Robbins Lab

Structure Function Relationships for the Contractile Proteins and Signaling Proteins in the Heart

Figures

Confocal Microscopy

Shown is a confocal micrograph in which the ability of the genetically engineered All-P troponin I is compared to the normal pattern of protein. It is important to confirm that the transgenically encoded protein is inserting into the sarcomere correctly and this micrograph confirms that.

Model of cTnC conformational changes

Model of cTnC conformational changes

A. cTnC bound to dephosphorylated cTnI. Cardiac TnC is depicted as a (light blue) dumbbell-shaped molecule composed of two globular lobes, connected by a central linker. Each lobe comprises the two Ca2+ binding EF hands. cTnC binds to TnI in an anti-parallel manner: the C lobe of TnC (C') interacts with the N terminal residues of TnI, and the N lobe of TnC (N') interacts with the C terminal region. The various mouse cTnI domains, including the cardiac specific N-terminus (1-32), amino terminus (33-80) (N-domain), predicted coil (81-129), inhibitory region (In. region), regulatory (R) domain (147-167) and carboxyl terminus (168-211) are shown. The F/G loop of cTnC is also shown. PKA and PKC phosphorylation sites are depicted as circles and triangles respectively. The cardiac specific N-terminus residues 1-32 contacts the N-domain of cTnC in the absence of phosphorylation. In the presence of cTnI and Ca2+, the regulatory domain of cTnC exists in a more "open" conformation.

B. cTnI-AllP causes the cTnC to adopt a more "closed" conformation. PKA phosphorylation at Ser23, 24 of cTnI alters cTnC regulatory domain equilibria, but does not affect the C-terminus conformation of cTnC Interaction with the cTnI-AllP protein induced chemical shifts changes within the Ca2+-saturated C- and N-domains of cTnC, substantially altering the N-domain conformation. As a result, the regulatory domain of cTnC assumed a more "closed" conformation.

The overall 5-year goal of this part of the laboratory's program is to integrate specific signaling pathways with cardiac and cardiomyocyte function in the adult and during development, emphasizing the pathological implications of aberrant signaling and resultant stress on the cardiomyocyte. The laboratory uses the general approach of targeted mutagenesis along with cardiac-specific loss- and gain-of-function approaches, which can be modulated precisely. Each project is driven by specific hypotheses and experiments so that these methodologies are appropriate. Since the modifications can be controlled in a tissue and even cardiac-compartment-specific fashion, as well as at different developmental times, very precise genetic manipulations can be carried out effectively. The cardiac-specific gain-of-function approach is particularly powerful if coupled with the use of inducible systems and a novel and robust, cardiac-specific inducible promoter, developed in the Robbins laboratory. A comprehensive analysis of the resultant mice can be a daunting task for a single lab, both in terms of time and expense, but the use of divisional cores greatly enhances the cost-effectiveness of the research. The HistoPathology/Physiology Core provides a coherent centralized facility for generating cytological analyses where gross or subtle manifestations of the pathology or cytopathology resulting from the various targeting experiments can be discerned, integrated, and valuable correlations made between the different projects. Integration of these data directly with physiology aids in their integrative interpretation as well as pointing the way to the next experiments in order to extend the descriptive data into mechanism and function. The lab also has the capability of studying the entire functional spectrum of the modified cells, organ or whole animals, using a variety of invasive and noninvasive techniques, while another core provides a common source of cells suitable for transfection as well as the necessary adenovirus constructs and stocks that can be shared between the different projects.

