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• W2004333037 abstract "Thin filament proteins tropomyosin (Tm), troponin T (TnT), and troponin I (TnI) form an allosteric regulatory complex that is required for normal cardiac contraction. Multiple isoforms of TnT, Tm, and TnI are differentially expressed in both cardiac development and disease, but concurrent TnI, Tm, and TnT isoform switching has hindered assignment of cellular function to these transitions. We systematically incorporated into the adult sarcomere the embryonic/fetal isoforms of Tm, TnT, and TnI by using gene transfer. In separate experiments, greater than 90% of native TnI and 40–50% of native Tm or TnT were specifically replaced. The Ca2+ sensitivity of tension development was markedly enhanced by TnI replacement but not by TnT or Tm isoform replacement. Titration of TnI replacement from >90% to <30% revealed a dominant functional effect of slow skeletal TnI to modulate regulation. Over this range of isoform replacement, TnI, but not Tm or TnT embryonic isoforms, influenced calcium regulation of contraction, and this identifies TnI as a potential target to modify contractile performance in normal and diseased myocardium. Thin filament proteins tropomyosin (Tm), troponin T (TnT), and troponin I (TnI) form an allosteric regulatory complex that is required for normal cardiac contraction. Multiple isoforms of TnT, Tm, and TnI are differentially expressed in both cardiac development and disease, but concurrent TnI, Tm, and TnT isoform switching has hindered assignment of cellular function to these transitions. We systematically incorporated into the adult sarcomere the embryonic/fetal isoforms of Tm, TnT, and TnI by using gene transfer. In separate experiments, greater than 90% of native TnI and 40–50% of native Tm or TnT were specifically replaced. The Ca2+ sensitivity of tension development was markedly enhanced by TnI replacement but not by TnT or Tm isoform replacement. Titration of TnI replacement from >90% to <30% revealed a dominant functional effect of slow skeletal TnI to modulate regulation. Over this range of isoform replacement, TnI, but not Tm or TnT embryonic isoforms, influenced calcium regulation of contraction, and this identifies TnI as a potential target to modify contractile performance in normal and diseased myocardium. tropomyosin troponin T troponin I slow skeletal TnI cardiac TnI C-terminal, FLAG epitope-tagged cTnI embryonic eTnT adult TnT normal goat serum Triton X-100 Sarcomeric thin filament proteins form an allosteric regulatory complex that governs Ca2+-activated contraction in the myocardium (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, 3Farah C.S. Reinach F.C. FASEB J. 1995; 9: 755-767Crossref PubMed Scopus (476) Google Scholar). Prior to contraction, intracellular Ca2+levels are low and the thin filament regulatory proteins block strong actin-myosin interactions to inhibit force generation. Upon release of Ca2+ from intracellular stores to initiate contraction, Ca2+ binding to troponin C (TnC) causes conformational changes within the regulatory apparatus: tropomyosin (Tm),1 troponin I (TnI), and troponin T (TnT). Consequently, strong interactions between actin and myosin are permitted and force is generated. This precise interplay between Ca2+ and the thin filament regulatory complex is essential for orchestrating normal beat-to-beat regulation of cardiac performance. The isoform content of the thin filament regulatory complex changes during cardiac development (Fig. 1) and in numerous cardiac disease states. Specifically, α- and β-Tm isoform ratios are altered during embryonic/fetal myocardial development, with the ratio of α-Tm gene to β-Tm gene expression increasing from 5:1 in fetal hearts to 60:1 in adult rodent and human myocardium (4Schiaffino S. Reggiani C. Physiol. Rev. 1996; 76: 371-423Crossref PubMed Scopus (1277) Google Scholar). Moreover, differential splicing of cardiac TnT gene transcripts results in expression of an embryonic TnT (eTnT/TnT2) isoform in the developing myocardium followed by a transition to a more basic, lower molecular weight adult TnT (aTnT/TnT4) in adult myocardium (5Anderson P.A. Moore G.E. Nassar R.N. Circ. Res. 1988; 63: 742-747Crossref PubMed Scopus (40) Google Scholar, 6Anderson P.A. Malouf N.N. Oakeley A.E. Pagani E.D. Allen P.D. Circ. Res. 1991; 69: 1226-1233Crossref PubMed Scopus (313) Google Scholar, 7Anderson P.A. Malouf N.N. Oakeley A.E. Pagani E.D. Allen P.D. Basic Res. Cardiol. 1992; 87 Suppl 1: 117-127PubMed Google Scholar, 8McAuliffe J.J. Robbins J. Pediatr. Res. 1991; 29: 580-585Crossref PubMed Scopus (23) Google Scholar, 9Townsend P.J. Barton P.J. Yacoub M.H. Farza H. J. Mol. Cell. Cardiol. 1995; 27: 2223-2236Abstract Full Text PDF PubMed Scopus (85) Google Scholar). In the mammalian heart two distinct TnI genes are expressed (4Schiaffino S. Reggiani C. Physiol. Rev. 1996; 76: 371-423Crossref PubMed Scopus (1277) Google Scholar, 10Murphy A.M. Jones L. Sims H.F. Strauss A.W. Biochemistry. 