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

Robbins Lab

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

The overall 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, 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 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.
A particular focus of the lab is the sarcomeric protein known as myosin binding protein C (MyBP-C). This is one of the most frequently mutated proteins found in human familial hypertrophic cardiomyopathy (FHC) and these alleles exhibit autosomal dominance. It is not clear as to whether the ensuing pathology is due to functional haploinsufficiency, the action(s) of a poison peptide or some combination. Complicating an understanding of the role or roles of truncated forms of cMyBP are our recent data showing the production of a stable 29kD fragment from cMyBP-C as a result of ischemia reperfusion injury and / or general cardiovascular stress. The fragment is stable but its functional capacity is unknown. Preliminary data indicate that it retains a capacity to influence contractile function, leading us to hypothesize that cMyBP-C fragments can play an important role in determining cardiomyocyte functionality.
Our objective is to explicitly define the functional and mechanistic outcomes of a series of cMyBP-C truncations that cause disease, and compare those data with the data obtained using the 29kD cMyBP-C fragment that is endogenously produced under stressed conditions. The central hypothesis is that while haploinsufficiency may play a role in the pathology of some of these mutations, a majority function as poison peptides, possibly through depression of thick-thin filament cycling. We are testing the hypothesis that the 29kD cMyBP-C fragment can function as a poison peptide by actively interfering with normal thick-thin filament interaction. Using a combination of isolated systems, in vitro cell culture and developmental stage-specific transgenesis, the function of this fragment will be determined. We are also testing the hypothesis that cMyBP-C truncations lead to pathology through haploinsufficiency or, more frequently, production of a poison peptide. A series of cMyBP-C’s carrying nonsense codons defined as causing human FHC will be made and used to replace endogenous cMyBP-C. The impact of the mutations on the cardiomyocyte’s general stress response and ensuing pathology will be determined and the pathogenic mechanism(s) determined.
Related Publications

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2010

Maloyan A, Sayegh J, Osinska H, Chua BH, Robbins J. Manipulation of death pathways in desmin-related cardiomyopathy. Circ. Res. May, 2010.
James J, Hor K, Moga MA, Martin L, Robbins J. Effects of myosin heavy chain manipulation in experimental heart failure. J. Mol. Cell. Cardiology. May, 2010.
Heineke J, Wollert KC, Osinska H, Sargent M, York AJ, Robbins J, Molkentin J. Calcineurin protects the heart in a murine model of dilated cardiomyopathy. J. Mol. Cell. Cardiology. 48: 1080-1087. 2010.
Terrell D, Robbins J. Protein conformation-based disease: getting to the heart of the matter. Annual Review Physiol. 72: 1-3. 2010.
Bardswell SC, Cuello F, Rowland A, Sadayappan S, Robbins J, Gautel M, Walker JW, Kentish JC, Avkirin M. Distinct sarcomeric substrates are responsible for protein kinase D-mediated regulation of cardiac myofilament Ca²⁺ sensitivity and crossbridge cycling. J. Biol. Chem. 285: 5674-5682. 2010.

