Publications by authors named "Jean-Claude Perriard"

33 Publications

Stabilised beta-catenin in postnatal ventricular myocardium leads to dilated cardiomyopathy and premature death.

Basic Res Cardiol 2010 Sep 8;105(5):597-608. Epub 2010 Apr 8.

Institute of Cell Biology, ETH Zurich-Hönggerberg, Switzerland.

Beta-catenin is a component of the intercalated disc in cardiomyocytes, but can also be involved in signalling and activation of gene transcription. We wanted to determine how long-term changes in beta-catenin expression levels would affect mature cardiomyocytes. Conditional transgenic mice that either lacked beta-catenin or that expressed a non-degradable form of beta-catenin in the adult ventricle were created. While mice lacking beta-catenin in the ventricle do not have an overt phenotype, mice expressing a non-degradable form develop dilated cardiomyopathy and do not survive beyond 5 months. A detailed analysis could reveal that this phenotype is correlated with a distinct localisation of beta-catenin in adult cardiomyocytes, which cannot be detected in the nucleus, no matter how much protein is present. Our report is the first study that addresses long-term effects of either the absence of beta-catenin or its stabilisation on ventricular cardiomyocytes and it suggests that beta-catenin's role in the nucleus may be of little significance in the healthy adult heart.
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http://dx.doi.org/10.1007/s00395-010-0101-8DOI Listing
September 2010

The role of cell death and myofibrillar damage in contractile dysfunction of long-term cultured adult cardiomyocytes exposed to doxorubicin.

Cytotechnology 2009 Nov 5;61(1-2):25-36. Epub 2009 Nov 5.

Swiss Cardiovascular Center, Inselspital, Bern University Hospital, 3010, Bern, Switzerland.

In failing hearts cardiomyocytes undergo alterations in cytoskeleton structure, contractility and viability. It is not known presently, how stress-induced changes of myofibrils correlate with markers for cell death and contractile function in cardiomyocytes. Therefore, we have studied the progression of contractile dysfunction, myofibrillar damage and cell death in cultured adult cardiomyocytes exposed to the cancer therapy doxorubicin. We demonstrate, that long-term cultured adult cardiomyocytes, a well-established model for the study of myofibrillar structure and effects of growth factors, can also be used to assess contractility and calcium handling. Adult rat ventricular myocytes (ARVM) were isolated and cultured for a total of 14 days in serum containing medium. The organization of calcium-handling proteins and myofibrillar structure in freshly isolated and in long-term cultured adult cardiomyocytes was studied by immunofluorescence and electron microscopy. Excitation contraction-coupling was analyzed by fura 2 and video edge detection in electrically paced cardiomyocytes forming a monolayer, and cell death and viability was measured by TUNEL assay, LDH release, MTT assay, and Western blot for LC3. Adult cardiomyocytes treated with Doxo showed apoptosis and necrosis only at supraclinical concentrations. Treated cells displayed merely alterations in cytoskeleton organization and integrity concomitant with contractile dysfunction and up-regulation of autophagosome formation, but no change in total sarcomeric protein content. We propose, that myofibrillar damage contributes to contractile dysfunction prior to cell death in adult cardiomyocytes exposed to clinically relevant concentrations of anthracyclines.
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http://dx.doi.org/10.1007/s10616-009-9238-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2795142PMC
November 2009

Activation of a HIF1alpha-PPARgamma axis underlies the integration of glycolytic and lipid anabolic pathways in pathologic cardiac hypertrophy.

Cell Metab 2009 Jun;9(6):512-24

Institute of Cell Biology and Competence Center for Systems Physiology and Metabolic Diseases, ETH Zurich, Zurich, Switzerland.