1991; 30: 707-712Crossref PubMed Scopus (99) Google Scholar). Early in cardiac development the slow skeletal TnI (ssTnI) isoform is expressed. In the neonatal heart, ssTnI is down-regulated and the cardiac TnI (cTnI) gene is expressed. In the adult myocardium only the cTnI isoform is detected. In concert with Tm, TnI, and TnT isoform switching during development, the cardiac contractile apparatus transitions from being highly sensitive to being less sensitive to Ca2+ (11Fabiato A. Fabiato F. Ann. N. Y. Acad. Sci. 1978; 307: 491-522Crossref PubMed Scopus (336) Google Scholar, 12Metzger J.M. Lin W.I. Samuelson L.C. J. Cell Biol. 1994; 126: 701-711Crossref PubMed Scopus (63) Google Scholar). This process effectively tunes the myofilaments to adaptive alterations in intracellular calcium handling (11Fabiato A. Fabiato F. Ann. N. Y. Acad. Sci. 1978; 307: 491-522Crossref PubMed Scopus (336) Google Scholar). Additionally, in the diseased adult myocardium, embryonic TnT and Tm isoforms have been shown to be re-expressed. For example, in pressure overload hypertrophy β-Tm expression is increased, and in failing human myocardium the eTnT isoform is re-expressed (5Anderson P.A. Moore G.E. Nassar R.N. Circ. Res. 1988; 63: 742-747Crossref PubMed Scopus (40) Google Scholar, 6Anderson P.A. Malouf N.N. Oakeley A.E. Pagani E.D. Allen P.D. Circ. Res. 1991; 69: 1226-1233Crossref PubMed Scopus (313) Google Scholar, 7Anderson P.A. Malouf N.N. Oakeley A.E. Pagani E.D. Allen P.D. Basic Res. Cardiol. 1992; 87 Suppl 1: 117-127PubMed Google Scholar, 9Townsend P.J. Barton P.J. Yacoub M.H. Farza H. J. Mol. Cell. Cardiol. 1995; 27: 2223-2236Abstract Full Text PDF PubMed Scopus (85) Google Scholar, 13Mesnard-Rouiller L. Mercadier J.J. Butler-Browne G. Heimburger M. Logeart D. Allen P.D. Samson F. J. Mol. Cell Cardiol. 1997; 29: 3043-3055Abstract Full Text PDF PubMed Scopus (48) Google Scholar, 14Greig A. Hirschberg Y. Anderson P.A. Hainsworth C. Malouf N.N. Oakeley A.E. Kay B.K. Circ. Res. 1994; 74: 41-47Crossref PubMed Scopus (39) Google Scholar). In myotonic dystrophy resulting from a CTG expansion in the TnT gene, increases in CUG-binding protein expression and binding to cardiac TnT pre-mRNA result in increased production of the longer fetal eTnT transcript (15Ladd A.N. Charlet N. Cooper T.A. Mol. Cell. Biol. 2001; 21: 1285-1296Crossref PubMed Scopus (341) Google Scholar, 16Philips A.V. Timchenko L.T. Cooper T.A. Science. 1998; 280: 737-741Crossref PubMed Scopus (699) Google Scholar). Collectively, these studies provide correlative evidence that thin filament regulatory isoform composition influences Ca2+-activated contractile function. The structural complexities of the multimeric thin filament assembly and the overlapping pattern of TnI, Tm and TnT isoform switching in cardiac development have made it difficult to assign specific functional outcomes to each regulatory isoform transition (4Schiaffino S. Reggiani C. Physiol. Rev. 1996; 76: 371-423Crossref PubMed Scopus (1277) Google Scholar). The cellular consequences of disease-mediated disregulation in TnT and Tm isoform expression in cardiac myocytes also remain unknown. Cardiac transgenic studies have proved to be extremely valuable in understanding organ and organismal outcomes to modifications in thin filament regulatory isoforms (17Muthuchamy M. Pieples K. Rethinasamy P. Hoit B. Grupp I.L. Boivin G.P. Wolska B. Evans C. Solaro R.J. Wieczorek D.F. Circ. Res. 1999; 85: 47-56Crossref PubMed Scopus (128) Google Scholar, 18James J. Robbins J. Am. J. Physiol. 1997; 273: H2105-H2118PubMed Google Scholar). One potential consideration in using this approach lies in distinguishing, at the cellular level, the primary effects of transgene expression from possible secondary/compensatory effects, including the cardiac histopathology and maladaptive growth that can arise in transgenic mice (19MacGowan G.A. Du C. Wieczorek D.F. Koretsky A.P. Am. J. Physiol. Heart Circ. Physiol. 2001; 281: H2539-H2548Crossref PubMed Google Scholar, 20Muthuchamy M. Boivin G.P. Grupp I.L. Wieczorek D.F. J. Mol. Cell Cardiol. 1998; 30: 1545-1557Abstract Full Text PDF PubMed Scopus (54) Google Scholar). In addition, to date there has been no single study comparing the functional significance of a systematic manipulation of cardiac-expressed Tm, TnT, and TnI thin filament regulatory isoforms in adult cardiac myocytes. We tested the hypothesis that systematic replacement of each embryonic isoform would uniquely modify adult myocyte contractile responses to Ca2+. To this end, recombinant viral vectors were used for acute genetic engineering of the thin filament regulatory complex. This system has been demonstrated to cause stoichiometric replacement of native thin filament regulatory proteins (TnI, TnT, or Tm) (21Michele D.E. Albayya F.P. Metzger J.M. Nat. Med. 1999; 5: 1413-1417Crossref PubMed Scopus (78) Google Scholar, 22Michele D.E. Albayya F.P. Metzger J.M. J. Cell Biol. 1999; 145: 1483-1495Crossref PubMed Scopus (63) Google Scholar, 23Michele D.E. Metzger J.M. Trends Cardiovasc. Med. 2000; 10: 177-182Crossref PubMed Scopus (10) Google Scholar, 24Michele D.E. Metzger J.M. J. Mol. Med. 2000; 78: 543-553Crossref PubMed Scopus (37) Google Scholar, 25Rust E.M. Albayya F.P. Metzger J.M. J. Clin. Invest. 