Publications

Sadayappan, S., Finley, N., Howarth, J. W., Osinska, H., Klevitsky, R., Lorenz, J. N., Rosevear, P. R., J. G., Robbins, J. (2008) Role of the acidic N¹-Region of cardiac troponin I in regulating myocardial function. FASEB
Journal. (in press)
 
Pattison, JS., Waggoner, J. R., James, J., Martin, L., Gulick, J., Osinska, H., Klevitsky, R., Kranias, E. G., Robbins, J. (2008) Phospholamban overexpression in transgenic rabbits. Transgenic Res. (in press)
 
Wolf, C. M., Arad, M., Ahmad, F., A., Bernstein, S. A., Toka, O., Morley, G., Robbins, J., Seidman, J. G., Seidman, C. E., Berul, C. I. (2008) Reversibility of PRKAG2 glycogen storage cardiomyopathy and electrophysiologic manifestations. Circulation. 117: 144-154
 
Pinz, I., Robbins, J., Benjamin, I. J., Ingwall, J. (2008) Unmasking different mechanical and energetic roles for the small heat shock proteins CryAB and HSPB2 using genetically modified mouse hearts. FASEB J. 22: 84-92
 
Krenz, M., Sadayappan, S., Osinska, H. E., Henry, J. A., Beck, S., Warshaw, D. M., Robbins, J. (2007) Distribution and structure-function relationship of myosin heavy chain isoforms in the adult mouse heart. J. Biol. Chem. 282: 24057-24064
 
Hambleton, M., York, A., Sargent, M. A., Kaiser, R. A., Lorenz, J. N., Robbins, J., Molkentin, J.D. (2007) Inducible and myocyte-specific inhibition of PKCa enhances cardiac contractility and protects against infarction-induced heart failure. Am. J. Physiol. Heart Circ. Physiol. 293: H3768-H3771
 
Heineke J., Auger-Messier M., Xu J., Oka T., Sargent M.A., York A, Klevitsky R., Vaikunth S., Duncan S.A., Aronow B.J., Robbins J., Crombleholm T.M., Molkentin J.D. (2007) Cardiomyocyte GATA4 functions as a stress-responsive regulator of angiogenesis in the murine heart. J. Clin. Invest. 117: 3198-3210
 
Nagyama, T., Takimoto, E., Sadayappan, S., Mudd, J. O., Seidman, J. G., Robbins, J., Kass, D. A. (2007) PKA-phosphorylation dependent and independent regulation of in vivo contraction/relaxation kinetics by myosin binding protein C. Circulation 116: 2399-2408
 
Diwan, A., Krenz, M., Sayed, F. M., Wanasapura, J., Ren, X., Matkovich, S. J., Koesters, A. G., Li, H., Kirshenbaum, L. A., Robbins, J., Jones, W. K., Dorn, G. W. II (2007) Inhibition of ischemic cardiomyocyte apoptosis through targeted ablation of bnip3 restrains post-infarction remodeling. J. Clin. Invest. 117: 2825-2833
 
Oka, T., Xu, J., Kaiser, R. A., Melendez, J., Hambleton, M., Sargent, M. A,, Lorts, A., Brunskill, E. W., Dorn, G. W., 2nd, Conway, S. J., Aronow, B. J., Robbins, J., Molkentin, J. D. (2007) Genetic manipulation of periostin expression reveals a role in cardiac hypertrophy and ventricular remodeling. Circ. Res. 101: 313-321

Oka T, Xu J, Kaiser RA, Melendez J, Hambleton M, Sargent MA, Lorts A, Brunskill EW, Dorn GW 2nd, Conway SJ, Aronow BJ, Robbins J, Molkentin JD. (2007) Genetic manipulation of periostin expression reveals a role in cardiac hypertrophy and ventricular remodeling. Circ Res 101: 313-321

Maloyan A, Gulick J, Glabe CG, Kayed R, Robbins J. (2007) Exercise reverses pre-amyloid oligomer and prolongs survival in αB-crystallin-based desmin related cardiomyopathy. Proc Natl Acad Sci, USA 104: 5995-6000

Nakamura T, Colbert M, Krenz M, Molkentin JD, Hahn HS, Dorn II GW, Robbins J. (2007) Mediating ERK1/2 signaling rescues congenital heart defects in a mouse model of Noonan syndrome. J Clin Invest 117: 2123-2132

Hsieh PCH, Davis ME, MacGillivray C, Gannon J, Molkentin JD, Robbins J, Lee RT. (2007) Evidence that stem cells refresh adult mammalian cardiomyocytes following injury: A genetic fate-mapping study. Nature Med 13, 970-974

Yasuda S-I, Coutu P, Sakthievel S, Robbins J, Metzger JM. (2007) Cardiac transgenic and gene transfer strategies converge to support a dominant effect of troponin I in regulating relaxation in cardiac myocytes. Circ Res 101: 377-386.