Development of cardiac hypertrophy and progression to heart failure entails profound changes in myocardial metabolism, characterized by a switch from fatty acid utilization to glycolysis and lipid accumulation. We report that hypoxia-inducible factor (HIF)1alpha and PPARgamma, key mediators of glycolysis and lipid anabolism, respectively, are jointly upregulated in hypertrophic cardiomyopathy and cooperate to mediate key changes in cardiac metabolism. In response to pathologic stress, HIF1alpha activates glycolytic genes and PPARgamma, whose product, in turn, activates fatty acid uptake and glycerolipid biosynthesis genes. These changes result in increased glycolytic flux and glucose-to-lipid conversion via the glycerol-3-phosphate pathway, apoptosis, and contractile dysfunction. Ventricular deletion of Hif1alpha in mice prevents hypertrophy-induced PPARgamma activation, the consequent metabolic reprogramming, and contractile dysfunction. We propose a model in which activation of the HIF1alpha-PPARgamma axis by pathologic stress underlies key changes in cell metabolism that are characteristic of and contribute to common forms of heart disease.
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http://dx.doi.org/10.1016/j.cmet.2009.05.005DOI Listing
June 2009

Essential role of developmentally activated hypoxia-inducible factor 1alpha for cardiac morphogenesis and function.

Circ Res 2008 Nov 10;103(10):1139-46. Epub 2008 Oct 10.

Institute of Cell Biology, Eidgenössische Technische Hochschule, Zürich, Switzerland.

Development of the mammalian heart is governed by precisely orchestrated interactions between signaling pathways integrating environmental cues and a core cardiac transcriptional network that directs differentiation, growth and morphogenesis. Here we report that in mice, at about embryonic day (E)8.5 to E10.0, cardiac development proceeds in an environment that is hypoxic and characterized by high levels of hypoxia-inducible factor (HIF)1alpha protein. Mice lacking HIF1alpha in ventricular cardiomyocytes exhibit aborted development at looping morphogenesis and embryonic lethality between E11.0 to E12.0. Intriguingly, HIF1alpha-deficient hearts display reduced expression of the core cardiac transcription factors Mef2C and Tbx5 and of titin, a giant protein that serves as a template for the assembly and organization of the sarcomere. Chromatin immunoprecipitation experiments revealed that Mef2C, Tbx5, and titin are direct target genes of HIF1alpha in vivo. Thus, hypoxia signaling controls cardiac development through HIF1alpha-mediated transcriptional regulation of key components of myofibrillogenesis and the cardiac transcription factor network, thereby providing a mechanistic basis of how heart development, morphogenesis, and function is coupled to low oxygen tension during early embryogenesis.
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http://dx.doi.org/10.1161/01.RES.0000338613.89841.c1DOI Listing
November 2008

Genetic selection system allowing monitoring of myofibrillogenesis in living cardiomyocytes derived from mouse embryonic stem cells.

Eur J Histochem 2008 Jan-Mar;52(1):1-10

Department of Cardiology, University of Lausanne, Lausanne, Switzerland.

Embryonic stem (ES) cell-derived cardiomyocytes recapitulate cardiomyogenesis in vitro and are a potential source of cells for cardiac repair. However, this requires enrichment of mixed populations of differentiating ES cells into cardiomyocytes. Toward this goal, we have generated bicistronic vectors that express both the blasticidin S deaminase (bsd) gene and a fusion protein consisting of either myosin light chain (MLC)-3f or human alpha-actinin 2A and enhanced green fluorescent protein (EGFP) under the transcriptional control of the alpha-cardiac myosin heavy chain (alpha-MHC) promoter. Insertion of the DNase I-hypersensitive site (HS)-2 element from the beta-globin locus control region, which has been shown to reduce transgene silencing in other cell systems, upstream of the transgene promoter enhanced MLC3f-EGFP gene expression levels in mouse ES cell lines. The alpha-MHC-alpha-actinin-EGFP, but not the alpha-MHC-MLC3f-EGFP, construct resulted in the correct incorporation of the newly synthesized fusion protein at the Z-band of the sarcomeres in ES cell-derived cardiomyocytes. Exposure of embryoid bodies to blasticidin S selected for a relatively pure population of cardiomyocytes within 3 days. Myofibrillogenesis could be monitored by fluorescence microscopy in living cells due to sarcomeric epitope tagging. Therefore, this genetic system permits the rapid selection of a relatively pure population of developing cardiomyocytes from a heterogeneous population of differentiating ES cells, simultaneously allowing monitoring of early myofibrillogenesis in the selected myocytes.
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http://dx.doi.org/10.4081/1180DOI Listing
July 2008

Deletion of the protein kinase A/protein kinase G target SMTNL1 promotes an exercise-adapted phenotype in vascular smooth muscle.