1999; 103: 1459-1467Crossref PubMed Scopus (82) Google Scholar, 26Rust E.M. Westfall M.V. Metzger J.M. Mol. Cell. Biochem. 1998; 181: 143-155Crossref PubMed Scopus (32) Google Scholar, 27Westfall M.V. Albayya F.P. Metzger J.M. J. Biol. Chem. 1999; 274: 22508-22516Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 28Westfall M.V. Albayya F.P. Turner I.I. Metzger J.M. Circ. Res. 2000; 86: 470-477Crossref PubMed Scopus (50) Google Scholar, 29Westfall M.V. Metzger J.M. News Physiol. Sci. 2001; 16: 278-281PubMed Google Scholar). Self-protein (e.g. expression of adult isoforms, cTnI, aTnT, α-Tm) or reporter viral gene transfer have further demonstrated the specificity of this approach in regard to myocyte contractile structure-function (22Michele D.E. Albayya F.P. Metzger J.M. J. Cell Biol. 1999; 145: 1483-1495Crossref PubMed Scopus (63) Google Scholar, 25Rust E.M. Albayya F.P. Metzger J.M. J. Clin. Invest. 1999; 103: 1459-1467Crossref PubMed Scopus (82) Google Scholar, 26Rust E.M. Westfall M.V. Metzger J.M. Mol. Cell. Biochem. 1998; 181: 143-155Crossref PubMed Scopus (32) Google Scholar, 30Westfall M.V. Rust E.M. Metzger J.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5444-5449Crossref PubMed Scopus (97) Google Scholar). Using this system, adult cardiac myocytes were systematically transduced with eight different vectors harboring Tm, TnI, or TnT isoform expression cassettes (Fig. 1). We then directly compared Ca 2+-activated isometric force responses of single adult myocytes following specific TnT, TnI, or Tm isoform replacement. Results demonstrate that ssTnI acts in a dominant manner with respect to the functional significance of cardiac thin filament isoforms in modulating regulation: TnI > Tm and TnT. Eight recombinant adenoviral vectors were used in this study: cTnI; cTnIFLAG (C-terminal FLAG epitope-tagged cTnI); ssTnI, using rat cDNAs; α-Tm (α tropomyosin); α-TmFLAG (C-terminal FLAG epitope-tagged α-Tm); β-Tm (β-tropomyosin), from human cDNAs; eTnT (embryonic cardiac troponin T/also termed TnT2); and aTnT (adult cardiac troponin T/also termed TnT4), from rat cDNA (Fig. 1,A and B). The general strategy and protocols for shuttle plasmid and vector construction, virus production, and purification have been described earlier (22Michele D.E. Albayya F.P. Metzger J.M. J. Cell Biol. 1999; 145: 1483-1495Crossref PubMed Scopus (63) Google Scholar, 25Rust E.M. Albayya F.P. Metzger J.M. J. Clin. Invest. 1999; 103: 1459-1467Crossref PubMed Scopus (82) Google Scholar, 30Westfall M.V. Rust E.M. Metzger J.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5444-5449Crossref PubMed Scopus (97) Google Scholar, 31Westfall M.V. Rust E.M. Albayya F. Metzger J.M. Methods Cell Biol. 1997; 52: 307-322Crossref PubMed Google Scholar). In each case, transcription was driven by the CMV promoter/enhancer. The polyadenylation signal derived from SV40. These methods have been previously described in detail (31Westfall M.V. Rust E.M. Albayya F. Metzger J.M. Methods Cell Biol. 1997; 52: 307-322Crossref PubMed Google Scholar). Briefly, hearts were removed from adult female Sprague-Dawley rats, mounted on a modified Langendorff perfusion apparatus, perfused with Krebs-Henseleit buffer (KHB) (in mm/liter, 118 NaCl, 4.8 KCl, 25 HEPES, 1.25 K2HPO4, 1.25 MgSO4, 11 glucose) containing 1 mmCa2+ for 5 min, and then perfused with KHB lacking added Ca2+. After 5 min, collagenase (0.5 mg/ml) and hyaluronidase (0.2 mg/ml) were added to the Ca2+-free KHB perfusate, and after 15 min of perfusion the Ca2+concentration was gradually increased to 1 mm and perfused for an additional 15 min. The heart was removed to a sterile beaker, and the ventricles were gently minced in enzyme solution (1 mm Ca2+-KHB + 0.5 mg/ml collagenase + 0.2 mg/ml hyaluronidase). Minced tissue was gently swirled in a 37 °C water bath for 4 × 5 min. The final 2 digests were collected, centrifuged at 45 × g for 10 s, and pelleted cells were resuspended in 1 mm Ca2+-KHB + 20 mg/ml bovine serum albumin before gradually increasing the solution Ca2+ to 1.75 mm. Cells were then resuspended in Dulbecco's modified Eagle's medium + 5% fetal bovine serum + penicillin/streptomycin, counted, and plated on 18-mm2 laminin-coated glass coverslips at 2 × 104 myocytes/coverslip for 2 h. For cardiac myocyte gene transfer, medium was gently replaced with 200 μl of adenovirus diluted in Dulbecco's modified Eagle's medium + P/S to the desired titer. The multiplicity of infection ranged from 250–500 plaque forming units (pfu/rod-shaped myocyte) to optimize gene transfer efficiency for each vector tested. One hour later, 2 ml of Dulbecco's modified Eagle's medium + P/S was added to each coverslip. Medium was replaced with fresh Dulbecco's modified Eagle's medium + P/S 24 h later and every 2–3 days thereafter for up to 7 days post-gene