Nakayama H, Chen X, Baines CP, Klevitsky R, Zhang L, Zhang H, Jaleel N, Chua BHL, Hewett TE, Robbins J, Houser SR, Molkentin JD. (2007) Ca2+- and mitochondrial-dependent cardiomyocyte necrosis as a primary mediator of heart failure. J Clin Invest 117, 2123-2132

Purcell NH, Wilkins BJ, York A, Robbins J, Molkentin J. (2007) Genetic inhibition of cardiac ERK 1/2 promotes stress-induced apoptosis but has no effect on hypertrophy in vivo. Proc Natl Acad Sci, USA 104, 14074-14079

Galvez AS, Diwan A, Odley AM, Hahn HS, Osinska H, Melendez JG, Robbins J, Lynch RA, Marreez Y, Dorn GW. (2007). Cardiomyocyte degeneration with calpain deficiency reveals a critical role in protein homeostasis. Circ Res 100, 1071-1078

Nishizawa T, Shen Y-T, Rossi F, Hong C, Robbins J, Ishikawa J, Sadoshima J, Vatner D, Vatner S. (2007) Altered autonomic control in conscious transgenic rabbits with overexpressed cardiac Gsα.  Am J Physiol 292, H971-975

Hoyer K, Krenz M, Robbins J, Ingwall JS. (2007) Shifts in the myosin heavy chain isozymes in the murine heart result in increased energy efficiency. J Mol Cell.Cardiol 42, 214-221

Yutzey KE, Robbins J. Principles of genetic murine models for cardiac disease. (2007) Circulation 115: 792-799.

Sadayappan S, Osinska H, Klevitsky R, Lorenz JN, Sargent M, Molkentin JD, Seidman CE, Seidman JG, Robbins J. (2006) Cardiac myosin binding protein-C phosphorylation is cardioprotective. Proc Natl Acad Sci, USA 103:16918-16923

Wang XJ, Robbins J. (2006) Heart failure and protein quality control. Circ Res 99:1315-1328

Funakoshi H, Chan TO, Good JC, Libonati JR, Piuhola J, Chen X, MacDonnell SM, Lee LL, Hermann DE, Zhang J, Martini J, Palmer T, Sanbe A, Robbins J, Houser SR, Koch WJ, Feldman AM. (2006) Regulated over-expression of the A1 adenosine receptor in mice results in adverse but reversible changes in cardiac morphology and function. Circulation 114: 2240-2250

Wang H, Grant JE, Doede CM, Sadayappan S, Robbins J, Walker JW. (2006) PKC sensitizes cardiac myofilaments to Ca2+ by phosphorylating troponin I on threonine-144. J Mol Cell Cardiol  41:823-833

Nakamura T, Colbert M, Robbins J. (2006) Neural crest cells retain multipotential characteristics in the developing valves and label the cardiac conduction system. Circ Res 98: 1547-1554

Dias FA, Walker LA, Arteaga GM, Walker JS, Vijayan K, Pena JR, Ke Y, Fogaca RT, Sanbe A, Robbins J, Wolska BM. (2006) The effect of myosin regulatory light chain phosphorylation on the frequency-dependent regulation of cardiac function. J Mol Cell Cardiol 41: 330-339

Tallini YN, Ohkura M, Choi B-R, Ji G, Imoto K, Doran R, Lee J, Plan P, Wilson J,  Xin H-B, Sanbe A, Gulick J, Mathai J, Robbins J, Salama G, Nakai G, Kotlikoff MI. (2006) Imaging cellular signals in the heart in vivo: cardiac expression of the high signal Ca2+ indicator GCaMP2.  Proc Natl Acad Sci, USA. 103: 4753-4758