J Biol Chem 2008 Apr 29;283(17):11850-9. Epub 2008 Feb 29.

Department of Pharmacology, Medicine, Duke University, Medical Center, Durham, North Carolina 27710, USA.

In vivo protein kinases A and G (PKA and PKG) coordinately phosphorylate a broad range of substrates to mediate their various physiological effects. The functions of many of these substrates have yet to be defined genetically. Herein we show a role for smoothelin-like protein 1 (SMTNL1), a novel in vivo target of PKG/PKA, in mediating vascular adaptations to exercise. Aortas from smtnl1(-/-) mice exhibited strikingly enhanced vasorelaxation before exercise, similar in extent to that achieved after endurance training of wild-type littermates. Additionally, contractile responses to alpha-adrenergic agonists were greatly attenuated. Immunological studies showed SMTNL1 is expressed in smooth muscle and type 2a striated muscle fibers. Consistent with a role in adaptations to exercise, smtnl1(-/-) mice also exhibited increased type 2a fibers before training and better performance after forced endurance training compared smtnl1(+/+) mice. Furthermore, exercise was found to reduce expression of SMTNL1, particularly in female mice. In both muscle types, SMTNL1 is phosphorylated at Ser-301 in response to adrenergic signals. In vitro SMTNL1 suppresses myosin phosphatase activity through a substrate-directed effect, which is relieved by Ser-301 phosphorylation. Our findings suggest roles for SMTNL1 in cGMP/cAMP-mediated adaptations to exercise through mechanisms involving direct modulation of contractile activity.
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http://dx.doi.org/10.1074/jbc.M708628200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2431077PMC
April 2008

Myomesin 3, a novel structural component of the M-band in striated muscle.

J Mol Biol 2008 Feb 22;376(2):338-51. Epub 2007 Nov 22.

Institute of Cell Biology, Swiss Federal Institute of Technology, ETH-Hönggerberg, CH-8093 Zurich, Switzerland.

The M-band is the cytoskeletal structure that cross-links the myosin and titin filaments in the middle of the sarcomere. Apart from the myosin tails and the C-termini of titin, only two closely related structural proteins had been detected at the M-band so far, myomesin and M-protein. However, electron microscopy studies revealed structural features that do not correlate with the expression of these two proteins, indicating the presence of unknown constituents in the M-band. Using comparative sequence analysis, we have identified a third member of this gene family, myomesin 3, and characterised its biological properties. Myomesin 3 is predicted to consist of a unique head domain followed by a conserved sequence of either fibronectin- or immunoglobulin-like domains, similarly to myomesin 3 and M-protein. While all three members of the myomesin family are localised to the M-band of the sarcomere, each member shows its specific expression pattern. In contrast to myomesin, which is ubiquitously expressed in all striated muscles, and M-protein, whose expression is restricted to adult heart and fast-twitch skeletal muscle, myomesin 3 can be detected mainly in intermediate speed fibers of skeletal muscle. In analogy to myomesin, myomesin 3 targets to the M-band region of the sarcomere via its N-terminal part and forms homodimers via its C-terminal domain. However, despite the high degree of homology, no heterodimer between distinct members of the myomesin gene family can be detected. We propose that each member of the myomesin family is a component of one of the distinct ultrastructures, the M-lines, which modulate the mechanical properties of the M-bands in different muscle types.
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http://dx.doi.org/10.1016/j.jmb.2007.11.048DOI Listing
February 2008

Molecular basis of the C-terminal tail-to-tail assembly of the sarcomeric filament protein myomesin.