transfer. Glass micropipettes were used to collect cardiac myocytes for subsequent gel analysis. The non-ionic detergent Triton X-100 (TX-100) was used to permeabilize the sarcolemma for comparison to membrane-intact myocytes prior to loading. SDS-PAGE was run at a constant current of 20 mA for 4–5 h, then transblotted onto polyvinylidene difluoride membrane (0.45 μm, Millipore) for 2000 volt-hours and fixed in PBS containing 0.25% glutaraldehyde. Western blots were carried out by initially blocking nonspecific binding sites with Tris-buffered saline containing 5% nonfat dry milk for 2 h. Blots were then incubated with either an anti-cardiac TnT monoclonal antibody (Chemicon mAb 1695, 1:500), an anti-striated muscle TnT monoclonal antibody (Sigma T-6277, clone JLT-12, 1:200), an anti-Tm monoclonal antibody (Sigma T-2780, clone TM311, 1:1 × 106), or an anti-TnI antibody (Chemicon mAb 1691, 1:500) for 2 h. To further validate isoform expression, anti-TnI antibody M32259 (Fitzgerald Industries International, Inc, Concord, MA) and anti-Tm antibodies CH-1 were used. Primary antibody was detected by peroxidase-conjugated goat anti-mouse IgG (1:1000) and an enhanced chemiluminescence detection assay (Amersham Biosciences). Myocytes on coverslips were fixed in 3% paraformaldehyde, washed with PBS, and treated with 50 mm ammonium chloride to remove excess aldehydes. Nonspecific binding of antibodies was minimized with blocking solution containing PBS with 20% normal goat serum (NGS) and 0.5% TX-100. Primary anti-TnI antibody (Chemicon mAb 1691) or anti-FLAG antibody were diluted in PBS with 0.5% TX-100, and 2% NGS was added to cells for 1.5 h at room temperature. Cells were then washed with PBS + 0.5% TX-100 and blocked in PBS with 20% normal goat serum in 0.5% TX-100. Secondary antibody conjugated to Texas Red, (Molecular Probes, T-862) diluted in PBS with 0.5% TX-100 and 2% NGS was added for 1 h. Coverslips were washed in PBS for 4 × 5 min, mounted, and sealed. Immunofluorescence labeling of myocytes was viewed on a Nikon Diaphot 200 microscope fitted with a Noran confocal laser imaging system and Silicon Graphics Indy work station. A force transducer (Cambridge Technology, Model 403A; sensitivity, 5 μg; 1–99% response time, 1 ms) and moving coil galvanometer (Cambridge Technology 6350 optical scanner) were attached to the chamber via three-way positioners. The temperature of the experimental chamber was controlled (15 °C) using thermoelectric modules (Melcor Inc.) coupled to a recirculating water bath heat sink. The chamber was positioned on an anti-vibration table. Coverslips were washed several times in relaxing solution (see below) 2–6 days post-gene transfer, and single, rod-shaped, cardiac myocytes were then attached to the glass micropipettes (32Metzger J.M. Biophys. J. 1995; 68: 1430-1442Abstract Full Text PDF PubMed Scopus (72) Google Scholar). Sarcomere length for each cardiac myocyte was set at 2.15 μm by viewing the myocyte using light microscopy and then adjusting the overall length of the preparation by way of three-way translators. Relaxing and activating solutions contained (in mm/liter) 7 EGTA, 1 free Mg2+, 4 MgATP, 14.5 creatine phosphate, 20 imidazole, and added KCl to yield a total ionic strength of 180 mm/liter. Solution pH was adjusted to 7.00 or 6.20 with KOH/HCl. The pCa (i.e. −log Ca2+) of the relaxing solution was set at 9.0, and thepCa of the solution for maximal activation was 4.0. At eachpCa, steady-state isometric tension developed, after which the fiber was rapidly (< 0.5 ms) slackened to obtain the tension baseline. The myocyte was then relaxed, and the difference between developed tension and the tension baseline following the slack step was measured as total tension. To obtain active tension, resting tension measured at pCa 9.0 was subtracted from total tension. The myocyte was transferred to relaxing solution after each activation at a given pCa. Tension-pCa relationships were constructed by expressing tensions (P) at various submaximal Ca2+ concentrations as fractions of the tension at maximal activation, Po (i.e. isometric tension at pCa 4.0), that bracketed the submaximal activations. To derive values for the Hill coefficient (nH) and midpoint from the tension-pCa relationship (termed K orpCa50), data were fit using the Marquardt-Levenberg non-linear least squares fitting algorithm using the Hill equation in the form: P = (Ca2+)nH/(KnH+ (Ca2+)nH), where Pis tension as a fraction of maximum tension (Po), K is the Ca2+ that yields one-half maximum tension, and nH is the Hill coefficient. Analysis of variance was used to examine whether there were significant differences in pCa50(−log Ca2+ at half maximal tension),nH, or maximum tension using Student-Neuman-Keuls multiple comparison test. A probability level ofp < 0.05 indicated significance. Isolated cardiac myocytes, transduced with recombinant virus harboring adult cardiac TnT (aTnT/TnT4) (14Greig A. Hirschberg Y. Anderson P.A. Hainsworth C. Malouf N.N. Oakeley A.E. Kay B.K. Circ. Res. 1994; 74: 41-47Crossref PubMed Scopus (39) Google Scholar) or embryonic cardiac TnT (eTnT, TnT2) (14Greig A. Hirschberg Y. Anderson P.A. Hainsworth C. Malouf N.N. Oakeley A.E. Kay B.K. Circ. Res. 1994; 74: 41-47Crossref PubMed Scopus (39) Google Scholar), were examined for TnT expression, followed by single myocyte isometric force recordings. On average, eTnT replacement (calculated as eTnT/(eTnT+aTnT) was about 45% (Fig.2A). Expression of eTnT was not detected in myocytes transduced with aTnT (Fig. 2A) or with reporter vectors (not shown). As demonstrated earlier (25Rust E.M. Albayya F.P. Metzger J.M. J. Clin. Invest. 