2009

Nakamura T, Gulick J, Colbert MC, Robbins J. Protein tyrosine phosphatase activity in the neural crest is essential for normal heart and skull development. Proc. Natl. Acad. Sci., U S A. 106:11270-11275. 2009.
Nakamura T, Gulick J, Pratt R, Robbins J. Noonan syndrome is associated with enhanced pERK activity, the repression of which can prevent craniofacial malformations. Proc. Natl. Acad. Sci., U S A. 106: 15436-15441. 2009.
Maloyan A, Osinska H, Lammerding J, Lee RT, Cingolani OH, Kass D, Lorenz JN, Robbins J. Biochemical and mechanical dysfunction in a mouse model of desmin-related myopathy. Circ. Res. 104: 1021-1028. 2009.
Xiang F-L, Hammoud L, Robbins J, Feng Q. Cardiomyocyte-specific overexpression of human stem cell factor improves cardiac function and survival after myocardial infarction in mice. Circulation. 120: 1065-1074. 2009.
Itoh H, MacGillivray C,Kwon H-S, Lammerding J, Robbins J, Lee R, So P. Three-dimensional cardiac architecture determined by two-photon microtomy. J. Biomedical Optics. Jul-Aug, 2009.
Nicolaou P, Rodriguez P, Zhou X, Ren X, Qian J, Sadayappan S, Mitton B, Pathak A, Robbins J, Hajjar R, Jones K, Kranias EG. Inducible expression of active protein phosphatase-1 inhibitor-1 enhances basal cardiac function and protects against ischemic/reperfusion injury. Circ Res. 104: 1012-1020. 2009.
Suzuki T, Palmer BM, James J, Yuan Wang Y, Chen Z, VanBuren P, Maughan DW, Robbins J, LeWinter MM. Effects of cardiac myosin isoform variation on myofilament function and crossbridge kinetics in transgenic rabbits. Circulation, Heart Failure. Jul, 2009.
Sadayappan S, Klevitsky R, Lorenz JN, Sargent M, Gulick J, Molkentin JD, Robbins J. Cardiac myosin binding protein-C phosphorylation in a beta-myosin heavy chain background. Circulation. 119: 1253-1262. 2009.
Scruggs SB, Hinken AC, Thawornkaiwong A, Robbins J, Walker LA, de Tombe P, Geenen D. L, Buttrick PM, Solaro RJ. Ablation of ventricular myosin regulatory light chain phosphorylation in mice causes cardiac dysfunction in situ and affects neighboring myofilament protein phosphorylation. J Biol Chem. 285: 5097-5106. 2009.
Wu X, Blair NS, Sargent M, York AJ, Robbins J, Shull G, Molkentin JD. Plasma membrane Ca²⁺-ATPase isoform 4 antagonizes cardiac hypertrophy in association with calcineurin inhibition in rodents. J Clin Invest. 119: 976-985. 2009.
Waggoner J, Ginsburg K, Bersohn M, Mitton B, Haghighi K, Robbins J, Bers D, Kranias E. Phospholamban overexpression in rabbit ventricular myocytes does not alter sarcoplasmic reticulum Ca transport. Am J Physiol. 296: H698-703. 2009.
Gulick J, Robbins J. Cell-type specific transgenesis in the mouse. Methods in Molecular Biology. 561: 91-106. 2009.

2008

Krenz M, Gulick J, Osinska HE, Colbert MC, Molkentin JD, Robbins J. Role of ERK1/2 signaling in congenital valve malformations in Noonan syndrome. Proc Natl Acad Sci, U S A. 105: 18930-18936. 2008.
Pinz I, Ostroy SE, Hoyer K, Osinska H, Robbins J, Molkentin JD, Ingwall JS. Calcineurin-induced energy wasting in a transgenic mouse model of heart failure. Am J Physiol Heart Circ Physiol. 294: H1459-1466. 2008.
Molkentin J, Robbins J. With great power comes great responsibility: using mouse genetics to study cardiac hypertrophy and failure. JMCC. 46: 130-136. 2008.
Moga MA, Nakamura T, Robbins J. Genetic approaches for changing the heart. JMCC. 45: 148-155. 2008.
Yano N, Tseng A, Zhao TC, Robbins J, Padbury J, Tseng YT. Temporally controlled overexpression of cardiac-specific PI3Kalpha induces enhanced myocardial contractility--a new transgenic model. Am J Physiol Heart Circ Physiol. 295: 1690-1694. 2008.
Lowey S, Lesko LM, Rovner AS, Hodges AR, White SL, Low RB, Gulick J, Robbins J. Functional effects of the hypertrophic cardiomyopathy R403Q mutation are different in an alpha- or beta-myosin heavy chain backbone. J. Biol. Chem. 283: 20579-2089. 2008.
Sadayappan S, Finley N, Howarth J W, Osinska H, Klevitsky R, Lorenz JN, Rosevear PR, JG, Robbins J. Role of the acidic N’ region of cardiac troponin I in regulating myocardial function. FASEB Journal. 22: 1246-1257. 2008.
Sadayappan S, Robbins J. The death of transcriptional chauvinism in the control and regulation of cardiac contractility. NY Acad Sci. 1123: 1-9. 2008.
Pattison JS, Waggoner JR, James J, Martin L, Gulick J, Osinska H, Klevitsky R, Kranias EG, Robbins J. Phospholamban overexpression in transgenic rabbits. Transgenic Res. 17: 157-170. 2008.
Wolf CM, Arad M, Ahmad F, Sanbe A, Bernstein SA, Toka O, Morley G, Robbins J, Seidman JG, Seidman CE, Berul CI. Reversibility of PRKAG2 glycogen storage cardiomyopathy and electrophysiologic manifestations. Circulation. 117: 144-154. 2008.
Pinz I, Robbins J, Benjamin IJ, Ingwall J. 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. 2008.

