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• W2034175826 abstract "Adrenergic stimulation induces positive changes in cardiac contractility and relaxation. Cardiac troponin I is phosphorylated at different sites by protein kinase A and protein kinase C, but the effects of these post-translational modifications on the rate and extent of contractility and relaxation during β-adrenergic stimulation in the intact animal remain obscure. To investigate the effect(s) of complete and chronic cTnI phosphorylation on cardiac function, we generated transgenic animals in which the five possible phosphorylation sites were replaced with aspartic acid, mimicking a constant state of complete phosphorylation (cTnI-AllP). We hypothesized that chronic and complete phosphorylation of cTnI might result in increased morbidity or mortality, but complete replacement with the transgenic protein was benign with no detectable pathology. To differentiate the effects of the different phosphorylation sites, we generated another mouse model, cTnI-PP, in which only the protein kinase A phosphorylation sites (Ser23/Ser24) were mutated to aspartic acid. In contrast to the cTnIAllP, the cTnI-PP mice showed enhanced diastolic function under basal conditions. The cTnI-PP animals also showed augmented relaxation and contraction at higher heart rates compared with the nontransgenic controls. Nuclear magnetic resonance amide proton/nitrogen chemical shift analysis of cardiac troponin C showed that, in the presence of cTnI-AllP and cTnI-PP, the N terminus exhibits a more closed conformation, respectively. The data show that protein kinase C phosphorylation of cTnI plays a dominant role in depressing contractility and exerts an antithetic role on the ability of protein kinase A to increase relaxation. Adrenergic stimulation induces positive changes in cardiac contractility and relaxation. Cardiac troponin I is phosphorylated at different sites by protein kinase A and protein kinase C, but the effects of these post-translational modifications on the rate and extent of contractility and relaxation during β-adrenergic stimulation in the intact animal remain obscure. To investigate the effect(s) of complete and chronic cTnI phosphorylation on cardiac function, we generated transgenic animals in which the five possible phosphorylation sites were replaced with aspartic acid, mimicking a constant state of complete phosphorylation (cTnI-AllP). We hypothesized that chronic and complete phosphorylation of cTnI might result in increased morbidity or mortality, but complete replacement with the transgenic protein was benign with no detectable pathology. To differentiate the effects of the different phosphorylation sites, we generated another mouse model, cTnI-PP, in which only the protein kinase A phosphorylation sites (Ser23/Ser24) were mutated to aspartic acid. In contrast to the cTnIAllP, the cTnI-PP mice showed enhanced diastolic function under basal conditions. The cTnI-PP animals also showed augmented relaxation and contraction at higher heart rates compared with the nontransgenic controls. Nuclear magnetic resonance amide proton/nitrogen chemical shift analysis of cardiac troponin C showed that, in the presence of cTnI-AllP and cTnI-PP, the N terminus exhibits a more closed conformation, respectively. The data show that protein kinase C phosphorylation of cTnI plays a dominant role in depressing contractility and exerts an antithetic role on the ability of protein kinase A to increase relaxation. Recent studies have demonstrated that changes in the phosphorylation states of key cardiac regulatory proteins can have dramatic effects on normal cardiac function (1Bodor G.S. Oakeley A.E. Allen P.D. Crimmins D.L. Ladenson J.H. Anderson P.A. Circulation. 