1999; 103: 1459-1467Crossref PubMed Scopus (82) Google Scholar), total TnT content was not altered in aTnT- or eTnT-transduced myocytes. In addition, in myocytes membrane-permeabilized just prior to Western analysis the detection of eTnT was unchanged compared with membrane-intact myocytes (Fig. 2, A and B). The finding of unchanged total TnT content, together with similar detection of eTnT in intact and permeabilized myocytes, provides evidence of sarcomeric replacement of TnT isoforms. Following TnT gene transfer, steady-state isometric force production was determined in single cardiac myocytes. Maximum isometric tension and the slope (Hill coefficient) and position (pCa50) of the tension-pCa relationship were not significantly different among the control, aTnT, or eTnT groups (Fig. 2, C and D). Adult cardiac myocytes were transduced with vectors harboring rat ssTnI or cTnI expression cassettes. In agreement with previous work (27Westfall M.V. Albayya F.P. Metzger J.M. J. Biol. Chem. 1999; 274: 22508-22516Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 28Westfall M.V. Albayya F.P. Turner I.I. Metzger J.M. Circ. Res. 2000; 86: 470-477Crossref PubMed Scopus (50) Google Scholar, 29Westfall M.V. Metzger J.M. News Physiol. Sci. 2001; 16: 278-281PubMed Google Scholar), ssTnI replacement of endogenous cTnI was greater than 90% at day six post-gene transfer (Fig.3, A and B) with no detected alterations in the expression pattern or stoichiometry of other sarcomeric proteins (data not shown). In addition, ssTnI or cTnI expression did not alter total TnI content and is evidence of stoichiometric replacement of TnI in the sarcomere. To further establish the efficiency and localization of newly expressed TnIs in this setting, we expressed an epitope-tagged TnI in the myocytes (cTnIFLAG). Results showed cTnIFLAG was detected in greater than 95% of the cardiac myocytes (Fig. 3C) with expression discretely localized to the cardiac thin filament as shown previously (22Michele D.E. Albayya F.P. Metzger J.M. J. Cell Biol. 1999; 145: 1483-1495Crossref PubMed Scopus (63) Google Scholar). In our previous work (27Westfall M.V. Albayya F.P. Metzger J.M. J. Biol. Chem. 1999; 274: 22508-22516Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 28Westfall M.V. Albayya F.P. Turner I.I. Metzger J.M. Circ. Res. 2000; 86: 470-477Crossref PubMed Scopus (50) Google Scholar, 30Westfall M.V. Rust E.M. Metzger J.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5444-5449Crossref PubMed Scopus (97) Google Scholar, 33Westfall M.V. Turner I. Albayya F.P. Metzger J.M. Am. J. Physiol. Cell Physiol. 2001; 280: C324-C332Crossref PubMed Google Scholar), we showed that replacement of >90% of cTnI by ssTnI causes marked increases in the Ca2+sensitivity of tension development, with an average increase in Ca2+ sensitivity of 0.26 pCa units. Left unknown is whether ssTnI replacement can be titrated to <50%, which is similar to maximum replacement obtained for eTnT (Fig. 2, Aand B), and still cause alterations in Ca2+-activated tension. Based on the efficiency and synchronization of myofilament gene transfer (Fig. 3C) and the normal turnover rate of sarcomeric proteins (34Martin A.F. J. Biol. Chem. 1981; 256: 964-968Abstract Full Text PDF PubMed Google Scholar), it is possible to construct a functional dose-response relationship for ssTnI by examining single myocytes at specific time points after gene transfer. Accordingly, in myocytes at days 2 and 3 post-gene transfer, ssTnI replacement averaged at the single myocyte level 14.4 and 20.4%, respectively (Fig. 3,A and B). Even at these relatively low levels of replacement there was a significant increase (p < 0.01) in the Ca2+ sensitivity of tension, indicating a dominant effect of ssTnI to modulate myofilament regulation of contraction (Fig. 3D). The magnitude increase in Ca2+ sensitivity of tension was 0.12 and 0.20pCa units at days 2 and 3, respectively (p< 0.01). For controls, we examined calcium-activated tension in untreated myocytes and myocytes after cTnI gene transfer at days 2 and 3 in primary culture. Results showed that control and cTnI myocytes were unchanged and not different from each other (Fig. 3D). This is in agreement with our earlier finding of stable contractile function in adult myocytes in short-term primary culture (26Rust E.M. Westfall M.V. Metzger J.M. Mol. Cell. Biochem. 