Other Significant Publications

Krenz M, Sadayappan S, Osinska HE, Henry JA, Beck S, Warshaw DM, Robbins J. Distribution and structure-function relationship of myosin heavy chain isoforms in the adult mouse heart. J. Biol. Chem. 282: 24057-24064. 2007.
Hambleton M, York A, Sargent MA, Kaiser RA, Lorenz JN, Robbins J, Molkentin JD. Inducible and myocyte-specific inhibition of PKCalpha enhances cardiac contractility and protects against infarction-induced heart failure. Am. J. Physiol. Heart Circ. Physiol. 293: H3768-H3771. 2007.
Nagyama T, Takimoto E, Sadayappan S, Mudd JO, Seidman JG, Robbins J, Kass DA. PKA-phosphorylation dependent and independent regulation of in vivo contraction/relaxation kinetics by myosin binding protein C. Circulation. 116: 2399-2408. 2007.
Diwan A, Krenz M, Sayed FM, Wanasapura J, Ren X, Matkovich SJ, Koesters AG, Li H, Kirshenbaum LA, Robbins J, Jones WK, Dorn GW 2^nd. Inhibition of ischemic cardiomyocyte apoptosis through targeted ablation of bnip3 restrains post-infarction remodeling. J. Clin. Invest. 117: 2825-2833. 2007.
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. Genetic manipulation of periostin expression reveals a role in cardiac hypertrophy and ventricular remodeling. Circ. Res. 101: 313-321. 2007.
Nakamura T, Colbert M, Krenz M, Molkentin JD, Hahn HS, Dorn GW 2nd, Robbins J. Mediating ERK1/2 signaling rescues congenital heart defects in a mouse model of Noonan syndrome. J Clin Invest. 117: 2123-2132. 2007.
Hsieh PCH, Davis ME, MacGillivray C, Gannon J, Molkentin JD, Robbins J, Lee RT. Evidence from a genetic fate-mapping study that stem cells refresh adult mammalian cardiomyocytes after injury. Nature Med. 13, 970-974. 2007.
Yasuda S-I, Coutu P, Sadayappan S, Robbins J, Metzger JM. Cardiac transgenic and gene transfer strategies converge to support an important role for troponin I in regulating relaxation in cardiac myocytes. Circ Res. 101: 377-386. 2007.
Purcell NH, Wilkins BJ, York A, Robbins J, Molkentin J. Genetic inhibition of cardiac ERK1/2 promotes stress-induced apoptosis and heart failure but has no effect on hypertrophy in vivo.Proc. Natl. Acad. Sci. U S A. 104, 14074-14079. 2007.
Galvez AS,Diwan A, Odley AM, Hahn HS, Osinska H, Melendez JG, Robbins J,Lynch RA, Marreez Y, Dorn GW. Cardiomyocyte degeneration with calpain deficiency reveals a critical role in protein homeostasis. Circ. Res.100, 1071-1078. 2007.
Nishizawa T, Shen Y-T, Rossi F, Hong C, Robbins J, Ishikawa J, Sadoshima J, Vatner D, Vatner S. Altered autonomic control in conscious transgenic rabbits with overexpressed cardiac Gsa. Am. J. Physiol. 292, H971-975. 2007.
Hoyer K, Krenz M, Robbins J, Ingwall JS. Shifts in the myosin heavy chain isozymes in the mouse heart result in increased energy efficiency. J. Mol. Cell. Cardiol. 42, 214-221. 2007.
Yutzey KE, Robbins J. Principles of genetic murine models for cardiac disease. Circulation 115: 792-799. 2007.

click to enlarge

Cardiomyocyte-specific expression of directed genes. Shown is a summary of the alpha myosin heavy chain promoter sequences that can drive cardiomyocyte-specific expression of any gene product chosen by the laboratory. The top two panels show expression driven by the promoter during fetal development, specifically in the developing atria: this expression is restricted only to the atria before birth. After birth, this particular promoter drives expression throughout, but only in, the heart.
click to enlarge

Shown are the three filament systems that make up the contractile unit of the cardiac muscle cell: myosin (green), actin (orange) and titin (pink-white). The myosin binding protein C’s position in this apparatus is shown, as is its potential for signaling via reversible phosphorylation on distinct sites in the protein.

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Robbins Lab

Robbins Lab

Related Publications

2010

2009

2008

Other Significant Publications