1997; 96: 1495-1500Crossref PubMed Scopus (190) Google Scholar, 2van der Velden J. Papp Z. Boontje N.M. Zaremba R. de Jong J.W. Janssen P.M. Hasenfuss G. Stienen G.J. Cardiovasc. Res. 2003; 57: 505-514Crossref PubMed Scopus (114) Google Scholar). Phosphorylation of cardiac troponin I (cTnI), 1The abbreviations used are: cTnI, cardiac troponin I; TnI, troponin I; cTnC, cardiac troponin C; cTnT, cardiac troponin T; cTnI-AllP, five phosphorylation sites of cTnI (Ser23, Ser24, Ser43, Ser45, and Thr144) mutated to aspartic acid; cTnI-PP, two phosphorylation sites (Ser23/Ser24) mutated to aspartic acid; WT, wild type; PKA, protein kinase A; PKC, protein kinase C; NTG, nontransgenic; TG, transgenic; bpm, beats/min; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; LV, left ventricular. 1The abbreviations used are: cTnI, cardiac troponin I; TnI, troponin I; cTnC, cardiac troponin C; cTnT, cardiac troponin T; cTnI-AllP, five phosphorylation sites of cTnI (Ser23, Ser24, Ser43, Ser45, and Thr144) mutated to aspartic acid; cTnI-PP, two phosphorylation sites (Ser23/Ser24) mutated to aspartic acid; WT, wild type; PKA, protein kinase A; PKC, protein kinase C; NTG, nontransgenic; TG, transgenic; bpm, beats/min; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; LV, left ventricular. a thin filament regulatory protein, may be particularly important in modulating cardiac function. cTnI, together with cardiac troponin T (cTnT) and cardiac troponin C (cTnC), form the troponin complex. The protein binds to both cTnC and actin and is a critical component in activating contraction as it serves as the Ca2+-sensing apparatus. Within the cardiac isoform's amino-terminal extension, serines are present at residues 23 and 24 (Ser23/Ser24), which serve as substrates for protein kinase A (PKA), which is activated in response to β-adrenergic stimulation of the heart (3Chandra M. Dong W.J. Pan B.S. Cheung H.C. Solaro R.J. Biochemistry. 1997; 36: 13305-13311Crossref PubMed Scopus (92) Google Scholar). Several investigations report that PKA-mediated phosphorylation of cTnI results in a reduction in myofilament Ca2+ sensitivity (4Kranias E.G. Solaro R.J. Nature. 1982; 298: 182-184Crossref PubMed Scopus (173) Google Scholar), an increase in cross-bridge cycling (5Kentish J.C. McCloskey D.T. Layland J. Palmer S. Leiden J.M. Martin A.F. Solaro R.J. Circ. Res. 2001; 88: 1059-1065Crossref PubMed Scopus (258) Google Scholar), and increased binding of cTnI to the thin filament. Cardiac TnI is also a substrate for protein kinase C (PKC) phosphorylation at Ser43/Ser45 and Thr144 (position 143 in the human protein) (6Noland Jr., T.A. Guo X. Raynor R.L. Jideama N.M. Averyhart-Fullard V. Solaro R.J. Kuo J.F. J. Biol. Chem. 1995; 270: 25445-25454Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). However, the substrate specificity of these sites is not absolute, since PKC can phosphorylate the PKA sites (7Swiderek K. Jaquet K. Meyer H.E. Schachtele C. Hofmann F. Heilmeyer Jr., L.M. Eur. J. Biochem. 1990; 190: 575-582Crossref PubMed Scopus (50) Google Scholar, 8Jideama 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, 9Noland Jr., T.A. Raynor R.L. Jideama N.M. Guo X. Kazanietz M.G. Blumberg P.M. Solaro R.J. Kuo J.F. Biochemistry. 1996; 35: 14923-14931Crossref PubMed Scopus (71) Google Scholar). Whereas PKA-mediated phosphorylation is thought to mainly affect the overall Ca2+ sensitivity of force development, PKC phosphorylation of TnI inhibits the actin-myosin interaction by decreasing maximum tension, Mg2+-ATPase activity, Ca2+ sensitivity, and thin filament sliding speed (10Burkart 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 (136) Google Scholar). These effects are important determinants in the enhanced relaxation that is critical to the response of the heart to adrenergic stimulation (11Wolska B.M. Arteaga G.M. Pena J.R. Nowak G. Phillips R.M. Sahai S. de Tombe P.P. Martin A.F. Kranias E.G. Solaro R.J. Circ. Res. 2002; 90: 882-888Crossref PubMed Scopus (49) Google Scholar, 12Montgomery D.E. Wolska B.M. Pyle W.G. Roman B.B. Dowell J.C. Buttrick P.M. Koretsky A.P. Del Nido P. Solaro R.J. Am. J. Physiol. 2002; 282: H2397-H2405Crossref PubMed Scopus (59) Google Scholar). Normally, there is little or no adrenergic drive in the human left ventricle (13Haber H.L. Simek C.L. Gimple L.W. Bergin J.D. Subbiah K. Jayaweera A.R. Powers E.R. Feldman M.D. Circulation. 