EMBO J 2008 Jan 6;27(1):253-64. Epub 2007 Dec 6.

EMBL-Hamburg c/o DESY, Hamburg, Germany.

Sarcomeric filament proteins display extraordinary properties in terms of protein length and mechanical elasticity, requiring specific anchoring and assembly mechanisms. To establish the molecular basis of terminal filament assembly, we have selected the sarcomeric M-band protein myomesin as a prototypic filament model. The crystal structure of the myomesin C-terminus, comprising a tandem array of two immunoglobulin (Ig) domains My12 and My13, reveals a dimeric end-to-end filament of 14.3 nm length. Although the two domains share the same fold, an unexpected rearrangement of one beta-strand reveals how they are evolved into unrelated functions, terminal filament assembly (My13) and filament propagation (My12). The two domains are connected by a six-turn alpha-helix, of which two turns are void of any interactions with other protein parts. Thus, the overall structure of the assembled myomesin C-terminus resembles a three-body beads-on-the-string model with potentially elastic properties. We predict that the found My12-helix-My13 domain topology may provide a structural template for the filament architecture of the entire C-terminal Ig domain array My9-My13 of myomesin.
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http://dx.doi.org/10.1038/sj.emboj.7601944DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2206126PMC
January 2008

Identification and characterization of layer-specific differences in extraocular muscle m-bands.

Invest Ophthalmol Vis Sci 2007 Mar;48(3):1119-27

Department of Physiology and Pennsylvania Muscle Institute, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA.

Purpose: To examine and characterize the expression of M-bands (or M-lines) in the orbital layer (OL) and global layer (GL) of adult rat extraocular muscles (EOMs).

Methods: Semiquantitative polymerase chain reaction (PCR), quantitative (q)PCR, immunohistochemistry, and confocal microscopy were used to analyze expression of the major gene and protein constituents of M-bands in freshly dissected and cryosectioned rectus extraocular muscles (EOMs) and tibialis anterior (TA) muscles. Electron microscopy (EM) was performed on perfusion-fixed EOMs and TA muscles in a layer-specific manner, to determine, characterize, and quantify laminar-specific differences in M-band expression.

Results: These studies demonstrate EOM layer-specific differences in the expression of M-bands and their major constituents, myomesin1 (Myom1) and myomesin2 (Myom2 or M-protein) at the structural, mRNA, and protein levels by using EM, semiquantitative PCR, qPCR, immunohistochemistry, and confocal microscopy. Differences in thick filament lattice order were quantified by using EM-based inter-thick-filament distance and variance measurements and were found to be TA > GL > OL.

Conclusions: The expression pattern of M-bands and their constituents in EOMs provides mechanistic insight for their allotypic and layer-specific viscoelastic properties. Modeling the differential expression of M-bands between EOMs and TA predicts increased elasticity but reduced force and eccentric contraction (ECC)-mediated damage in EOMs and suggests a potential mechanism for the clinical sparing of EOMs noted in Duchenne's muscular dystrophy (DMD).
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http://dx.doi.org/10.1167/iovs.06-0701DOI Listing
March 2007

Re-expression of proteins involved in cytokinesis during cardiac hypertrophy.

Exp Cell Res 2007 Apr 27;313(6):1270-83. Epub 2007 Jan 27.

Institute of Cell Biology, ETH Zürich-Hönggerberg, CH-8093 Zürich, Switzerland.

