A Mutant Tropomyosin That Causes Hypertrophic Cardiomyopathy Is Expressed In Vivo and Associated With an Increased Calcium Sensitivity

From the Institute of Human Physiology (R.B., M.A.P., C.R.), University of Pavia (Italy); the Institute of Biology and Genetics (D.A.C.), University of Genoa (Italy); the Department of Cardiovascular Medicine (C.S.R., H.W.), University of Oxford (UK); the Minneapolis (Minn) Heart Institute Foundation (B.J.M.); and the Department of Cardiology (P.S.), St Andrea Hospital, La Spezia, Italy.

Abstract—Mutant contractile protein genes that cause familial hypertrophic cardiomyopathy (FHC) are presumed to encode mutant proteins that interfere with contractile function. However, it has generally not been possible to show mutant protein expression and incorporation into the sarcomere in vivo. This study aimed to assess whether a mutant -fast tropomyosin (TM) responsible for FHC is actually expressed and determines abnormal contractile function. Since -fast TM is expressed in heart and skeletal muscle, samples from vastus lateralis muscles were studied from two FHC patients carrying an Asp¹⁷⁵Asn -fast TM mutation and two healthy control subjects. TM isoforms from whole biopsy samples and single fibers were identified by gel electrophoresis and Western blot analysis. An additional faster-migrating TM band was observed in both FHC patients. The aberrant TM was identified as the Asp¹⁷⁵Asn -fast TM by comigration with purified recombinant human Asp¹⁷⁵Asn -fast TM. Densitometric quantification of mutant and wild-type -fast TMs suggested equal expression of the two proteins. Contractile parameters of single skinned muscle fibers from FHC patients and healthy control subjects were compared. Calcium sensitivity was significantly increased in muscle fibers containing Asp¹⁷⁵Asn -fast Tm compared with fibers lacking the mutant TM. No discernible difference was found regarding cooperativity, maximum force, and maximum shortening velocity. This is the first demonstration that the mutant TM that causes FHC is indeed expressed and almost certainly incorporated into muscle in vivo and does result in altered contractile function; this confirms a dominant-negative, rather than null allele, action. Since the mutant TM was associated with increased calcium sensitivity, this mutation might be associated with an enhancement and not a depression of cardiac contractile performance. If so, this contrasts with the hypothesis that FHC mutations induce contractile impairment followed by compensatory hypertrophy.

Key Words: familial hypertrophic cardiomyopathy • tropomyosin • Ca²⁺ sensitivity • skinned muscle fiber

This article has been cited by other articles:

				L. Carrier, S. Schlossarek, M. S. Willis, and T. Eschenhagen The ubiquitin-proteasome system and nonsense-mediated mRNA decay in hypertrophic cardiomyopathy Cardiovasc Res, January 15, 2010; 85(2): 330 - 338. [Abstract] [Full Text] [PDF]

				S. Carballo, P. Robinson, R. Otway, D. Fatkin, J. D.H. Jongbloed, N. de Jonge, E. Blair, J. P. van Tintelen, C. Redwood, and H. Watkins Identification and Functional Characterization of Cardiac Troponin I As a Novel Disease Gene in Autosomal Dominant Dilated Cardiomyopathy Circ. Res., August 14, 2009; 105(4): 375 - 382. [Abstract] [Full Text] [PDF]

				J. Ochala, M. Li, M. Ohlsson, A. Oldfors, and L. Larsson Defective regulation of contractile function in muscle fibres carrying an E41K {beta}-tropomyosin mutation J. Physiol., June 15, 2008; 586(12): 2993 - 3004. [Abstract] [Full Text] [PDF]

				J. P. Sumida, E. Wu, and S. S. Lehrer Conserved Asp-137 Imparts Flexibility to Tropomyosin and Affects Function J. Biol. Chem., March 14, 2008; 283(11): 6728 - 6734. [Abstract] [Full Text] [PDF]