1993; 88: 1610-1619Crossref PubMed Scopus (84) Google Scholar). Whereas increased adrenergic drive is beneficial in initially supporting normal cardiac function, chronic β-adrenergic stimulation can exacerbate an underlying pathology. However, it is unclear whether constant stimulation at the myofibrillar level through the β-adrenergic pathway, as manifested by chronic phosphorylation of the myofibrillar protein targets, causes the deleterious effects. The present study was aimed at determining in vivo whether PKA and/or PKC phosphorylation of TnI were determining factors in mediating contraction or relaxation at base line or during cardiovascular stimulation. In a first iteration, rather than trying to segregate the effects of PKA and PKC phosphorylation, we chose to convert both the PKA and PKC sites in cTnI to aspartic acid to mimic a constant state of complete phosphorylation (cTnIAllP), since PKC can cross-phosphorylate the PKA sites (8Jideama 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). To investigate the long term effects of TnI charge modification via phosphorylation, we mimicked PKA and PKC cTnI phosphorylation by replacing the endogenous cardiac protein with transgenically encoded cTnI in which the five substrate residues, Ser23/Ser24 (PKA) and Ser43/Ser45/Thr144 (PKC), were replaced by aspartic acid, whose charge mimics the endogenous residues' phosphorylated state. To segregate the functional role of PKC-mediated phosphorylation from PKA effects, we also generated a transgenic (TG) mouse in which only the PKA sites (Ser23/Ser24) were mutated to aspartic acid (cTnI-PP). A comparison of their contractile functions and responses to β-adrenergic stimulation allowed us to determine that PKC phosphorylation can antagonize the effects of PKA-induced cTnI phosphorylation in the whole animal and that the primary in vivo function of PKC-mediated cTnI phosphorylation is to decrease contractility at base line and increase relaxation during β-adrenergic stimulation. Transgene Construction—The wild type (WT) cDNA for mouse cTnI was obtained by reverse transcription-PCR using total RNA isolated from the mouse cardiac ventricle. The cDNA (cTnI-WT) was subcloned into pBluescript and sequenced in full as described (14James J. Zhang Y. Osinska H. Sanbe A. Klevitsky R. Hewett T.E. Robbins J. Circ. Res. 2000; 87: 805-811Crossref PubMed Scopus (116) Google Scholar). The phosphorylation sites (Ser23/Ser24 by PKA; Ser43/Ser45 and Thr144 by PKC) were mutated to aspartic acid to mimic a constant state of phosphorylation using standard PCR-based methods (cTnI-AllP). In parallel, the PKA phosphorylation sites of cTnI (Ser23/Ser24) were mutated to aspartic acid to produce the cTnI-PP construct. The cTnI-AllP and cTnI-PP cDNAs were subcloned into the mouse α-myosin heavy chain promoter (Fig. 1), and the construct was purified from the plasmid backbone after NotI digestion. The DNAs were subsequently used to generate multiple lines of TG mice for each construct (15Palermo J. Gulick J. Colbert M. Fewell J. Robbins J. Circ. Res. 1996; 78: 504-509Crossref PubMed Scopus (115) Google Scholar). Except where noted, five or six mice, 12-15 weeks old of mixed gender were used in each experiment after preliminary experiments showed no gender differences. Molecular Analyses—For assessment of cTnI-AllP and cTnI-PP transcript size, Northern blot analyses were performed using 7.5 μg of total RNA, as described (14James J. Zhang Y. Osinska H. Sanbe A. Klevitsky R. Hewett T.E. Robbins J. Circ. Res. 2000; 87: 805-811Crossref PubMed Scopus (116) Google Scholar). Expression levels were determined by RNA dot blot analysis with γ-32P-labeled cTnI and human growth hormone probes using transcript-specific oligonucleotides (14James J. Zhang Y. Osinska H. Sanbe A. Klevitsky R. Hewett T.E. Robbins J. Circ. Res. 2000; 87: 805-811Crossref PubMed Scopus (116) Google Scholar). Protein Analyses—Enriched myofibrillar proteins were isolated using F60 buffer (60 mm KCl, 30 mm imidazole, 7.2 mm MgCl2, pH 7.0) with protease/phosphatase inhibitors (Mixture