Familial Hypertrophic Cardiomyopathy Mutations from Different Functional Regions of Troponin T Result in Different Effects on the pH and Ca2+ Sensitivity of Cardiac Muscle Contraction

To understand the molecular function of troponin T (TnT) in the Ca2+ regulation of muscle contraction as well as the molecular pathogenesis of familial hypertrophic cardiomyopathy (FHC), eight FHC-linked TnT mutations, which are located in different functional regions of human cardiac TnT (HCTnT), were produced, and their structural and functional properties were examined. Circular dichroism spectroscopy demonstrated different secondary structures of these TnT mutants. Each of the recombinant HCTnTs was incorporated into porcine skinned fibers along with human cardiac troponin I (HCTnI) and troponin C (HCTnC), and the Ca2+ dependent isometric force development of these troponin-replaced fibers was determined at pH 7.0 and 6.5. All eight mutants altered the contractile properties of skinned cardiac fibers. E244D potentiated the maximum force development without changing Ca2+ sensitivity. In contrast, the other seven mutants increased the Ca2+ sensitivity of force development but not the maximal force. R92L, R92W, and R94L also decreased the change in Ca2+ sensitivity of force development observed on lowering the pH from 7 to 6.5, when compared with wild type TnT. The examination of additional mutants, H91Q and a double mutant H91Q/R92W, suggests that mutations in a region including residues 91–94 in HCTnT can perturb the proper response of cardiac contraction to changes in pH. These results suggest that different regions of TnT may contribute to the pathogenesis of TnT-linked FHC through different mechanisms. To understand the molecular function of troponin T (TnT) in the Ca2+ regulation of muscle contraction as well as the molecular pathogenesis of familial hypertrophic cardiomyopathy (FHC), eight FHC-linked TnT mutations, which are located in different functional regions of human cardiac TnT (HCTnT), were produced, and their structural and functional properties were examined. Circular dichroism spectroscopy demonstrated different secondary structures of these TnT mutants. Each of the recombinant HCTnTs was incorporated into porcine skinned fibers along with human cardiac troponin I (HCTnI) and troponin C (HCTnC), and the Ca2+ dependent isometric force development of these troponin-replaced fibers was determined at pH 7.0 and 6.5. All eight mutants altered the contractile properties of skinned cardiac fibers. E244D potentiated the maximum force development without changing Ca2+ sensitivity. In contrast, the other seven mutants increased the Ca2+ sensitivity of force development but not the maximal force. R92L, R92W, and R94L also decreased the change in Ca2+ sensitivity of force development observed on lowering the pH from 7 to 6.5, when compared with wild type TnT. The examination of additional mutants, H91Q and a double mutant H91Q/R92W, suggests that mutations in a region including residues 91–94 in HCTnT can perturb the proper response of cardiac contraction to changes in pH. These results suggest that different regions of TnT may contribute to the pathogenesis of TnT-linked FHC through different mechanisms. Vertebrate striated muscles contract in response to increasing intracellular Ca2+ concentration where Ca2+-bound troponin (Tn) 1The abbreviations used are: Tn, troponin; HCTnT, human cardiac troponin T; TnI, troponin I; TnC, troponin C; Tm; tropomyosin; FHC, familial hypertrophic cardiomyopathy; MES, 2-N-morpholinoethanesulfonic acid; SCD, sudden cardiac death; WT, wild type. together with tropomyosin (Tm) activates the thin filaments and leads to interactions between the thick and thin filaments (1Zot A.S. Potter J.D. Annu. Rev. Biophys. Biophys. Chem. 1987; 16: 535-559Crossref PubMed Scopus (472) Google Scholar, 2Ohtsuki I. Maruyama K. Ebashi S. Adv. Protein Chem. 1986; 38: 1-67Crossref PubMed Scopus (207) Google Scholar). Tn, one of the regulatory proteins of striated muscle, is composed of three subunits, a Ca2+ binding subunit troponin C (TnC), an inhibitory subunit troponin I (TnI), and a Tm binding subunit troponin T (TnT). Although the activation and inhibition of muscle contraction are primarily achieved by TnC and TnI, respectively, these events cannot occur without TnT. In other words TnT not only anchors the Tn complex to the thin filament but also contributes to the Ca2+-dependent regulation of muscle contraction (3Perry S.V. J. Muscle Res. Cell Motil. 