				S. Morimoto Sarcomeric proteins and inherited cardiomyopathies Cardiovasc Res, March 1, 2008; 77(4): 659 - 666. [Abstract] [Full Text] [PDF]

				P. Gunning, G. O'neill, and E. Hardeman Tropomyosin-Based Regulation of the Actin Cytoskeleton in Time and Space Physiol Rev, January 1, 2008; 88(1): 1 - 35. [Abstract] [Full Text] [PDF]

				P. Robinson, P. J. Griffiths, H. Watkins, and C. S. Redwood Dilated and Hypertrophic Cardiomyopathy Mutations in Troponin and {alpha}-Tropomyosin Have Opposing Effects on the Calcium Affinity of Cardiac Thin Filaments Circ. Res., December 7, 2007; 101(12): 1266 - 1273. [Abstract] [Full Text] [PDF]

				J. Ochala, M. Li, H. Tajsharghi, E. Kimber, M. Tulinius, A. Oldfors, and L. Larsson Effects of a R133W beta-tropomyosin mutation on regulation of muscle contraction in single human muscle fibres J. Physiol., June 15, 2007; 581(3): 1283 - 1292. [Abstract] [Full Text] [PDF]

				I. Kulikovskaya, G. B. McClellan, R. Levine, and S. Winegrad Multiple Forms of Cardiac Myosin-binding Protein C Exist and Can Regulate Thick Filament Stability J. Gen. Physiol., April 30, 2007; 129(5): 419 - 428. [Abstract] [Full Text] [PDF]

				P. Robinson, S. Lipscomb, L. C. Preston, E. Altin, H. Watkins, C. C. Ashley, and C. S. Redwood Mutations in fast skeletal troponin I, troponin T, and {beta}-tropomyosin that cause distal arthrogryposis all increase contractile function FASEB J, March 1, 2007; 21(3): 896 - 905. [Abstract] [Full Text] [PDF]

				N. Frey, K. Brixius, R. H. G. Schwinger, T. Benis, A. Karpowski, H. P. Lorenzen, M. Luedde, H. A. Katus, and W. M. Franz Alterations of Tension-dependent ATP Utilization in a Transgenic Rat Model of Hypertrophic Cardiomyopathy J. Biol. Chem., October 6, 2006; 281(40): 29575 - 29582. [Abstract] [Full Text] [PDF]

				M. Canton, A. Skyschally, R. Menabo, K. Boengler, P. Gres, R. Schulz, M. Haude, R. Erbel, F. Di Lisa, and G. Heusch Oxidative modification of tropomyosin and myocardial dysfunction following coronary microembolization Eur. Heart J., April 1, 2006; 27(7): 875 - 881. [Abstract] [Full Text] [PDF]

				F. C. Chen and O. Ogut Decline of contractility during ischemia-reperfusion injury: actin glutathionylation and its effect on allosteric interaction with tropomyosin Am J Physiol Cell Physiol, March 1, 2006; 290(3): C719 - C727. [Abstract] [Full Text] [PDF]

				M. Mirza, S. Marston, R. Willott, C. Ashley, J. Mogensen, W. McKenna, P. Robinson, C. Redwood, and H. Watkins Dilated Cardiomyopathy Mutations in Three Thin Filament Regulatory Proteins Result in a Common Functional Phenotype J. Biol. Chem., August 5, 2005; 280(31): 28498 - 28506. [Abstract] [Full Text] [PDF]

				T. Hayashi, T. Arimura, M. Itoh-Satoh, K. Ueda, S. Hohda, N. Inagaki, M. Takahashi, H. Hori, M. Yasunami, H. Nishi, et al. Tcap gene mutations in hypertrophic cardiomyopathy and dilated cardiomyopathy J. Am. Coll. Cardiol., December 7, 2004; 44(11): 2192 - 2201. [Abstract] [Full Text] [PDF]