I and II; Sigma) as described (16Fewell 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). The percentage of cTnI-AllP replacement was estimated via SDS-PAGE (4-15 or 8-16% criterion gradient Tris-glycine precast gels; Bio-Rad), Western blots, and MALDI-TOF. The cTnI-PP replacement was calculated by MALDI-TOF, since the TG species could not be separated from endogenous TnI via gel electrophoresis. Two-dimensional gel electrophoresis was performed using the Immobiline DryS-trip kit (Amersham Biosciences) and ReadyStrip (Bio-Rad) according to the manufacturers' guidelines to identify shifts due to phosphorylation. Myofibrillar proteins from cTnI-AllP, cTnI-PP, and NTG hearts were prepared for isoelectric focusing by dilution of 50-75 μg in 185 μl of immobilized pH gradient rehydration buffer (Bio-Rad) containing 0.5% of the appropriate carrier ampholytes and subjected to isoelectric focusing on immobilized pH gradient strips (11 cm, pH 3-10, pH 4-7, or pH 7-10 linear gradient; Bio-Rad), followed by 10-20% gradient SDS-PAGE (Criterion gradient Tris-glycine precast gels; Bio-Rad). The gels were stained with SYPRO-Ruby (Bio-Rad), and protein spots were analyzed using HT Analyzer version 3.0 (Genomic Solutions) or transferred to polyvinylidene difluoride membranes (Bio-Rad) to quantitate the level of myofibrillar protein phosphorylation. Antibodies used for Western blot analysis were against cTnI (Cell Signaling Technology), α-tropomyosin, cTnT, actin (Sigma), myosin light chain 1v (17James J. Zhang Y. Wright K. Witt S. Glascock E. Osinska H. Klevitsky R. Martin L. Yager K. Sanbe A. Robbins J. J. Mol. Cell. Cardiol. 2002; 34: 873-882Abstract Full Text PDF PubMed Scopus (23) Google Scholar), and myosin light chain 2v (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Immunohistochemistry—Heart/body weights were first measured in order to determine whether hypertrophy had occurred. Hearts were then fixed in 4% paraformaldehyde. Five-μm cryostat sections were probed with polyclonal antibodies against cTnI followed by incubation with Alexa 488-conjugated secondary antibody. Specimens were examined using confocal microscopy. For paraffin sectioning, hearts from littermates were perfusion-fixed with 10% formalin, embedded, and stained with hematoxylin-eosin or Masson trichrome (14James J. Zhang Y. Osinska H. Sanbe A. Klevitsky R. Hewett T.E. Robbins J. Circ. Res. 2000; 87: 805-811Crossref PubMed Scopus (116) Google Scholar). Structural analyses at the light and electron microscopy levels were performed as described (18Sanbe 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). Using 2-4 mice of mixed gender, multiple sections were cut, and >50 fields were observed by a blinded observer. In Vitro PKA and PKC Phosphorylation—Total skinned myofibrils were isolated as described above in the presence of 1% Triton X-100 (Fisher) and protease/phosphatase inhibitors (Mixture I and II; Sigma). The phosphorylation assays contained 100 μg of freshly isolated total skinned myofibrils (19Yang Q. Hewett T.E. Klevitsky R. Sanbe A. Wang X. Robbins J. Cardiovasc. Res. 2001; 51: 80-88Crossref PubMed Scopus (19) Google Scholar). Myofibrils were incubated 120 min at 37 °C in 100 μl of 20 mm Tris-HCl, 150 mm NaCl, 20 mm MgCl2, 10 μm ATP, and 30 units of the PKA catalytic subunit (PKAce kit; Novagen). For PKC phosphorylation, 100 μg of skinned myofibrils were incubated 120 min at 37 °C in 200 μl of solution containing 10 μm ATP, 100 mm KCl, 10 mm imidazole, 2 mm EGTA, 1 mm MgCl2, 1 mm phenylmethylsulfonyl fluoride, 10 mm benzamidine, and purified ϵ-PKC (5 ng/μl) activated with phosphatidylserine (100 μg/ml), dioctanoylglycerol (10 μg/ml), and 50 nm phorbol 12-myristate 13-acetate (Sigma). The PKA inhibitor PKI 6-22 (Amide; Calbiochem) and PKC inhibitor myr-PKC (Sigma) were used for control studies. Myofibrils were washed three times in F60 and either solubilized in rehydration/sample buffer (Bio-Rad) for two-dimensional gel electrophoresis or suspended in F60 buffer for the Mg2+-ATPase assay. Myofibrillar Mg2+-ATPase Assays—Myofibrillar