Research Report 2000 - MDC

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Molecular Muscle Physiology Ingo L. Morano The aim of the group is to understand the molecular mechanisms involved in the regulation of cardiac and smooth muscle contraction. Contractility of both cell types is regulated by calcium ions (Ca 2+ ) which penetrate the cells through voltage-gated L-type Ca 2+ channels and, subsequently, induce the release of large amounts of Ca 2+ from the sarcoplasmic reticulum into the myoplasm through Calcium Release Channels (Ryanodin Receptors). Ca 2+ activates the contractile apparatus by binding to troponin C, allowing the molecular motor myosin to interact with thin filaments to produce force and shortening. Due to their key-roles in muscle, we are studying the expression regulation, posttranslational modifications, and functional roles of the subunits of L-type Ca 2+ channels, Ryanodine Receptors, and Type II myosin in cardiac and smooth muscle. Any structural change in these key proteins, by mutation, differential gene expression, alternative splicing of the transcripts, or post-translational modification, modulates cardiac and smooth muscle function. Understanding muscle contraction regulation at molecular and functional levels provides the opportunity to develop causal therapies for the treatment of cardiac and smooth muscle dysfunction. We are working with cardiac and smooth muscle preparations from transgenic/knockout animals and humans as well as cultures of cardiomyocytes and smooth muscle cells. 84 Understanding the molecular motor Essential myosin light chain isoforms regulate human heart contractility Type II myosin isoenzymes are hexamers of about 500 kDa consisting of two heavy chains (MHC) and 4 light chains (MLC), designated essential and regulatory MLC. Two genes coding for cardiac MHC are expressed, β-MHC and α-MHC, located as a tandem on chromosome 14. The cardiac-specific MLC isoforms are designated as atrium- and ventricle-specific essential (ALC-1 and VLC-1, respectively) and regulatory (ALC-2 and VLC-2, respectively) MLC. The cardiomyocytes of hypertrophied ventricles of patients with congenital heart diseases and hypertrophic cardiomyopathy reexpress ALC-1, while MHC isoenzymes are unchanged. This is in sharp contrast to the hypertrophied rodent ventricle which exhibits a change in MHC isoforms rather than MLC expression. The failing ventricles of patients with dilated cardiomypathy, however, hardly express ALC-1. Expression of the cardiac-specific basic-helix-loophelix transcription factors, eHAND and dHAND which bind to E-box elements in the ALC-1 promoter, is increased in hypertrophied human ventricle. Ventricular cross-bridges with ALC-1 have revealed a higher shortening velocity and rate of force development than normal cross-bridges without ALC-1. Maximal isometric force production per cross-sectional area as well as the Ca 2+ sensitivity of the force-Ca 2+ ratio are enhanced. Besides MHC, essential cardiac MLC isoforms also bind via their Nterminus to actin. Inhibition of the actin-MLC interaction by peptide competition increases force production and shortens the velocity of human heart fibers. We have suggested that interaction between actin and MLC represents a “molecular load” for the cross-bridge, thus depressing its cycling kinetics and force production. Regulation of smooth muscle contraction by recruitment of nonmuscle myosin. Prolonged smooth muscle activation produces an initial transient state (phase 1) of high maximal shortening velocity (Vmax) and ATP consumption which is followed by a sustained state (phase 2) of force generation with low Vmax and ATP consumption. Three different genes coding for MHC are expressed in smooth muscle cells, namely, one smooth-muscle-specific MHC (SM- MHC) and two genes coding for nonmuscle MHC. We have mutated the SM-MHC gene by gene targeting technology and found that non-muscle MHC expression remained normal. Smooth muscle from knock-out (KO) neonatal mice did not exhibit a phase 1, although surprisingly, a phase 2 was observed. Thus, the initial transient phase 1 is generated by SM- MHC recruitment while the sustained contraction state can be generated upon switching from SM- to nonmuscle-MHC activation. Non-muscle- MHC-dependent sustained force generation was sufficient for normal fetal development. However, phase 1 i.e. high smooth muscle contractility, becomes indispensable for survival and normal growth soon after birth, especially as far as homeostasis and circulation functions are concerned.