				G. Jagatheesan, S. Rajan, N. Petrashevskaya, A. Schwartz, G. Boivin, G. Arteaga, P. P. de Tombe, R. J. Solaro, and D. F. Wieczorek Physiological significance of troponin T binding domains in striated muscle tropomyosin Am J Physiol Heart Circ Physiol, October 1, 2004; 287(4): H1484 - H1494. [Abstract] [Full Text] [PDF]

				A. J. Kee, G. Schevzov, V. Nair-Shalliker, C. S. Robinson, B. Vrhovski, M. Ghoddusi, M. R. Qiu, J. J.-C. Lin, R. Weinberger, P. W. Gunning, et al. Sorting of a nonmuscle tropomyosin to a novel cytoskeletal compartment in skeletal muscle results in muscular dystrophy J. Cell Biol., August 30, 2004; 166(5): 685 - 696. [Abstract] [Full Text] [PDF]

				R. L. Verrier, A. V. Tolat, and M. E. Josephson T-Wave alternans for arrhythmia risk stratification in patients with idiopathic dilated cardiomyopathy J. Am. Coll. Cardiol., June 18, 2003; 41(12): 2225 - 2227. [Full Text] [PDF]

				R. J. Jongbloed, C. L. Marcelis, P. A. Doevendans, J. M. Schmeitz-Mulkens, W. G. Van Dockum, J. P. Geraedts, and H. J. Smeets Variable clinical manifestation of a novel missense mutation in the alpha-tropomyosin (TPM1) gene in familial hypertrophic cardiomyopathy J. Am. Coll. Cardiol., March 19, 2003; 41(6): 981 - 986. [Abstract] [Full Text] [PDF]

				A. Hinkle and L. S. Tobacman Folding and Function of the Troponin Tail Domain. EFFECTS OF CARDIOMYOPATHIC TROPONIN T MUTATIONS J. Biol. Chem., January 3, 2003; 278(1): 506 - 513. [Abstract] [Full Text] [PDF]

				D. Fatkin and R. M. Graham Molecular Mechanisms of Inherited Cardiomyopathies Physiol Rev, October 1, 2002; 82(4): 945 - 980. [Abstract] [Full Text] [PDF]

				D. E. Michele, C. A. Gomez, K. E. Hong, M. V. Westfall, and J. M. Metzger Cardiac Dysfunction in Hypertrophic Cardiomyopathy Mutant Tropomyosin Mice Is Transgene-Dependent, Hypertrophy-Independent, and Improved by {beta}-Blockade Circ. Res., August 9, 2002; 91(3): 255 - 262. [Abstract] [Full Text] [PDF]

				S. B. Marston, J. S. Ingwall, and S. B. Glueck Calcium, contractions, and tropomyosin Focus on "Divergent abnormal muscle relaxation by hypertrophic cardiomyopathy and nemaline myopathy mutant tropomyosins" Physiol Genomics, May 10, 2002; 9(2): 57 - 58. [Full Text] [PDF]

				D. E. Michele, P. Coutu, and J. M. Metzger Divergent abnormal muscle relaxation by hypertrophic cardiomyopathy and nemaline myopathy mutant tropomyosins Physiol Genomics, May 10, 2002; 9(2): 103 - 111. [Abstract] [Full Text] [PDF]

				H. Watkins Hypertrophic cardiomyopathy: from molecular and genetic mechanisms to clinical management Eur. Heart J. Suppl., October 1, 2001; 3(suppl_L): L43 - L50. [Abstract] [PDF]

				R. Rossi, R. Bottinelli, V. Sorrentino, and C. Reggiani Response to caffeine and ryanodine receptor isoforms in mouse skeletal muscles Am J Physiol Cell Physiol, August 1, 2001; 281(2): C585 - C594. [Abstract] [Full Text] [PDF]