Mg2+-ATPase activity was determined by measuring inorganic phosphate release (20Krenz 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). Skinned myofibrillar proteins were purified as described (16Fewell 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), treated with either PKA or PKC, and suspended in F60 buffer. Protein was determined by the Bradford method (Bio-Rad protein assay). The reaction mixture contained 0.2-0.4 mg/ml myofibrillar proteins, 60 mm imidazole, 6 mm MgCl2, 5 mm EGTA, and 5 mm ATP (pH 7.0). Assays were carried out in 96-well microtiter plates at pCa2+ concentrations ranging from pCa 8.0-4.0 at room temperature during the linear Pi release phase of the reaction. Incubations were performed in triplicate from five animals of each group. The 100-μl reaction was incubated at room temperature for 5 min and then quenched with 40 μl of 15% trichloroacetic acid. Precipitated proteins were pelleted at 4 °C, 80 μl of the supernatant (containing Pi) was transferred to microtiter plates, and 160 μl of FeSO4-ammonium-molybdate solution to react for color development was added, after which the production of inorganic phosphate was determined colorimetrically at 625 nm using a kinetic microplate reader (Molecular Devices). Ca2+-stimulated Mg2+-ATPase activity was measured by subtracting the activity at pCa 4.0 from the activity at pCa 8.0. In Vivo Catheterization and Hemodynamic Studies—Assessment of left ventricular (LV) function in the intact animal was performed essentially as previously described (21Lorenz J.N. Robbins J. Am. J. Physiol. 1997; 272: H1137-H1146PubMed Google Scholar). Mice were anesthetized with ketamine-thiobutabarbital, and following tracheotomy (PE-90), the right femoral artery and vein were cannulated with polyethylene tubing for measuring systemic arterial pressure and for the infusion of experimental drugs. To assess myocardial performance, a 1.4F Millar Mikro-Tip transducer (Millar Instruments) was inserted in the right carotid artery and advanced into the LV for pressure measurement and determining dP/dt. Cardiovascular responses to increasing doses of dobutamine were determined during 3-min constant infusions (0.1 μl/min/g body weight), and average values were determined during the final 30 s of each infusion. At the end of each dose response protocol, a bolus dose of propranolol (100 ng/g body weight) was administered to evaluate base-line cardiac function in the absence of endogenous β-adrenergic activity. In separate experiments designed to assess the phosphorylation status of Ca2+-handling proteins and contractile proteins during adrenergic stimulation in the cTnI-AllP mice, a maximum dobutamine dose of 32 ng/g body weight was infused, responses were monitored for 3 min, and the hearts were flash-frozen in liquid nitrogen. Myofibrillar proteins were prepared, and the phosphorylation status was analyzed on two-dimensional gels. Arterial blood pressure was determined by tail cuff measurement of nonsedated 12-week-old mice using a Visitech Systems (Apex, NC) Bp-2000 system. Closed chest invasive hemodynamic studies for force frequency relations were performed under ketamine/thiobutabarbital anesthesia. LV function was measured as above. Atrial pacing was accomplished using a 0.3-mm bipolar electrode placed at the wall of the right atrium via the right jugular vein. After base-line hemodynamic data were acquired, atrial pacing was instituted to overdrive the intrinsic heart rate (350-700 bpm) and, unless stated otherwise, maintained at a constant rate of 7.5 Hz (450 bpm) (22D'Angelo D.D. Sakata Y. Lorenz J.N. Boivin G.P. Walsh R.A. Liggett S.B. Dorn II, G.W. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8121-8126Crossref PubMed Scopus (531) Google Scholar). Data were collected and analyzed using a PowerLab system (AD Instruments, Colorado Springs, CO). NMR Spectroscopy—Full-length cDNA encoding mouse cTnI-WT and cTnI-AllP were generated by PCR and subcloned into the pET23a+ expression vector (Novagen). [15N,2H]cTnC, cTnI-WT, cTnI-AllP, and cTnI-PP proteins were expressed in bacteria and purified, and complex formation was carried out as described (23Finley N. Abbott M.B. Abusamhadneh E. Gaponenko V. Dong W. Gasmi-Seabrook G. Howarth J.W. Rance M. Solaro R.J. Cheung H.C. Rosevear P.R. FEBS Lett. 1999; 453: 107-112Crossref PubMed Scopus (81) Google Scholar, 24Gaponenko V. Abusamhadneh E. Abbott M.B. Finley N. Gasmi-Seabrook G. Solaro R.J. Rance M. Rosevear P.R. J. Biol. Chem. 1999; 274: 16681-16684Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Varian 600- or 800-MHz spectrometers (25Abbott M.B. Dong W.J. Dvoretsky A. DaGue B. Caprioli R.M. Cheung H.C. Rosevear P.R. Biochemistry. 2001; 40: 5992-6001Crossref PubMed Scopus (45) Google Scholar) were used, and spectral widths in the t1 and t2 dimensions were 3.3 and 12 kHz, respectively. Composite amide chemical shift differences were determined from the square root of the weighted sum of the squares of proton and nitrogen chemical shift differences. 15N chemical shift differences were weighted by a factor of one-seventh to scale their contributions to a magnitude similar to 1H chemical shift differences and the data processed as described (24Gaponenko V. Abusamhadneh E. Abbott M.B. Finley N. Gasmi-Seabrook G. Solaro R.J. Rance M. Rosevear P.R. J. Biol. Chem. 1999; 274: 16681-16684Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). The structural effects of PKA phosphorylation at Ser23/Ser24 of cTnI on cTnC conformation have been reported previously (23Finley N. Abbott M.B. Abusamhadneh E. Gaponenko V. Dong W. Gasmi-Seabrook G. Howarth J.W. Rance M. Solaro R.J. Cheung H.C. Rosevear P.R. FEBS Lett. 1999; 453: 107-112Crossref PubMed Scopus (81) Google Scholar, 25Abbott M.B. Dong W.J. Dvoretsky A. DaGue B. Caprioli R.M. Cheung H.C. Rosevear P.R. Biochemistry. 2001; 40: 5992-6001Crossref PubMed Scopus (45) Google Scholar). Statistical Analysis—All data are expressed as means ± S.E. Data were analyzed by fitting the data obtained for each individual and then averaging the derived Hill parameters. The Ca2+ sensitivities of Mg2+-ATPase activities were determined by fitting normalized Mg2+-ATPase activity-pCa curves with a Hill equation for a general cooperative model for substrate as the built-in Hill function in the Origin 7.5 NLSF tool (OriginLab Corp., Northampton, MA) using nonlinear regression analysis with the expression, y=Vmax×Ca2+n/(EC50n+Ca2+n)), where Vmax represents maximum activity, EC50 is the [Ca2+] at which 50% of maximum activity was produced, and Ca2+ is the substrate concentration. The steepness of the transition to maximal activation (n) is a measure of the cooperativity of the relation (i.e. Hill coefficient). The minimum and maximum Mg2+-ATPase and pCa50 activities were used as an index of myofibrillar Ca2+ sensitivity, and the Hill coefficient was used as an index of cooperative activation of the myofibrillar proteins. EC50 values were reported as pCa50, where pCa50=−logEC50. Comparisons between NTG and either cTnI-AllP or cTnI-PP TG littermates were evaluated using Student's t test, with significance defined as p < 0.05 using a two-tailed unpaired t test. Expression and Incorporation of cTnI-AllP and cTnI-PP—Cardiac TnI can serve as a substrate for both PKA and PKC (6Noland Jr., T.A. Guo X. Raynor R.L. Jideama N.M. Averyhart-Fullard V. Solaro R.J. Kuo J.F. J. Biol. Chem. 1995; 270: 25445-25454Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 7Swiderek K. Jaquet K. Meyer H.E. Schachtele C. Hofmann F. Heilmeyer Jr., L.M. Eur. J. Biochem. 1990; 190: 575-582Crossref PubMed Scopus (50) Google Scholar, 9Noland Jr., T.A. Raynor R.L. Jideama N.M. Guo X. Kazanietz M.G. Blumberg P.M. Solaro R.J. Kuo J.F. Biochemistry. 1996; 35: 14923-14931Crossref PubMed Scopus (71) Google Scholar, 26Venema R.C. Kuo J.F. J. Biol. Chem. 1993; 268: 2705-2711Abstract Full Text PDF PubMed Google Scholar, 27Wattanapermpool J. Guo X. Solaro R.J. J. Mol. Cell. Cardiol. 