1998; 181: 143-155Crossref PubMed Scopus (32) Google Scholar). In separate experiments, we evaluated the extent of ssTnI replacement by SDS-PAGE/silver staining and through use of a second anti-TnI antibody. In broad agreement with our main findings (Fig. 3, A and B), SDS-PAGE showed 27.5% replacement of cTnI by ssTnI at day 2 and 67% replacement at day 4. Similarly, blots probed with a second TnI antibody (Fitzgerald) showed 29% replacement of cTnI by ssTnI at day 2 after gene transfer (Fig. 3, E and F). Collectively, our findings, using two different TnI antibodies (Chemicon and Fitzgerald) and SDS-PAGE, demonstrate that the extent of TnI replacement is between 14–29% at day 2 and that this extent of TnI replacement achieves nearly the same magnitude shift in Ca2+ sensitivity of tension as we have reported in several previous studies with > 90% replacement (0.26 pCa units, Refs. 27Westfall M.V. Albayya F.P. Metzger J.M. J. Biol. Chem. 1999; 274: 22508-22516Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 28Westfall M.V. Albayya F.P. Turner I.I. Metzger J.M. Circ. Res. 2000; 86: 470-477Crossref PubMed Scopus (50) Google Scholar, 30Westfall M.V. Rust E.M. Metzger J.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5444-5449Crossref PubMed Scopus (97) Google Scholar, 33Westfall M.V. Turner I. Albayya F.P. Metzger J.M. Am. J. Physiol. Cell Physiol. 2001; 280: C324-C332Crossref PubMed Google Scholar). Tropomyosin isoforms alone or together with different TnT isoforms have been postulated to play a role in determining Ca2+ sensitivity of tension (35Greaser M.L. Moss R.L. Reiser P.J. J. Physiol. 1988; 406: 85-98Crossref PubMed Scopus (163) Google Scholar, 36Reiser P.J. Greaser M.L. Moss R.L. J. Physiol. 1992; 449: 573-588Crossref PubMed Scopus (63) Google Scholar, 37Schachat F.H. Diamond M.S. Brandt P.W. J. Mol. Biol. 1987; 198: 551-554Crossref PubMed Scopus (123) Google Scholar, 38McAuliffe J.J. Gao L.Z. Solaro R.J. Circ. Res. 1990; 66: 1204-1216Crossref PubMed Scopus (91) Google Scholar). In general agreement with the 5.5-day half-life for Tm (34Martin A.F. J. Biol. Chem. 1981; 256: 964-968Abstract Full Text PDF PubMed Google Scholar), gene transfer of β-Tm to adult cardiac myocytes resulted in 30–40% replacement of β-Tm at day six post-gene transfer (Fig. 4,A and B). These results were obtained by using two different anti-Tm antibodies (Fig. 4). In earlier work, total Tm protein content and the stoichiometry of Tm within the sarcomere were shown to be unchanged after Tm gene transfer (21Michele D.E. Albayya F.P. Metzger J.M. Nat. Med. 1999; 5: 1413-1417Crossref PubMed Scopus (78) Google Scholar, 22Michele D.E. Albayya F.P. Metzger J.M. J. Cell Biol. 1999; 145: 1483-1495Crossref PubMed Scopus (63) Google Scholar). Expression of β-Tm also was not different in intact and permeabilized myocytes (Fig. 4, A and B), an indication that β-Tm incorporated into the myofilaments. In functional studies on permeabilized single cardiac myocytes, neither maximum isometric tension nor the Ca2+ sensitivity of tension (Hill coefficient/pCa50) were affected by α-Tm, α-TmFLAG, or by β-Tm gene transfer (Fig. 4C legend). Thus, at this level of α-Tm replacement by β-Tm, which is similar to the Tm isoform switching observed during normal myocardial development, myofilament Ca2+ sensitivity is not significantly altered. Acidosis is an important contributor to cardiac dysfunction during myocardial ischemia (39Lee J.A. Allen D.G. J. Clin. Invest. 1991; 88: 361-367Crossref PubMed Scopus (92) Google Scholar). Differential thin filament isoform expression in cardiac development, and among slow and fast skeletal fibers, is thought to play an important role in defining the magnitude in acidic pH-mediated contractile dysfunction (30Westfall M.V. Rust E.M. Metzger J.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5444-5449Crossref PubMed Scopus (97) Google Scholar, 40Solaro R.J. Lee J.A. Kentish J.C. Allen D.G. Circ. Res. 1988; 63: 779-787Crossref PubMed Scopus (158) Google Scholar). We therefore compared the relative effects of Tm, TnI, and TnT isoforms on contractile function at pH 6.20, a level of acidification that accrues during acute myocardial ischemia (39Lee J.A. Allen D.G. J. Clin. Invest. 1991; 88: 361-367Crossref PubMed Scopus (92) Google Scholar). In concert with results obtained under physiological conditions, ssTnI replacement, but not Tm or TnT isoform replacement, had a significant effect on the Ca2+ sensitivity of tension under acidic pH conditions (Fig. 5). Our results demonstrate a functional difference among thin filament regulatory protein isoforms for modulation of Ca2+ sensitivity of tension. Stoichiometric replacement of < 30% of native cTnI with ssTnI caused significant alterations in myofilament Ca2+ sensitivity of tension development. By contrast, 30–50% isoform replacement with either eTnT or β-Tm had no significant influence on the Ca2+ sensitivity of tension. This comparative analysis provides direct evidence that ssTnI has a dominant modulatory effect on the regulation of contraction in cardiac