2 mm DTT, 50 μg/ml leupeptin, 1% Triton X-100, and 0.2 mm phenylmethylsulfonyl fluoride. The cell suspension was lysed in a 50-ml hand-held homogenizer chilled on ice. The lysate was centrifuged at 50,000 × g and 4 °C for 60 min. The supernatant was loaded onto a HP Q-Sepharose Fast Flow column (Amersham Biosciences). PKC-α was eluted with a 0.0–0.4 m KCl gradient in PKC-Buffer A (25 mm Tris-HCl, pH 7.5, 1 mm EGTA, 1 mm EDTA, and 1 mm DTT). Active PKC fractions were pooled, adjusted to 2 m KCl, and loaded onto a POROS 20 HP2 column (Per-Septive Biosystems) and eluted with linear salt gradient from 2 to 0 m KCl in Buffer A. Active PKC fractions were concentrated in an Ultra-free-15 centrifugal filter device (Millipore). Activity of PKC-α was assayed by measuring the initial rate of [32P]phosphate incorporation into 5 μm cTn (modified from Ref. 27Medkova M. Cho W. Biochemistry. 1998; 37: 4892-4900Crossref PubMed Scopus (78) Google Scholar). Phosphorylation of cTnT by PKC-α—Phosphorylation of cTnT-wt and cTnT-T197A/S201A/T287A mutant (to exclusively phosphorylate Thr206) was accomplished by using 50 ng of PKC-α/μg cTnT in the presence of 0.3 mm phosphatidylserine (Avanti Polar-Lipids), 0.02 mm diacylglycerol (Avanti Polar-Lipids), 0.5 mm CaCl2, 0.4 mm ATPγS (Sigma), 20 mm Hepes pH 7.4 and 300 mm KCl at 30 °C for 1–3 h. The phosphorylated cTnT was then denatured in the Solubilization Buffer (see below) and used to reconstitute the cTn complex. Recombinant cTn Complex Reconstitution—The recombinant heterotrimeric cTn complex was reconstituted by mixing equimolar amounts of cTnT, cTnI and cTnC in a solubilization buffer containing 6 m urea, 50 mm Tris pH 8.0, 1 m KCl, 5 mm MgCl2, 1 mm CaCl2, 1 mm DTT (28Kobayashi T. Zhao X. Wade R. Collins J.H. Biochemistry. 1999; 38: 5386-5391Crossref PubMed Scopus (15) Google Scholar). The solution was subjected to sequential dialysis using 0.7 m KCl, 0.4 m KCl, and finally 0.2 m KCl in the above buffer minus urea. Next the cTn complex was further separated from the monomeric troponins by anion exchange on a RESOURCE-Q column (Amersham Biosciences) connected to a FPLC system. The cTn complex identity and purity was verified by 12% SDS-PAGE and then concentrated to about 2 ml using a Centriprep 10K (Millipore) and dialyzed against the Exchange Buffer containing 20 mm MOPS pH 6.5, 200 mm KCl, 5 mm EGTA, 5 mm MgCl2, 1 mm DTT (19Brenner B. Kraft T. Yu L.C. Chalovich J.M. Biophys. J. 1999; 77: 2677-2691Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 20She M. Trimble D. Yu L.C. Chalovich J.M. J. Muscle Res. Cell Motil. 2000; 21: 737-745Crossref PubMed Scopus (21) Google Scholar). Next, the cTn complex was aliquoted and stored at –80 °C until it was used for exchange experiments. Exchange of Recombinant cTn in Left Ventricular Fiber Bundles— Fiber bundles were prepared from hearts of 5–6 months old, FVBN mice purchased from Harlan or Charles Rivers laboratories. The mice were anesthetized with ether and hearts were quickly removed and rinsed free of blood in ice-cold saline (0.9% NaCl). Muscle strips (∼150–200 μm wide and 3–4 mm long) were dissected from left ventricular papillary muscle. Then the fiber bundles were detergent-treated at 4 °C, in a High Relax Buffer containing 20 mm MOPS pH 7.0, 50 mm potassium propionate, 6.8 mm MgCl2, 10 mm EGTA, 25 μm CaCl2, 12 mm phosphocreatine, 5 mm Na2ATP, 10 UI/ml creatine kinase, 0.5 mm DTT, a mixture of protease inhibitors and 1% Triton-X100 (21Janssen P.M. de Tombe P.P. Am. J. Physiol. 