Nishizawa T, Vatner SF, Hong C, Shen Y-T, Hardt S, Robbins J, Ishikawa Y, Sadoshima J, Vatner D. (2006) Overexpressed Gsαin rabbits. J Mol Cell Cardiol 41: 44-50

Gaffin RD, Gokulan K, Sacchettini JC, Hewett TE, Klevitsky R, Robbins J, Sarin V, Zawieja DC, Meininger GA, Muthuchamy M. (2006) End-to-end interactions of TM and cardiac function. Am. J Physiol 291: 552-563

Sadayappan S, Gulick J, Osinska H, Martin LA, Hahn HS, Dor GW II, Klevitsky R, Seidman CE, Seidman JG, Robbins J. (2005) Cardiac myosin binding protein-C phosphorylation and cardiac function. Circ Res 97: 1156-1163

Sanbe A, Osinska H, Villa C, Gulick, Klevitsky R, Glabe CG, Kayed R, Robbins J. (2005) Reversal of amyloid-induced heart disease in desmin related cardiomyopathy. Proc Natl Acad Sci, USA. 102: 13592-13597

Maloyan A, Sanbe A, Osinska H, Westfall M, Robinson D, Imahashi K-I, Murphy E, Robbins J. (2005) Mitochondrial dysfunction and apoptosis underlie the pathogenic process in alpha-B-crystallin desmin related cardiomyopathy. Circulation 112: 3451-3461

Yutzey KE, Colbert M, Robbins J. (2005) Ras-related signaling pathways in valve development: ebb and flow. Physiology 20: 390-397

Krenz M, Yutzey KE, Robbins J. (2005) The Noonan syndrome mutation Q79R in Shp2 increases proliferation of valve primordia mesenchymal cells via ERK1/2 signaling. Circ Res 97: 813-820

Miller MS, Palmer BM, Ruch S, Murray LA, Farman GP, Wang Y, Robbins J, Irving TC, Maughan DW. (2005) The essential light chain N-terminal extension alters force and fiber kinetics in mouse cardiac muscle. J Biol Chem 280: 34427-34434

Gulick J, Robbins J. (2005) Inducible cardiac-specific transgenesis. Current Protocols in Molecular Biology Unit 23.12

den Engelsman J, Gerrits D, de Jong WW, Robbins J, Kato K, Boelens WC. (2005) Nuclear import of αB-crystallin is phosphorylation-dependent and hampered by hyperphosphorylation of the myopathy-related mutant R120G. J Biol Chem 280: 36657-36663

James J, Martin L, Krenz M, Quatman C, Jones F, Klevitsky R, Gulick J, Robbins J. (2005) Forced expression of alpha-myosin heavy chain in the rabbit ventricle results in cardioprotection under cardiomyopathic conditions. Circulation 111:2339-46.

Sanbe A, James J, Tuzcu V, Nas S, Martin L, Gulick J, Osinska H, Sakthivel S, Klevitsky R, Ginsburg KS, Bers DM, Zinman B, Lakatta EG, Robbins J. (2005) Transgenic rabbit model for human troponin I-based hypertrophic cardiomyopathy. Circulation 111:2330-8.

Miller MS, Palmer BM, Ruch S, Martin LA, Farman GP, Wang Y, Robbins J, Irving TC, Maughan DW. (2005) The essential light chain N-terminal extension alters force and fiber kinetics in mouse cardiac muscle. J Biol Chem 280:34427-34.

Gaffin RD, Gokulan K, Sacchettini JC, Hewett TE, Klevitsky R, Robbins J, Sarin V, Zawieja DC, Meininger GA, Muthuchamy M. (2006) Changes in the end-to-end interactions of tropomyosin affect mouse cardiac muscle dynamics. Am J Physiol Heart Circ Physiol.