1998; 19: 575-602Crossref PubMed Scopus (260) Google Scholar). Therefore, any functional and structural defects in these Tn subunits may cause alteration of the Ca2+ regulation of muscle contraction. Familial hypertrophic cardiomyopathy (FHC) is an autosomal dominant disorder of heart muscle, is genetically heterogeneous, and results from mutations in genes encoding almost every major sarcomeric protein, including β-myosin heavy chain (4Geisterfer-Lowrance A.A. Kass S. Tanigawa G. Vosberg H.P. McKenna W. Seidman C.E. Seidman J.G. 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Full Text Full Text PDF PubMed Scopus Google Scholar). the demonstrated properties in the Ca2+ sensitivity of force development and as well as other properties of FHC, as function T. D. J. F. L. J. Y. Y. Potter J.D. J. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, K. F. T. M. M. Potter J.D. J. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). results at the molecular and that altered Ca2+ regulation of muscle contraction by TnT mutations the TnT-linked To the functional of the mutations on muscle contraction eight different HCTnT mutations, R92L, R92W, and are located in and different regions of the HCTnT R92W, and were to cause a of FHC by a of C. F. McKenna W.J. 2001; PubMed Scopus Google Scholar, B. K. Seidman C. Watkins H. J. 1997; PubMed Scopus Google Scholar, A. C. F. L. McKenna W.J. 1999; PubMed Scopus Google Scholar, C. H. Y. S. T. K. J. Cell. 1997; Full Text PDF PubMed Scopus Google Scholar). a R. Seidman J.G. Seidman C.E. PubMed Scopus Google Scholar). a a The to a with a of but SCD, the of is C. F. McKenna W.J. 2001; PubMed Scopus Google Scholar, Carrier L. H. G. Bercovici J. Richard P. Hainque B. S. O. A. A.A. M. M. K. 1996; PubMed Scopus Google Scholar). are and the by these mutations are Y. Toshima H. Kimura A. Harada H. Koyanagi T. Nishi H. M. Imaizumi T. J. 1996; Full Text PDF PubMed Scopus Google Scholar, H. McKenna W.J. Thierfelder L. R. A. P. A. Seidman J.G. Seidman C.E. N. J. 1995; PubMed Scopus Google Scholar). To of these TnT mutations, recombinant HCTnT mutants were incorporated into porcine cardiac skinned fibers together with and the Tn by the of Ca2+-dependent isometric force development at pH 7.0 and 6.5. Circular dichroism spectroscopy was to the secondary structural the TnT from a between the of the FHC in the TnT and on force. the of TnT to mutations in the and the of the altered mutants on Ca2+-dependent force in increased Ca2+ sensitivity. These results suggest that different TnT mutations the Ca2+ regulation of muscle contraction through different and that these may contribute to the molecular pathogenesis of of FHC by the TnT and of of human cardiac TnI, and TnC was D. R. J. M. G. Potter J.D. J. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). To the mutations R92L, R92W, and E244D as well as the additional TnT mutations H91Q and H91Q/R92W, a chain was and were in the in the TnT were by and of human cardiac Tn subunits were in and were used the of the and D. R. J. M. G. Potter J.D. J. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). recombinant HCTnTs were from a a by on the the were a were with a in a the a in a the and in a the Circular of HCTnT of HCTnT and the mutants were a with a of at were including The of the HCTnT mutants were determined the with wild type HCTnT as a The protein concentration was to and of were the were and the of the was a in the were the where is the in is the and is the in The was the at Biophys. Res. PubMed Scopus Google Scholar), where is the of in of and of muscles were from the of of fibers were and with the 7 and I and including at of force fibers in were and between a force and a were skinned with in at maximum force was at the fibers were to increasing Ca2+ concentration from to and the was at pH 7.0 and at pH on the The were with the as D. R. J. M. G. Potter J.D. J. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Tn and in The Tn which from that of and M. I. J. PubMed Scopus Google Scholar), was to the Tn in the porcine fibers with human subunits D. R. J. M. G. Potter J.D. J. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, M. I. J. PubMed Scopus Google Scholar). the Ca2+-dependent force development was the fibers were with an of HCTnT mutants in a and were in the TnT fibers were with the without protein and force at and force development is to the of the porcine TnI and To the of by different TnT mutants the regulation was where and are the force development of fibers at and of the TnT fibers was also compared with that of HCTnT fibers the of TnT mutations on isometric force fibers were with an complex Ca2+-dependent force development of fibers was determined at pH 7.0 and at pH 6.5. were as the The of the between the HCTnT mutants was determined by of by a was to TnT the of on the maximum force the was of the of TnT of HCTnT mutants were in the of The of TnT was of the to mutations, and in increased to and R92W, and also increased the of and E244D were to that of In the of compared with not of TnT in mutations in TnT the of TnT the thin in the of TnT Tm other thin filament proteins the of Tn in skinned fibers. that the into the porcine skinned cardiac muscle D. R. J. M. G. Potter J.D. J. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). is with in which including the Tm binding region of TnT were and a a decreased Tm T. S. Biophys. J. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, A. J. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). To the of TnT mutants the regulation of force development fibers were with TnT mutant and The of Tn complex that in the fibers TnT well with the regulation TnT D. R. J. M. G. Potter J.D. J. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, S. F. R. I. J. 1998; PubMed Google Scholar). These from the of force can used as an the of Tn demonstrated that porcine cardiac Tn can by recombinant HCTnT and HCTnT fibers almost Ca2+ regulation were with and TnC D. R. J. M. G. Potter J.D. J. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). with observed Ca2+ regulation of force development HCTnT regulation in the The regulation and fibers was with the that the of HCTnT with porcine Tn was not altered by these The of regulation of fibers was also not different from that of and not any Ca2+ regulation of force development the fibers were with The fibers increased regulation but were not different when were compared with fibers. In contrast, the regulation of force development was increased when fibers were with R92W, that the to at to at and to at the of The mutants were to with the Tn, as by the Ca2+ regulation of force of of fibers at pH Ca2+ of the isometric force of skinned development TnT was the where and are force at and in are the maximum force development of fibers with TnI and All of maximum force development were in the of the maximum force development of fibers at pH 7.0 as of wild wild wild wild wild wild wild wild wild wild wild wild wild wild wild wild wild wild of the isometric force of skinned development TnT was the where and are force at and in are the maximum force development of fibers with TnI and All of maximum force development were in the of the maximum force development of fibers at pH 7.0 as wild wild wild in a of on the and observed the maximum force development was decreased by HCTnT compared with the maximum force of porcine cardiac fibers. was in to a between porcine and human cardiac the of maximum force decreased when porcine fibers were with porcine cardiac TnT when human cardiac fibers were with recombinant HCTnT D. R. J. M. G. Potter J.D. J. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, K. D. Potter J.D. Biophys. J. Scholar). The maximum force development of HCTnT was not by the and the maximum force of fibers was of that of fibers. the of mutants on the maximum force development TnT as well as and the of force development is as the of the maximum force development to which was as the maximum force development of fibers by TnT of and fibers were to that of the of and E244D fibers were the E244D fibers. the regulation was fibers were with a of the maximum force development a of these TnT to the was not different from the maximum force development of the TnT fibers the E244D fibers to that of fibers with The E244D fibers with increased maximal force development compared with the the maximum force All including not the of force development force development at of fibers. of TnT on the at pH development were determined at pH 7.0 and in the fibers that with HCTnT the HCTnT mutants and the development of FHC mutants and fibers at pH The of and the maximum force are in All mutants, with the of increased the Ca2+ sensitivity of force development in the of R94L The of were also increased by R92W, and In contrast, E244D not change the but increased maximal force development as of of fibers at pH at pH 7.0 at pH of wild wild wild wild wild wild wild wild wild wild wild wild wild wild wild at pH 7.0 at pH wild wild in a The Ca2+ sensitivity and the maximum force development of fibers were when pH was from 7.0 to to fibers decreased Ca2+ sensitivity and maximal force development at pH compared with pH The Ca2+ sensitivity of force development of seven of the eight mutant and