1995; 27: 1383-1391Abstract Full Text PDF PubMed Scopus (86) Google Scholar). However, most of the data bearing on the functional outcome(s) of phosphorylation, with rare exceptions (28Takimoto E. Soergel D.G. Janssen P.M. Stull L.B. Kass D.A. Murphy A.M. Circ. Res. 2004; 94: 496-504Crossref PubMed Scopus (117) Google Scholar, 29Pi Y. Zhang D. Kemnitz K.R. Wang H. Walker J.W. J. Physiol. 2003; 552: 845-857Crossref PubMed Scopus (101) Google Scholar), have been obtained in vitro or in isolated systems, with substitution of a noncardiac protein or incomplete replacement (12Montgomery D.E. Wolska B.M. Pyle W.G. Roman B.B. Dowell J.C. Buttrick P.M. Koretsky A.P. Del Nido P. Solaro R.J. Am. J. Physiol. 2002; 282: H2397-H2405Crossref PubMed Scopus (59) Google Scholar). To investigate the role of cTnI phosphorylation in cardiac function, we generated two sets of mice in which either the PKA sites (cTnI-PP) or PKA and PKC sites (cTnI-AllP) were altered, allowing us to determine the additive and potential antithetic effects of the two on in vivo hemodynamics and the force-frequency response. The cardiac isoform of TnI contains a unique 32-amino acid sequence at the N terminus, in which two serines at residues 23 and 24 are substrates for PKA. Whereas Ser43/Ser45 and Thr144 are phosphorylated by PKC (Fig. 1A), it is well documented that PKC can cross-phosphorylate the PKA sites as well (6Noland Jr., T.A. Guo X. Raynor R.L. Jideama N.M. Averyhart-Fullard V. Solaro R.J. Kuo J.F. J. Biol. Chem. 1995; 270: 25445-25454Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 7Swiderek K. Jaquet K. Meyer H.E. Schachtele C. Hofmann F. Heilmeyer Jr., L.M. Eur. J. Biochem. 1990; 190: 575-582Crossref PubMed Scopus (50) Google Scholar, 8Jideama 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). Therefore, to rigorously explore the long term effects of chronic β-adrenergic stimulation, the five sites that can be phosphorylated by PKA and/or PKC were mutated to aspartic acid to mimic the fully charged state of cTnI (Fig. 1A). This transgene was termed cTnI-AllP. To compare and contrast the in vivo effects of PKA-mediated phosphorylation in the absence of PKC-induced modification of cTnI, we generated another construct (cTnI-PP) in which only the PKA phosphorylation sites of cTnI (Ser23/Ser24) were mutated to aspartic acid (Fig. 1A). Each cDNA was then linked to the mouse α-myosin heavy chain promoter and used to generate TG mice. Lines in which replacement was essentially complete were generated with both constructs (Fig. 1, B-E), with replacement confirmed either by SDS-PAGE and Western blots (lane 112; cTnI-AllP) or MALDI-TOF 2S. M. Helmke, S. Sakthivel, M. W. Duncan, and J. Robbins, unpublished data. (lanes 112 and 62; cTnI-AllP and cTnI-PP, respectively). As described for other contractile proteins, increases observed at the RNA level are not translated into absolute increases of protein (20Krenz 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). Rather, levels of the endogenous protein are reduced such that contractile protein stoichiometry is conserved, resulting in replacement of the endogenous protein with the transgenically encoded species. The degree of cTnI-AllP replacement was confirmed by Western blot analyses using a cTnI antibody that recognizes both the endogenous and TG species (Fig. 1C) with line 112 (cTnI-AllP). For cTnI-PP, which could not be resolved from endogenous cTnI on SDS-PAGE (Fig. 1D), MALDI-TOF showed greater than 95% replacement. 2S. M. Helmke, S. Sakthivel, M. W. Duncan, and J. Robbins, unpublished data. Lines 112 and 62 were chosen for detailed analyses on the basis of essentially complete replacement of endogenous cTnI with the TG species. Both the cTnI-AllP and cTnI-PP proteins were incorporated normally into the sarcomere (Fig. 2A). Heart/body weight ratios did not differ between NTG (0.0053 ± 0.0008, n = 36) and cTnI-AllP (0.0057 ± 0.0007, n = 28, p = 0.07) or NTG (0.00525 ± 0.0005, n = 18) and cTnI-PP (0.0054 ± 0.0008, n = 15, p = 0.41) littermates. The transcriptional patterns of molecular markers for hypertrophy such as β-myosin, atrial natriuretic factor, and α-skeletal actin were identical for cTnIAllP, cTn" @default.
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