1997; 273: H2415-H2422PubMed Google Scholar). Following the detergent treatment the fiber bundles were transferred to a bath containing a recombinant cTn complex in Exchange Buffer (see above) and incubated overnight at 4 °C. The extent of recombinant cTn exchange was determined after isometric tension and actomyosin Mg-ATPase activity measurements (see below) by immunoblot analysis of control versus exchanged fiber bundles using anti-Myc antibody (clone 9E10, Santa Cruz Biotechnology) followed by treatment of the same membrane (after c-Myc removal) with anti-TnT antibody (clone JLT-12, Sigma). The presence of the 9-amino acid Myc tag at the N terminus of recombinant cTnT (29Tardiff J.C. Factor S.M. Tompkins B.D. Hewett T.E. Palmer B.M. Moore R.L. Schwartz S. Robbins J. Leinwand L.A. J. Clin. Invest. 1998; 101: 2800-2811Crossref PubMed Scopus (165) Google Scholar) allowed us to separate it from the endogenous mouse cTnT on a 15% SDS-PAGE (acrylamide:bis-acrylamide ratio 200:1). Isometric Tension and Actomyosin Mg-ATPase Activity Measurements—The method of de Tombe and Stienen used in these experiments was described in detail elsewhere (21Janssen P.M. de Tombe P.P. Am. J. Physiol. 1997; 273: H2415-H2422PubMed Google Scholar). The fiber bundles (control or exchanged) were attached to a displacement generator at one end and a force transducer at the other end using aluminum T-clips. The sarcomere length was adjusted to 2.3 μm using a laser diffraction pattern and the cross-sectional area was determined based on an elliptical model. Fiber bundles were equilibrated for 5 min in Relaxing Buffer (100 mm BES, pH 7.1, 8.37 mm MgCl2, 5.80 mm Na2ATP, 20 mm EGTA, 42.5 mm potassium propionate), followed by 2 min in Pre-Activating Buffer (100 mm BES pH 7.1, 7.78 mm MgCl2, 5.80 mm Na2ATP, 0.50 mm EGTA, 19.5 mm HDTA, 43.6 mm potassium propionate) and then immersed into a bath containing a Maximal Activating Solution (100 mm BES, pH 7.1, 7.63 mm MgCl2, 5.87 mm Na2ATP, 20 mm Ca2+-EGTA, 43.6 mm potassium propionate). Relaxing, pre-activating, and maximum activating solutions also contained 900 μm NADH, 5 mm sodium azide, 10 mm phospho(enol)pyruvate, 1 mg/ml pyruvate kinase (500 units/mg), 0.12 mg/ml lactate dehydrogenase (870 units/mg), 10 μm oligomycin B, 20 μm P1P5-di(adenosine-5′)pentaphosphate, and 10 μm leupeptin, 1 μm pepstatin, 1 mm DTT, and 10 μm phenylmethylsulfonyl fluoride. After each contraction, fiber bundles were incubated for 1 min in Relaxing Buffer, followed by 2 min in Pre-Activating Buffer. The final contraction was induced in Activating solution containing maximally activating [Ca2+]. Only those fibers able to generate greater than 80% of initial tension in their final contraction were kept for analysis. The isometric tension and actomyosin Mg-ATPase activity were determined simultaneously at 20 °C in the presence of variable Ca2+ concentrations as described (30Janssen P.M. de Tombe P.P. Am. J. Physiol. 