				E. Blair, C. Redwood, H. Ashrafian, M. Oliveira, J. Broxholme, B. Kerr, A. Salmon, I. Ostman-Smith, and H. Watkins Mutations in the {{gamma}}2 subunit of AMP-activated protein kinase cause familial hypertrophic cardiomyopathy: evidence for the central role of energy compromise in disease pathogenesis Hum. Mol. Genet., May 1, 2001; 10(11): 1215 - 1220. [Abstract] [Full Text] [PDF]

				O. M. Hernandez, P. R. Housmans, and J. D. Potter Plasticity in Skeletal, Cardiac, and Smooth Muscle: Invited Review: Pathophysiology of cardiac muscle contraction and relaxation as a result of alterations in thin filament regulation J Appl Physiol, March 1, 2001; 90(3): 1125 - 1136. [Abstract] [Full Text] [PDF]

				J. MOGENSEN, P. S. ANDERSEN, U. STEFFENSEN, M. CHRISTIANSEN, H. EGEBLAD, N. GREGERSEN, and A. D. BØRGLUM Development and application of linkage analysis in genetic diagnosis of familial hypertrophic cardiomyopathy J. Med. Genet., March 1, 2001; 38(3): 193 - 198. [Full Text]

				C. C. Evans, J. R. Pena, R. M. Phillips, M. Muthuchamy, D. F. Wieczorek, R. J. Solaro, and B. M. Wolska Altered hemodynamics in transgenic mice harboring mutant tropomyosin linked to hypertrophic cardiomyopathy Am J Physiol Heart Circ Physiol, November 1, 2000; 279(5): H2414 - H2423. [Abstract] [Full Text] [PDF]

				C. Redwood, K. Lohmann, W. Bing, G. M. Esposito, K. Elliott, H. Abdulrazzak, A. Knott, I. Purcell, S. Marston, and H. Watkins Investigation of a Truncated Cardiac Troponin T That Causes Familial Hypertrophic Cardiomyopathy : Ca2+ Regulatory Properties of Reconstituted Thin Filaments Depend on the Ratio of Mutant to Wild-Type Protein Circ. Res., June 9, 2000; 86(11): 1146 - 1152. [Abstract] [Full Text] [PDF]

				A. M. Gordon, E. Homsher, and M. Regnier Regulation of Contraction in Striated Muscle Physiol Rev, April 1, 2000; 80(2): 853 - 924. [Abstract] [Full Text] [PDF]

				P. Sorajja, P.M. Elliott, and W.J. Mckenna The molecular genetics of hypertrophic cardiomyopathy: prognostic implications Europace, January 1, 2000; 2(1): 4 - 14. [PDF]

				L. S. Tobacman, D. Lin, C. Butters, C. Landis, N. Back, D. Pavlov, and E. Homsher Functional Consequences of Troponin T Mutations Found in Hypertrophic Cardiomyopathy J. Biol. Chem., October 1, 1999; 274(40): 28363 - 28370. [Abstract] [Full Text] [PDF]

				C. S. Redwood, J. C. Moolman-Smook, and H. Watkins Properties of mutant contractile proteins that cause hypertrophic cardiomyopathy Cardiovasc Res, October 1, 1999; 44(1): 20 - 36. [Abstract] [Full Text] [PDF]

				M. V. Westfall, F. P. Albayya, and J. M. Metzger Functional Analysis of Troponin I Regulatory Domains in the Intact Myofilament of Adult Single Cardiac Myocytes J. Biol. Chem., August 6, 1999; 274(32): 22508 - 22516. [Abstract] [Full Text] [PDF]

				H. Nakaura, S. Morimoto, F. Yanaga, M. Nakata, H. Nishi, T. Imaizumi, and I. Ohtsuki Functional changes in troponin T by a splice donor site mutation that causes hypertrophic cardiomyopathy Am J Physiol Cell Physiol, August 1, 1999; 277(2): C225 - C232. [Abstract] [Full Text] [PDF]