Cardiomyocytes stop dividing after birth and postnatal heart growth is only achieved by increase in cell volume. In some species, cardiomyocytes undergo an additional incomplete mitosis in the first postnatal week, where karyokinesis takes place in the absence of cytokinesis, leading to binucleation. Proteins that regulate the formation of the actomyosin ring are known to be important for cytokinesis. Here we demonstrate for the first time that small GTPases like RhoA along with their downstream effectors like ROCK I, ROCK II and Citron Kinase show a developmental stage specific expression in heart, with high levels being expressed in cardiomyocytes only at stages when cytokinesis still occurs (i.e. embryonic and perinatal). This suggests that downregulation of many regulatory and cytoskeletal components involved in the formation of the actomyosin ring may be responsible for the uncoupling of cytokinesis from karyokinesis in rodent cardiomyocytes after birth. Interestingly, when the myocardium tries to adapt to the increased workload during pathological hypertrophy a re-expression of proteins involved in DNA synthesis and cytokinesis can be detected. Nevertheless, the adult cardiomyocytes do not appear to divide despite this upregulation of the cytokinetic machinery. The inability to undergo complete division could be due to the presence of stable, highly ordered and functional sarcomeres in the adult myocardium or could be because of the inefficiency of degradation pathways, which facilitate the division of differentiated embryonic cardiomyocytes by disintegrating myofibrils.
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http://dx.doi.org/10.1016/j.yexcr.2007.01.009DOI Listing
April 2007

Nkx2.5 cell-autonomous gene function is required for the postnatal formation of the peripheral ventricular conduction system.

Dev Biol 2007 Mar 23;303(2):740-53. Epub 2006 Dec 23.

Institut de Biologie du Développement de Marseille-Luminy, IBDML, Université de la Méditerranée, CNRS UMR6216, Campus de Luminy, Marseille, France.

The ventricular conduction system is responsible for rapid propagation of electrical activity to coordinate ventricular contraction. To investigate the role of the transcription factor Nkx2.5 in the morphogenesis of the ventricular conduction system, we crossed Nkx2.5(+/-) mice with Cx40(eGFP/+) mice in which eGFP expression permits visualization of the His-Purkinje conduction system. Major anatomical and functional disturbances were detected in the His-Purkinje system of adult Nkx2.5(+/-)/Cx40(eGFP/+) mice, including hypoplasia of eGFP-positive Purkinje fibers and the disorganization of the Purkinje fiber network in the ventricular apex. Although the action potential properties of the individual eGFP-positive cells were normal, the deficiency of Purkinje fibers in Nkx2.5 haploinsufficient mice was associated with abnormalities of ventricular electrical activation, including slowed and decremented conduction along the left bundle branch. During embryonic development, eGFP expression in the ventricular trabeculae of Nkx2.5(+/-) hearts was qualitatively normal, with a measurable deficiency in eGFP-positive cells being observed only after birth. Chimeric analyses showed that maximal Nkx2.5 levels are required cell-autonomously. Reduced Nkx2.5 levels are associated with a delay in cell cycle withdrawal in surrounding GFP-negative myocytes. Our results suggest that the formation of the peripheral conduction system is time- and dose-dependent on the transcription factor Nkx2.5 that is cell-autonomously required for the postnatal differentiation of Purkinje fibers.
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http://dx.doi.org/10.1016/j.ydbio.2006.12.044DOI Listing
March 2007

Neuregulin-1 beta attenuates doxorubicin-induced alterations of excitation-contraction coupling and reduces oxidative stress in adult rat cardiomyocytes.

J Mol Cell Cardiol 2006 Nov 26;41(5):845-54. Epub 2006 Sep 26.

Swiss Cardiovascular Center, University Hospital, CH-3010 Bern, Switzerland.