muscle. In its simplest form, the cardiac thin filament can be considered a linear array of 24 regulatory units. A regulatory unit is defined as seven actin monomers, one Tm dimer, and one heterotrimeric troponin complex consisting of troponin C, troponin T, and TnI (41Moss R.L. Circ. Res. 1992; 70: 865-884Crossref PubMed Google Scholar). There is considerable evidence that these discrete regulatory units are coupled functionally. Nearest neighbor and long-range interactions have been demonstrated among thin filament regulatory units (41Moss R.L. Circ. Res. 1992; 70: 865-884Crossref PubMed Google Scholar, 42Brandt P.W. Roemer D. Schachat F.H. J. Mol. Biol. 1990; 212: 473-480Crossref PubMed Scopus (32) Google Scholar, 43Brandt P.W. Diamond M.S. Rutchik J.S. Schachat F.H. J. Mol. Biol. 1987; 195: 885-896Crossref PubMed Scopus (81) Google Scholar). Our finding that 14–29% TnI isoform replacement can cause significant alterations in function supports the concept of long-range regulatory unit interactions. We previously showed that TnI replacement is a stochastic process along the thin filaments, even at early time points after gene transfer (22Michele D.E. Albayya F.P. Metzger J.M. J. Cell Biol. 1999; 145: 1483-1495Crossref PubMed Scopus (63) Google Scholar). As an example, 14% TnI isoform replacement would represent an average replacement of 3–4 regulatory units with ssTnI of a total of 24 in each thin filament. Stated differently, 1 in 7 regulatory units, on average, is switched from cTnI to ssTnI. With stochastic TnI replacement, a dominant effect of ssTnI is proposed to extend across 3–4 native cTnI regulatory units from both sides of each new ssTnI regulated unit. Similar types of long-range interaction have been inferred by the partial removal of troponin C subunits, where as little as 5% manipulation in troponin C content has been hypothesized to exert an effect on calcium-activated tension (41Moss R.L. Circ. Res. 1992; 70: 865-884Crossref PubMed Google Scholar, 42Brandt P.W. Roemer D. Schachat F.H. J. Mol. Biol. 1990; 212: 473-480Crossref PubMed Scopus (32) Google Scholar). Mechanistically, the near neighbor effects of TnI could be mediated via tropomyosin. Recent studies on thin filament regulation reveal multiple transition states/binding interactions of tropomyosin on actin under the influence of calcium and myosin binding (44Lehman W. Hatch V. Korman V. Rosol M. Thomas L. Maytum R. Geeves M.A. Van Eyk J.E. Tobacman L.S. Craig R. J. Mol. Biol. 2000; 302: 593-606Crossref PubMed Scopus (217) Google Scholar). In the present study, there were further increases in Ca2+ sensitivity as TnI isoform replacement rose from day 2 to day 3 after ssTnI gene transfer (Fig. 3). The calcium sensitivity data at physiological and acidic pH at < 30% TnI replacement are in general agreement with previous gene transfer studies in which nearly all cTnI had been replaced by ssTnI (27Westfall M.V. Albayya F.P. Metzger J.M. J. Biol. Chem. 1999; 274: 22508-22516Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 28Westfall M.V. Albayya F.P. Turner I.I. Metzger J.M. Circ. Res. 2000; 86: 470-477Crossref PubMed Scopus (50) Google Scholar, 29Westfall M.V. Metzger J.M. News Physiol. Sci. 2001; 16: 278-281PubMed Google Scholar). This suggests apparent saturation of TnI isoform-dependent functional effects even though 70–80% regulatory units remained coupled with native cTnI. Collectively, these results establish a dominant gain-in-function by ssTnI. Because a change in only about 1 in 7 regulatory units is required to bring about functional effects, this may impact on strategies to influence contractile performance in diseased myocardium. Our finding that up to 45% replacement of α-Tm with the β-Tm isoform has no significant effect on Ca2+-activated tension is in apparent contrast to earlier studies on transgenic mice (45Muthuchamy M. Grupp I.L. Grupp G. O'Toole B.A. Kier A.B. Boivin G.P. Neumann J. Wieczorek D.F. J. Biol. Chem. 1995; 270: 30593-30603Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). Cardiac bundles isolated from β-Tm transgenic mice show increased Ca2+ sensitivity when activated under physiological conditions; however, in agreement with our findings, β-Tm had no significant effect on myofilament pH sensitivity (46Palmiter K.A. Kitada Y. Muthuchamy M. Wieczorek D.F. Solaro R.J. J. Biol. Chem. 1996; 271: 11611-11614Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). One factor to consider in comparing these results is the extent of Tm replacement. In transgenic studies, about 55% or greater Tm replacement was achieved in vivo (17Muthuchamy M. Pieples K. Rethinasamy P. Hoit B. Grupp I.L. Boivin G.P. Wolska B. Evans C. Solaro R.J. Wieczorek D.F. Circ. Res. 1999; 85: 47-56Crossref PubMed Scopus (128) Google Scholar, 45Muthuchamy M. Grupp I.L. Grupp G. O'Toole B.A. Kier A.B. Boivin G.P. Neumann J. Wieczorek D.F. J. Biol. Chem. 1995; 270: 30593-30603Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar), a value greater than we have obtained by Tm gene transfer. Based on biochemical and transgenic studies (45Muthuchamy M. Grupp I.L. Grupp G. O'Toole B.A. Kier A.B. Boivin G.P. Neumann J. Wieczorek D.F. J. Biol. Chem. 1995; 270: 30593-30603Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar), it appears that significant αβ heterodimer formation is necessary for Tm isoform-dependent effects on myofilament Ca2+sensitivity. Another issue to consider is that transgenic manipulation of Tm can and does result in organ-level maladaptive growth (19MacGowan G.A. Du C. Wieczorek D.F. Koretsky A.P. Am. J. Physiol. Heart Circ. Physiol. 