1997; 272: H1892-H1897Crossref PubMed Google Scholar). Data were analyzed using Labview (National Instruments, Austin, Texas). Tension-, actomyosin Mg-ATPase activity-[Ca2+] relations were fit by a nonlinear fit procedure to a modified Hill equation shown in Equation 1, P=Max·[Ca2+]H/([Ca2+]H+EC50H)(Eq. 1) where P is the parameter of interest (isometric tension, actomyosin Mg-ATPase activity); Max is the maximum value at saturating [Ca2+]; EC50 is the [Ca2+] at which 50% of Max is reached; and H represents the slope of the relationship (Hill coefficient). Cosedimentation Assay—The binding affinity of cTn complexes containing cTnT-wt or mutants to bovine cardiac tropomyosin (Tm) was assessed using a cosedimentation assay (31Hill L.E. Mehegan J.P. Butters C.A. Tobacman L.S. J. Biol. Chem. 1992; 267: 16106-16113Abstract Full Text PDF PubMed Google Scholar). Variable amounts of cTn complex (wt or mutant) in the presence of 1 μm Tm were incubated in 10 mm MOPS pH 7.0, 5 mm MgCl2, 100 mm KCl and of either 1 mm EGTA or 100 μm CaCl2, at 4 °C (conditions shown to promote sedimentation of cTn-Tm complex, (31Hill L.E. Mehegan J.P. Butters C.A. Tobacman L.S. J. Biol. Chem. 1992; 267: 16106-16113Abstract Full Text PDF PubMed Google Scholar)). The Tm-bound cTn was determined by cosedimentation at 74,000 rpm for 45 min using a TLA rotor. Sedimentation supernatants and pellets were analyzed by 15% SDS-PAGE. Densitometric analysis of the gels was carried out using NIH Image software. Materials—Triton X-100 was purchased from Pierce. Ammonium sulfate (enzyme grade) and formic acid (sequencing grade) were from Fisher. 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (phosphatidylserine) and 1,2-sn-dioleoylglycerol (diacylglycerol) were purchased from Avanti Polar-Lipids, Inc. Unless mentioned otherwise all the other chemicals were from Sigma. Animal Care—All animals were handled in accordance with the guidelines of the Animal Care Committee at the University of Illinois, Chicago. Statistical Analysis—All values are presented as mean ± S.E., and values of p < 0.05 were the criteria for statistical significance. Data was analyzed using a one-way ANOVA and post-hoc Dunnett's t test. Recombinant cTn Incorporation into the Myofilament Lattice—To assess the functional role of each cTnT phosphorylation site we used a modified version (see "Materials and Methods") of the technique of whole troponin exchange introduced by Brenner et al. (19Brenner B. Kraft T. Yu L.C. Chalovich J.M. Biophys. J. 1999; 77: 2677-2691Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). This method allows the gentle replacement of the endogenous cTn with a recombinant cTn in muscle fiber bundles. The exchange procedure introduces no major alteration in the structure and properties of the fiber bundles, since the myofilament is never depleted of cTn. Fig. 1 shows the Western blot analysis of representative fibers used in our measurements (1 fiber bundle per lane). The samples were separated on 15% SDS-PAGE, and after transfer to a nitrocellulose membrane probed with an anti-c-Myc antibody (data not shown) and then with anti-cTnT antibody. Lanes 1, 5, 6, and 10 show representative native (no exchange) fibers bundles that have been used in control measurements, whereas the other lanes contain fibers that had undergone exchange with recombinant cTn-wt or Tn-T3SE. The middle lanes 3 and 8 serve as standards for recombinant cTn. The endogenous mouse cTnT and recombinant mouse cTnT have different mobilities on the SDS-PAGE due to the presence of a c-Myc tag at the N terminus of recombinant cTnT. The c-Myc tag retards the recombinant cTnT migration on the gel allowing us to assess the exchange efficiency. The Western blot analysis demonstrated that exchange of endogenous cTn with recombinant cTn was basically 100% since the fibers bundles treated with the recombinant cTn showed no band