Treatment of metastatic breast cancer with doxorubicin (Doxo) in combination with trastuzumab, an antibody targeting the ErbB2 receptor, results in an increased incidence of heart failure. Doxo therapy induces reactive oxygen species (ROS) and alterations of calcium homeostasis. Therefore, we hypothesized that neuregulin-1 beta (NRG), a ligand of the cardiac ErbB receptors, reduces Doxo-induced alterations of EC coupling by triggering antioxidant mechanisms. Adult rat ventricular cardiomyocytes (ARVM) were isolated and treated for 18-48 h. SERCA protein was analyzed by Western blot, EC coupling parameters by fura-2 and video edge detection, gene expression by RT-PCR, and ROS by DCF-fluorescence microscopy. At clinically relevant doses Doxo reduced cardiomyocytes contractility, SERCA protein and SR calcium content. NRG, similarly as the antioxidant N-acetylcystein (NAC), did not affect EC coupling alone, but protected against Doxo-induced damage. NRG and Doxo showed an opposite modulation of glutathione reductase gene expression. NRG, similarly as NAC, reduced peroxide- or Doxo-induced oxidative stress. Specific inhibitors showed, that the antioxidant action of NRG depended on signaling via the ErbB2 receptor and on the Akt- and not on the MAPK-pathway. Therefore, NRG attenuates Doxo-induced alterations of EC coupling and reduces oxidative stress in ARVM. Inhibition of the ErbB2/NRG signaling pathway by trastuzumab in patients concomitantly treated with Doxo might prevent beneficial effects of NRG in the myocardium.
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http://dx.doi.org/10.1016/j.yjmcc.2006.08.002DOI Listing
November 2006

Tissue-transplant fusion and vascularization of myocardial microtissues and macrotissues implanted into chicken embryos and rats.

Tissue Eng 2006 Sep;12(9):2541-53

Institute for Chemical and Bio-Engineering, Swiss Federal Institute of Technology, Zurich, Switzerland.

Cell-based therapies and tissue engineering initiatives are gathering clinical momentum for next-generation treatment of tissue deficiencies. By using gravity-enforced self-assembly of monodispersed primary cells, we have produced adult and neonatal rat cardiomyocyte-based myocardial microtissues that could optionally be vascularized following coating with human umbilical vein endothelial cells (HUVECs). Within myocardial microtissues, individual cardiomyocytes showed native-like cell shape and structure, and established electrochemical coupling via intercalated disks. This resulted in the coordinated beating of microtissues, which was recorded by means of a multi-electrode complementary metal-oxide-semiconductor microchip. Myocardial microtissues (microm3 scale), coated with HUVECs and cast in a custom-shaped agarose mold, assembled to coherent macrotissues (mm3 scale), characterized by an extensive capillary network with typical vessel ultrastructures. Following implantation into chicken embryos, myocardial microtissues recruited the embryo's capillaries to functionally vascularize the rat-derived tissue implant. Similarly, transplantation of rat myocardial microtissues into the pericardium of adult rats resulted in time-dependent integration of myocardial microtissues and co-alignment of implanted and host cardiomyocytes within 7 days. Myocardial microtissues and custom-shaped macrotissues produced by cellular self-assembly exemplify the potential of artificial tissue implants for regenerative medicine.
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http://dx.doi.org/10.1089/ten.2006.12.2541DOI Listing
September 2006

alpha-E-catenin inactivation disrupts the cardiomyocyte adherens junction, resulting in cardiomyopathy and susceptibility to wall rupture.

Circulation 2006 Sep 21;114(10):1046-55. Epub 2006 Aug 21.

Department of Medicine, University of California-San Diego, La Jolla, CA 92093-0613, USA.

Background: alpha-E-catenin is a cell adhesion protein, located within the adherens junction, thought to be essential in directly linking the cadherin-based adhesion complex to the actin cytoskeleton. Although alpha-E-catenin is expressed in the adherens junction of the cardiomyocyte intercalated disc, and perturbations in its expression are observed in models of dilated cardiomyopathy, its role in the myocardium remains unknown.

Methods And Results: To determine the effects of alpha-E-catenin on cardiomyocyte ultrastructure and disease, we generated cardiac-specific alpha-E-catenin conditional knockout mice (alpha-E-cat cKO). alpha-E-cat cKO mice displayed progressive dilated cardiomyopathy and unique defects in the right ventricle. The effects on cardiac morphology/function in alpha-E-cat cKO mice were preceded by ultrastructural defects in the intercalated disc and complete loss of vinculin at the intercalated disc. alpha-E-cat cKO mice also revealed a striking susceptibility of the ventricular free wall to rupture after myocardial infarction.