fibers were that of fibers. The Ca2+ sensitivity of the fibers was not different from that of fibers. the maximal force development was increased by of the of lowering pH on the Ca2+ sensitivity of fibers was R92L, R92W, and R94L fibers sensitivity to changes in Ca2+ lowering of the pH and at pH 7.0 at pH and respectively, that these mutants pH in of Ca2+ sensitivity. In contrast, the of and fibers and were to fibers The pH of and E244D fibers were that of fibers. of to the molecular of Ca2+ regulation of muscle contraction. the molecular function of TnT is not as well as that of TnC different mutations in the HCTnT J.A. Harada K. Potter J.D. Cell. Thierfelder et (9Thierfelder L. Watkins H. MacRae C. Lamas R. McKenna W. Vosberg H.P. Seidman J.G. Seidman C.E. Cell. 1994; 77: 701-712Abstract Full Text PDF PubMed Scopus (873) Google the TnT mutations in FHC that is by TnT mutations a of an altered molecular function of that to cardiac muscle the functional of these TnT mutations of the molecular function of TnT as well as the of et D. A. J. Clin. Invest. 1996; PubMed Scopus Google the functional of TnT in and et S. F. R. I. J. 1998; PubMed Google that the mutants, and increased Ca2+ sensitivity of isometric force development of skinned cardiac fibers when were with the Tn including from that mutants can Ca2+ regulation of muscle contraction J.A. Harada K. Potter J.D. Cell. Scholar, Potter J.D. J. 2001; PubMed Scopus Google Scholar, Potter J.D. 2001; 11: PubMed Scopus Google Scholar). 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Chem. 1999; Full Text Full Text PDF PubMed Scopus Google that the complex the TnT increased These results suggest that Ca2+ sensitivity of and force development of skinned fibers by increasing Ca2+ of Although not in the of TnT mutations on the between TnT and the other Tn subunits suggest that the and the region of TnT where the to as the in Ca2+ sensitivity and maximum force spectroscopy demonstrated secondary structures of TnT mutants in the of and of The and TnT mutants in the of in not was not to protein of these at The of by concentration was mutant other TnT proteins from different that a TnT the of secondary compared with the other mutants T. S. Biophys. J. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, A. J. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). All TnT mutants secondary and the of not when was to the and to in that these mutations may not the secondary of TnT the structural with TnT mutants may that mutations cause interactions between TnT and TnI, that these are TnT to into in the of the other troponin Ca2+ sensitivity of force the mutants, R92L, R92W, and were to decreased to Tn complex in the fibers. et T. S. Biophys. J. 2001; Full Text Full Text PDF PubMed Scopus Google that mutations in the region Tm binding TnT of Tm binding to results that R92L, R92W, and R94L in fibers with their the which not well with porcine cardiac Tn complex D. R. J. M. G. Potter J.D. J. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). that not et T. S. Biophys. J. 2001; Full Text Full Text PDF PubMed Scopus Google that the of Tm binding to actin but not The mutant also decreased The of Tm the thin is is located in the essential TnT region Tm binding A. J. Chem. Full Text Full Text PDF PubMed Scopus Google is that an to at the binding in the with the mutant in In contrast, in the of the TnT mutants and and the mutant E244D increased maximum force development of fibers without their TnT In the of TnI and TnC, and increased the maximum force development by compared with WT, E244D increased maximum force development by the fibers maximum force development with the complex the and fibers not decreased the maximum force development These results together with the that these mutations in the different structural and functional regions suggest that the by which E244D the isometric force development different from the The region including residues and is the of the Tm binding region of and in the regulation of force development is not is that in region thin filament the the observed of force at Ca2+ et F. S. I. J. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google that E244D increased the maximum et H. F. I. S. J. 1999; PubMed Scopus Google that of E244D into skinned cardiac fibers also potentiated the maximum force development and These results are with that the region including in TnT with TnI R. S. U. S. A. 1998; PubMed Scopus Google Scholar). The of human cardiac Tn including the TnC the of TnI and the of TnT by et S. A. K. Y. 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