Sadayappan S, Gulick J, Osinska H, Martin LA, Hahn HS, Dorn GW, 2nd, Klevitsky R, Seidman CE, Seidman JG, Robbins J. (2005) Cardiac myosin-binding protein-C phosphorylation and cardiac function. Circ Res 97:1156-63.

Nakayama M, Yan X, Price RL, Borg TK, Ito K, Sanbe A, Robbins J, Lorell BH. (2005) Chronic ventricular myocyte-specific overexpression of angiotensin II type 2 receptor results in intrinsic myocyte contractile dysfunction. Am J Physiol Heart Circ Physiol 288:H317-27.

Kruger M, Zittrich S, Redwood C, Blaudeck N, James J, Robbins J, Pfitzer G, Stehle R. (2005) Effects of the mutation R145G in human cardiac troponin I on the kinetics of the contraction-relaxation cycle in isolated cardiac myofibrils. J Physiol 564:347-57.

Krenz M, Robbins J. (2004) Impact of beta-myosin heavy chain expression on cardiac function during stress. J Am Coll Cardiol 44:2390-7.

Sakthivel, S., Finley, N. L., Rosevear, P. R., Lorenz, J. N., Gulick, J., Kim, S., VanBuren, P., Martin, L. A., Robbins, J. (2005) In vivo and in vitro analysis of cardiac troponin I phosphorylation. J. Biol. Chem. 280: 703-714

Kumar, A., Crawford, K., Flick, R., Klevitsky, R., Lorenz, J. N., Bove, K. E., Robbins, J. and Lessard, J. L. (2004) Transgenic overexpression of cardiac actin in the mouse heart suggests coregulation of cardiac, skeletal and vascular actin expression. Transgenic Research 13:531-540

Gaffin, R. D., Tong, C. W., Zawieja, D. C., Hewett, T. E., Klevitsky, R., Robbins, J., Muthuchamy, M. (2004) Charged residue alterations in the inner core domain and carboxy terminus of α-tropomyosin differentially affect mouse cardiac muscle contractility. Am J Physiol Heart Circ Physiol. (In press)

Gaffin, R. D., Gokulan, K., Sacchettini, J. C., Hewett, T. E., Klevitsky, R., Robbins, J., Muthuchamy, M. (2004) Charged residue changes in the carboxy-terminus of α-tropomyosin alter cardiac muscle contractility. Am J Physiol Heart Circ Physiol. (In press)

Odley, A., Hahn, H. A., Lynch, R. A., Marreez, Y., Osinska, H., Robbins, J., Dorn G. W. II (2004) Regulation of cardiac contractility by Rab4-modulated β2 adrenergic receptor recycling. Proc. Natl. Acad. Sci., USA. 101:7082-7087

Krenz, 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. (2003) Analysis of myosin heavy chain functionality in the heart. J. Biol Chem. 278:17466-17474

Ishibashi, Y., Takahashi, M., Isomatsu, Y., Qiao, F., Iijima, Y., Shiraishi, H., Simsic, J. M., Baicu, C. F., Robbins, J., Zile, M. R., Cooper, G. (2003) Role of Microtubules versus Myosin Heavy Chain Isoforms in the Contractile Dysfunction of Hypertrophied Murine Cardiocytes Am J Physiol Heart Circ Physiol 285: H1270-H1285

Robbins, J., Benson, D. W. (2002) Structure-function relationships in myosin binding protein-C. Circ. Res. 91: 656-658.

James, J., Zhang, Y., Osinska, H., Sanbe, A., Klevitsky, R., Hewett, T. E., Robbins, J., (2000) Transgenic modeling of a cardiac troponin I mutation linked to familial hypertrophic cardiomyopathy. Circ Res. 87, 805-811

Contact Dr. Robbins

Jeffrey Robbins
Molecular Cardiovascular Biology
ML 7020
Children's Hospital
3333 Burnet Ave
Cincinnati, OH 45229-3039
jeff.robbins@cchmc.org