				M. Muthuchamy, K. Pieples, P. Rethinasamy, B. Hoit, I. L. Grupp, G. P. Boivin, B. Wolska, C. Evans, R. J. Solaro, and D. F. Wieczorek Mouse Model of a Familial Hypertrophic Cardiomyopathy Mutation in {alpha}-Tropomyosin Manifests Cardiac Dysfunction Circ. Res., July 9, 1999; 85(1): 47 - 56. [Abstract] [Full Text] [PDF]

				J. M. Metzger, P. A. Wahr, D. E. Michele, F. Albayya, and M. V. Westfall Effects of Myosin Heavy Chain Isoform Switching on Ca2+-Activated Tension Development in Single Adult Cardiac Myocytes Circ. Res., June 11, 1999; 84(11): 1310 - 1317. [Abstract] [Full Text] [PDF]

				E. N. Olson and J. D. Molkentin Prevention of Cardiac Hypertrophy by Calcineurin Inhibition : Hope or Hype? Circ. Res., April 2, 1999; 84(6): 623 - 632. [Full Text] [PDF]

				F. Yanaga, S. Morimoto, and I. Ohtsuki Ca2+ Sensitization and Potentiation of the Maximum Level of Myofibrillar ATPase Activity Caused by Mutations of Troponin T Found in Familial Hypertrophic Cardiomyopathy J. Biol. Chem., March 26, 1999; 274(13): 8806 - 8812. [Abstract] [Full Text] [PDF]

				H. L. Sweeney, H. S. Feng, Z. Yang, and H. Watkins Functional analyses of troponin T mutations that cause hypertrophic cardiomyopathy: Insights into disease pathogenesis and troponin function PNAS, November 24, 1998; 95(24): 14406 - 14410. [Abstract] [Full Text] [PDF]

				B. J. Maron, J. H. Moller, C. E. Seidman, G. M. Vincent, H. C. Dietz, A. J. Moss, J. A. Towbin, H. M. Sondheimer, R. E. Pyeritz, G. McGee, et al. Impact of Laboratory Molecular Diagnosis on Contemporary Diagnostic Criteria for Genetically Transmitted Cardiovascular Diseases: Hypertrophic Cardiomyopathy, Long-QT Syndrome, and Marfan Syndrome : A Statement for Healthcare Professionals From the Councils on Clinical Cardiology, Cardiovascular Disease in the Young, and Basic Science, American Heart Association Circulation, October 6, 1998; 98(14): 1460 - 1471. [Full Text] [PDF]

				G. Bonne, L. Carrier, P. Richard, B. Hainque, and K. Schwartz Familial Hypertrophic Cardiomyopathy : From Mutations to Functional Defects Circ. Res., September 21, 1998; 83(6): 580 - 593. [Abstract] [Full Text] [PDF]

				J. Robbins {alpha}-Tropomyosin Knockouts : A Blow Against Transcriptional Chauvinism Circ. Res., January 23, 1998; 82(1): 134 - 136. [Full Text] [PDF]

				H.-J. Wang, Y.-C. Zhu, and T. Yao Effects of all-trans retinoic acid on angiotensin II-induced myocyte hypertrophy J Appl Physiol, May 1, 2002; 92(5): 2162 - 2168. [Abstract] [Full Text] [PDF]

				K. Elliott, H. Watkins, and C. S. Redwood Altered Regulatory Properties of Human Cardiac Troponin I Mutants That Cause Hypertrophic Cardiomyopathy J. Biol. Chem., July 14, 2000; 275(29): 22069 - 22074. [Abstract] [Full Text] [PDF]

				C. C. Witt, B. Gerull, M. J. Davies, T. Centner, W. A. Linke, and L. Thierfelder Hypercontractile Properties of Cardiac Muscle Fibers in a Knock-in Mouse Model of Cardiac Myosin-binding Protein-C J. Biol. Chem., February 9, 2001; 276(7): 5353 - 5359. [Abstract] [Full Text] [PDF]