Understanding calcium handling proteins pp700/AHNAK is a cardiac PKA target and binds to the β-subunit of Ca 2+ channels. Ca 2+ channels are multisubunit complexes composed of the poreforming α 1 subunit along with regulatory β and α 2/δ subunits. Coordinated upregulation of Ca 2+ channel subunit expression was observed in patients with hypertrophic, but not dilated, cardiomyopathy. Furthermore, we have identified fetal isoforms of both α 2 - and β-subunits. A novel calcium channel-associated protein of 700-kDa was detected in mammalian cardiomyocytes that undergoes substantial protein kinase A phosphorylation (pp700). Amino acid sequence analysis of pp700 revealed homology to AHNAK and pp700/AHNAK is preferentially localized in the plasma membrane of cardiomyocytes. We believe that both phosphorylation of pp700 and its coupling to Ca 2+ -channels play a physiological role in regulation of cardiac contractility. Selected Publications Wetzel, U., Lutsch, G., Haase, H., Ganten, U., and Morano, I.L. (1998) Alternative splicing of smooth muscle myosin jeavy chain in cardiac vessels of normotensive and hypertensive rats. Circ. Res. 83, 204-20967. Morano, I.L., Chai, G.-X., Walther, T., Baltas, L.G., Lamounier-Zepter, V., Kott, M., and Bader, M. (<strong>2000</strong>) Smooth muscle contraction without smooth muscle myosin. Nat. Cell Biol. in press. Haase, H., Podzuweit, T., Lutsch, G., Hohaus, G., Kostka, S., Lindschau, C., Kott, M., Kraft, R., and Morano, I.L. (1999) Signaling from βadrenoceptor to L-type calcium channel: identification of a novel cardiac protein kinase A target possessing similiarities to AHNAK. FASEB J. 13, 2161-2172. Ritter, O., Luther, H., Haase, H., Schulte, H.D., and Morano, I.L. (1999) Remodeling of the hypertrophied human myocardium by cardiac-specific bHLH transcription factors. J. Cell Biochem. 74, 551-561. Morano I.L. (1999) Tuning the human heart molecular motors by myosin light chains. J. Mol. Med. 77, 544- 555. Patent Applications 1998: “New substance to increase cardiac contractility” PCT/DE98/01240 1999: “A diagnostic marker for the human heart” DPA 199 19 205.7 1999: “Cardiovascular active Peptides” DPA 199 33 090.5 1999: “Drug for the treatment of cardiac insufficiency” DPA 199 38 255.7 Structure of the Group Group leader Prof. Dr. Ingo L. Morano Scientists Dr. Hannelore Haase Dr. Monika Kott Dr. Leonidas Baltas Dr. Udo Zacharzowski Dr. Ines Pagel Dr. Thomas Walther Graduate and undergraduate students Birgit Pfitzmaier Ulrike Wetzel Corina-Mihaela Isac Katarina Wetzel Valeria Lamounier-Zepter Katja Großmann Technical assistants Daniela Balzereit Annette Hohaus Hanna Sydow Ursula Klamm Christel Kemsies Secretariat Verona Kuhle Figure 30: Localization of pp700/AHNAKin rat cardiac tissue (A: logitudinal and B: crosssection). Nuclei are stained with DAPI (in blue). Arrow heads: plasma membrane; large arrows: intercalated discs; small arrows: capillaries (micrograph taken by G. Lutsch, <strong>MDC</strong>) 85
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Research Report 2000 Covers the Per
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Molecular Therapy Molecular and Dev
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The quantum theory, that provided a
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Introduction Clinical Research The
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Genome Research in Berlin- Brandenb
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External Evaluation Over the period
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Congresses In the years reported, t
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Awards A number of prestigious priz
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Genetics, Bioinformatics and Struct
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disorders. Various MDC groups have
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Steen R.G., Kwitek-Black A.E., Glen
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Selected Publications Binas, B., Da
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Technology transfer Based on the fu
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Selected Publications Müller, D.N.