Conclusions: These results demonstrate a clear functional role for alpha-E-catenin in the cadherin/catenin/vinculin complex in the myocardium in vivo. Ablation of alpha-E-catenin within this complex leads to defects in cardiomyocyte structural integrity that result in unique forms of cardiomyopathy and predisposed susceptibility to death after myocardial stress. These studies further highlight the importance of studying the role of alpha-E-catenin in human cardiac injury and cardiomyopathy in the future.
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http://dx.doi.org/10.1161/CIRCULATIONAHA.106.634469DOI Listing
September 2006

Probing the role of septins in cardiomyocytes.

Exp Cell Res 2006 May;312(9):1598-609

Institute of Cell Biology, ETH Zurich-Honggerberg, CH-8093 Zurich, Switzerland.

Heart growth in the embryo is achieved by division of differentiated cardiomyocytes. Around birth, cardiomyocytes stop dividing and heart growth occurs only by volume increase of the individual cells. Cardiomyocytes seem to lose their capacity for cytokinesis at this developmental stage. Septins are GTP-binding proteins that have been shown to be involved in cytokinesis from yeast to vertebrates. We wanted to determine whether septin expression patterns can be correlated to the cessation of cytokinesis during heart development. We found significant levels of expression only for SEPT2, SEPT6, SEPT7 and SEPT9 in heart, in a developmentally regulated fashion, with high levels in the embryonic heart, downregulation around birth and no detectable expression in the adult. In dividing embryonic cardiomyocytes, all septins localize to the cleavage furrow. We used drugs to probe for the functional interactions of SEPT2 in dividing embryonic cardiomyocytes. Differences in the effects on subcellular septin localization in cardiomyocytes were observed, depending whether a Rho kinase (ROCK) inhibitor was used or whether actin and myosin were targeted directly. Our data show a tight correlation of high levels of septin expression and the ability to undergo cytokinesis in cardiomyocytes. In addition, we were able to dissect the different contributions of ROCK signaling and the actomyosin cytoskeleton to septin localization to the contractile ring using cardiomyocytes as an experimental system.
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http://dx.doi.org/10.1016/j.yexcr.2006.01.029DOI Listing
May 2006

Infarct-remodeled myocardium is receptive to protection by isoflurane postconditioning: role of protein kinase B/Akt signaling.

Anesthesiology 2006 May;104(5):1004-14

Institute of Anesthesiology, University Hospital Zurich, Switzerland.

Background: Postinfarct remodeled myocardium exhibits numerous structural and biochemical alterations. So far, it is unknown whether postconditioning elicited by volatile anesthetics can also provide protection in the remodeled myocardium.

Methods: Myocardial infarct was induced in male Wistar rats by ligation of the left anterior descending coronary artery. Six weeks later, hearts were buffer-perfused and exposed to 40 min of ischemia followed by 90 min of reperfusion. Anesthetic postconditioning was induced by 15 min of 2.1 vol% isoflurane. In some experiments, LY294002 (15 microM), a phosphatidylinositol 3-kinase inhibitor, was coadministered with isoflurane. Masson's trichrome staining, immunohistochemistry, Western blot analysis, and reverse-transcription polymerase chain reaction served to confirm remodeling. In buffer-perfused hearts, functional recovery was recorded, and acute infarct size was measured using 1% triphenyltetrazolium chloride staining and lactate dehydrogenase release during reperfusion. Western blot analysis was used to determine phosphorylation of reperfusion injury salvage kinases including protein kinase B/Akt and its downstream targets after 15 min of reperfusion.