corresponding to endogenous cTnT. Mechanoenergetic Characteristics of Native and Recombinant cTn-wt Exchanged Fibers—In a first set of experiments, we determined the functional effect of introducing recombinant cTn-wt into the myofilament lattice of left ventricular fiber bundles. To determine the effect of cTn-wt incorporation we compared the mechanoenergetic characteristics of native (no exchange) and recombinant cTn-wt exchanged fiber bundles. The data (Fig. 2 and Table II) indicate that the basal mechanics and energetics of native versus cTn-wt were not significantly altered. Native and cTn-wt exchange fibers demonstrated no significant differences in myofilament Ca2+ sensitivity or Hill coefficients for any parameter (Table II).Table IIMechanical and energetic profiles of left ventricular papillary fiber bundlesIsometric TensionActomyosin Mg-ATPaseMaximumEC50Hill CoefficientMaximumEC50Hill CoefficientTension costNmN/mm2μ mpmol/s/mm3μ mNative42.3 ± 3.31.6 ± 0.14.9 ± 0.8369.2 ± 24.31.4 ± 0.15.3 ± 1.110.3 ± 0.78 (6)WT39.2 ± 1.01.7 ± 0.14.7 ± 0.6362.9 ± 15.91.5 ± 0.14.8 ± 0.89.6 ± 0.510 (7)T3SE14.9 ± 2.5ap < 0.05 as compared with wild-type exchange.22.8 ± 0.8ap < 0.05 as compared with wild-type exchange.2.5 ± 0.2ap < 0.05 as compared with wild-type exchange.181.1 ± 19.8ap < 0.05 as compared with wild-type exchange.20.9 ± 0.7ap < 0.05 as compared with wild-type exchange.2.0 ± 0.2ap < 0.05 as compared with wild-type exchange.10.7 ± 1.57 (4)T3E28.3 ± 1.6ap < 0.05 as compared with wild-type exchange.7.5 ± 0.7ap < 0.05 as compared with wild-type exchange.2.5 ± 0.5ap < 0.05 as compared with wild-type exchange.227.3 ± 17.8ap < 0.05 as compared with wild-type exchange.6.4 ± 0.3ap < 0.05 as compared with wild-type exchange.2.8 ± 0.8ap < 0.05 as compared with wild-type exchange.9.0 ± 0.97 (6)T2E24.3 ± 3.9ap < 0.05 as compared with wild-type exchange.15.7 ± 0.4ap < 0.05 as compared with wild-type exchange.2.2 ± 0.1ap < 0.05 as compared with wild-type exchange.230.2 ± 19.0ap < 0.05 as compared with wild-type exchange.12.0 ± 1.0ap < 0.05 as compared with wild-type exchange.1.8 ± 0.2ap < 0.05 as compared with wild-type exchange.9.4 ± 1.16 (5)T206E19.1 ± 1.3ap < 0.05 as compared with wild-type exchange.18.6 ± 0.7ap < 0.05 as compared with wild-type exchange.2.8 ± 0.6ap < 0.05 as compared with wild-type exchange.210.3 ± 14.4ap < 0.05 as compared with wild-type exchange.15.7 ± 0.8ap < 0.05 as compared with wild-type exchange.2.4 ± 0.5ap < 0.05 as compared with wild-type exchange.10.1 ± 1.06 (3)T2SE38.2 ± 1.82.3 ± 0.24.2 ± 0.2359.8 ± 16.02.1 ± 0.23.9 ± 0.19.2 ± 0.25 (3)STE38.8 ± 4.12.3 ± 0.14.5 ± 0.8350.6 ± 23.61.6 ± 0.13.9 ± 0.49.0 ± 0.66 (3)T197E42.4 ± 2.02.5 ± 0.14.9 ± 0.2392.9 ± 21.12.2 ± 0.14.2 ± 0.38.9 ± 0.45 (3)T3SA30.6 ± 2.0ap < 0.05 as compared with wild-type exchange.6.6 ± 0.3ap < 0.05 as compared with wild-type exchange.3.7 ± 0.7280.3 ± 17.06.2 ± 0.6ap < 0.05 as compared with wild-type exchange.3.7 ± 0.58.9 ± 0.67 (5)T3A36.8 ± 2.27.5 ± 0.1ap < 0.05 as compared with wild-type exchange.4.1 ± 0.3319.1 ± 14.66.9 ± 0.6ap < 0.05 as compared with wild-type exchange.3.3 ± 0.38.60 ± 0.46 (3)T206A21.1 ± 3.2ap < 0.05 as compared with wild-type exchange.12.0 ± 0.8ap < 0.05 as compared with wild-type exchange.2.9 ± 0.1ap < 0.05 as compared with wild-type exchange.229.9 ± 3.2ap < 0.05 as compared with wild-type exchange.10.8 ± 0.7ap < 0.05 as compared with wild-type exchange.2.6 ± 0.1ap < 0.05 as compare
Referência(s)