2001; 281: H2539-H2548Crossref PubMed Google Scholar, 20Muthuchamy M. Boivin G.P. Grupp I.L. Wieczorek D.F. J. Mol. Cell Cardiol. 1998; 30: 1545-1557Abstract Full Text PDF PubMed Scopus (54) Google Scholar), and this could impact subsequent assays of myocyte function. Similarly, eTnT replacement of 40–50% did not alter Ca2+-activated tension. Previously, correlations have been reported between developmental/disease-mediated transitions in cardiac TnT isoform expression and myocardial function (35Greaser M.L. Moss R.L. Reiser P.J. J. Physiol. 1988; 406: 85-98Crossref PubMed Scopus (163) Google Scholar, 37Schachat F.H. Diamond M.S. Brandt P.W. J. Mol. Biol. 1987; 198: 551-554Crossref PubMed Scopus (123) Google Scholar). As with Tm isoforms, higher levels of replacement of TnT isoforms may be necessary to detect changes in myofilament function. In this light, it is interesting to note that single missense mutations associated with hypertrophic cardiomyopathy (HCM) and placed in eTnT can significantly affect calcium sensitivity, even at very low levels (about 8%) of TnT replacement (25Rust E.M. Albayya F.P. Metzger J.M. J. Clin. Invest. 1999; 103: 1459-1467Crossref PubMed Scopus (82) Google Scholar). Similarly, HCM mutant Tms produce alterations in function at replacement levels comparable with the β-Tm replacement achieved in the present study (21Michele D.E. Albayya F.P. Metzger J.M. Nat. Med. 1999; 5: 1413-1417Crossref PubMed Scopus (78) Google Scholar). There are 39 amino acid differences between α- and β-Tm, including many in the C terminus thought to be critical for Tm and TnT interactions (47Smillie L. Biochemistry of Smooth Muscle Contraction. Academic Press, San Diego1996Google Scholar). In our functional assay, these 39 amino acid differences appear less significant than HCM-associated single missense mutations in Tm. However, regarding the developmental regulation of thin filament isoform expression, it is apparent that TnI has a much more dominant effect than TnT or Tm isoforms on developmental tuning/modulation of myofilament Ca2+ sensitivity. We cannot exclude the possibility that if higher levels of TnT or Tm replacement could be achieved in this system this would then cause alterations in the Ca2+ sensitivity of tension. This result, if obtained, would suggest that the threshold for thin filament isoforms to have a modulatory effect on regulation is markedly higher for Tm and TnT than for TnI isoforms. The finding of a dominant effect of ssTnI isoform replacement even at low levels on Ca2+ sensitivity may be useful in designing TnI proteins for addressing cardiac contractile dysfunction in disease. Based on our results, modified TnI appears to be the best candidate for improving myofilament Ca2+ sensitivity of tension under acidotic conditions during acute myocardial ischemia. In failing myocardium, or in cardiomyopathies associated with altered diastolic Ca2+levels, TnI chimeras with specified regulatory functions may be advantageous (27Westfall M.V. Albayya F.P. Metzger J.M. J. Biol. Chem. 1999; 274: 22508-22516Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 28Westfall M.V. Albayya F.P. Turner I.I. Metzger J.M. Circ. Res. 2000; 86: 470-477Crossref PubMed Scopus (50) Google Scholar, 29Westfall M.V. Metzger J.M. News Physiol. Sci. 2001; 16: 278-281PubMed Google Scholar). Modified TnIs may also serve as a potential strategy for correcting inherited diseases associated with mutated sarcomeric proteins because these mutations have been associated with changes in myofilament Ca2+ sensitivity (17Muthuchamy M. Pieples K. Rethinasamy P. Hoit B. Grupp I.L. Boivin G.P. Wolska B. Evans C. Solaro R.J. Wieczorek D.F. Circ. Res. 1999; 85: 47-56Crossref PubMed Scopus (128) Google Scholar, 21Michele D.E. Albayya F.P. Metzger J.M. Nat. Med. 1999; 5: 1413-1417Crossref PubMed Scopus (78) Google Scholar). It may be possible to affect disease progression if these Ca2+sensitivity shifts are prevented by targeted TnI replacement. Alternatively, pharmaceutical-based strategies to manipulate TnI function may be another approach for future investigation. Finally, although re-expression of embryonic isoforms of TnT and Tm may provide useful bio-markers of failing adult myocardium, our findings indicate that these alterations, at least up to 30–50% replacement, do not appear to contribute to altered calcium-activated tension of the diseased heart." @default.
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