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Selected Publications Hennies, H.C.
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Somatic genetic alterations in brea
Page 33 and 34: Clinical and Molecular Genetics of
Page 35 and 36: Selected Publications Toka, H.R., B
Page 37 and 38: Lbx1, c-met and the control of cell
Page 39 and 40: Selected Publications Moore, A., Mc
Page 41 and 42: Selected Publications Herz, J., Wil
Page 43 and 44: We aim to study the genetic epidemi
Page 45 and 46: Nucleation Nucleation as a pre-requ
Page 47 and 48: Selected Publications Damaschun, G.
Page 49 and 50: Selected Publications Yuan, T., Wal
Page 51 and 52: stability. By determining the struc
Page 53 and 54: Role of Protein Dynamics in Enzyme
Page 55 and 56: Modeling Nucleic Acid Structure and
Page 57 and 58: Conformation, Stability and Interac
Page 59 and 60: Protein Structure Analysis and Prot
Page 61 and 62: Cell Growth and Differentiation 61
Page 63 and 64: dendritic cells (DC). Adoptive tran
Page 65 and 66: developmental pathway that may invo
Page 67 and 68: Regulation of Transcription in Mamm
Page 69 and 70: Differentiation and Growth Control
Page 71 and 72: Cell cycle-dependent transcriptiona
Page 73 and 74: Cell Cycle Regulation Hans-Dieter R
Page 75 and 76: Epithelial Differentiation, Invasio
Page 77 and 78: Selected Publications Hartmann, G.,
Page 79 and 80: Selected Publications Behrens, J.,
Page 81 and 82: Selected Publications Karsten, U.,
Page 83: Depressed contractility in human he
Page 87 and 88: VSMCs. We have cloned and character
Page 89 and 90: Surgical Oncology Peter M. Schlag T
Page 91 and 92: Ubiquitin System and Endoplasmic Re
Page 93 and 94: P450 Cytochromes and the Endoplasmi
Page 95 and 96: Vascular Biology Hermann Haller Thi
Page 97 and 98: Selected Publications Gollasch, M.,
Page 99 and 100: Electron Microscopy Members of the
Page 101 and 102: Molecular Therapy 101
Page 103 and 104: Hematology, Oncology and Tumor Immu
Page 105 and 106: Selected Publications Sturm, I., K
Page 107 and 108: Selected Publications Westermann, J
Page 109 and 110: fractions. The hematopoietic potent
Page 111 and 112: Interaction of pharmacologically ac
Page 113 and 114: Molecular Basis of Congestive Heart
Page 115 and 116: Selected Publications Schneider, G.
Page 117 and 118: different murine and human tumor ce
Page 119 and 120: Molecular and Cell Biology of Hemat
Page 121 and 122: Phospholipids Dietrich Arndt Cytoto
Page 123 and 124: Regulation and Deregulation of Cell
Page 125 and 126: Evolution, Regulation and Genetic A
Page 127 and 128: Molecular and Developmental Neurosc
Page 129 and 130: Cellular Neurosciences Helmut Kette
Page 131 and 132: Growth Factor and Regeneration Gary
Page 133 and 134: Synapse Formation and Function Fran
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Developmental Neurobiology Fritz G.
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Selected Publications Volkmer, H.,
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Structure and Organization 139
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Prof. Dr. Hans Meyer President of t
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Supporting Divisions Safety The div
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Press and Public Relations Research
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Purchasing and Materials Management
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Computing The computing group of th
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Awards Thomas Biederer Boehringer-M
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Index A Abdul, Yetunde 90 Aguirre-A
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H Haase, Hannelore 85, 97, 100, 142
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Q Qin, Zhihai 107, 117 Quass, Petra
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Board of Trustees Chair Wolf-Michae
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