Results: Infarct hearts exhibited typical macroscopic and molecular changes of remodeling. Isoflurane postconditioning improved functional recovery and decreased acute infarct size, as determined by triphenyltetrazolium (35 +/- 5% in unprotected hearts vs. 8 +/- 3% in anesthetic postconditioning; P < 0.05) and lactate dehydrogenase release. This protection was abolished by LY294002, which inhibited phosphorylation of protein kinase B/Akt and its downstream targets glycogen synthase kinase 3beta, endothelial nitric oxide synthase, and p70S6 kinase.

Conclusions: Infarct-remodeled myocardium is receptive to protection by isoflurane postconditioning via protein kinase B/Akt signaling. This is the first time to demonstrate that anesthetic postconditioning retains its marked protection in diseased myocardium.
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http://dx.doi.org/10.1097/00000542-200605000-00017DOI Listing
May 2006

The sarcomeric M-band during development and in disease.

J Muscle Res Cell Motil 2005 ;26(6-8):375-9

The Randall Division of Cell & Molecular Biophysics and the Cardiovascular Division, King's College London, SE1 1UL, London, UK.

The C-terminus of connectin/titin at the M-band of the sarcomere interacts with several structural as well as potential signalling proteins. One of these is myomesin, which can also bind to myosin and has been suggested to function as an integral structural linker of the thick filaments into the sarcomere. Recent evidence that myomesin possesses the ability to form antiparallel dimers via its C-terminal domain has prompted us to propose a novel three-dimensional model for the sarcomeric M-band. A splice variant of myomesin, termed EH-myomesin, contains an additional segment that has disordered conformation and functions as an entropic spring. It is expressed in a subset of muscle types that are characterised by a broader operational range and are more resistant to damage caused by eccentric contraction. In addition, it is also re-expressed in dilated cardiomyopathy. DRAL/FHL-2 is another protein that interacts with the M-band portion of connectin/titin and which probably functions as an adaptor for the compartmentalisation of metabolic enzymes. Together these results suggest that the M-band is crucial for sarcomere function and maintenance and that its molecular composition can be adapted to divergent physiological needs in different muscle types, which may help to cope with pathological alterations.
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http://dx.doi.org/10.1007/s10974-005-9019-4DOI Listing
January 2007

Establishment of cardiac cytoarchitecture in the developing mouse heart.

Dev Biol 2006 Jan 9;289(2):430-41. Epub 2005 Dec 9.

Institute of Cell Biology, ETH Zurich-Hönggerberg, CH-8093 Zurich, Switzerland.

Cardiomyocytes are characterized by an extremely well-organized cytoarchitecture. We investigated its establishment in the developing mouse heart with particular reference to the myofibrils and the specialized types of cell-cell contacts, the intercalated discs (ICD). Early embryonic cardiomyocytes have a polygonal shape with cell-cell contacts distributed circumferentially at the peripheral membrane and myofibrils running in a random orientation in the sparse cytoplasm between the nucleus and the plasma membrane. During fetal development, the cardiomyocytes elongate, and the myofibrils become aligned. The restriction of the ICD components to the bipolar ends of the cells is a much slower process and is achieved for adherens junctions and desmosomes only after birth, for gap junctions even later. By quantifying the specific growth parameters of prenatal cardiomyocytes, we were able to identify a previously unknown fetal phase of physiological hypertrophy. Our results suggest (1) that myofibril alignment, bipolarization and ICD restriction happen sequentially in cardiomyocytes, and (2) that increase of heart mass in the embryo is not only achieved by hyperplasia alone but also by volume increase of the individual cardiomyocytes (hypertrophy). These observations help to understand the mechanisms that lead to the formation of a functional heart during development at a cellular level.
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http://dx.doi.org/10.1016/j.ydbio.2005.10.046DOI Listing
January 2006

Isoflurane postconditioning prevents opening of the mitochondrial permeability transition pore through inhibition of glycogen synthase kinase 3beta.

Anesthesiology 2005 Nov;103(5):987-95

Institute of Anesthesiology, University Hospital Zurich, Zwitzerland.

Background: Postischemic administration of volatile anesthetics activates reperfusion injury salvage kinases and decreases myocardial damage. However, the mechanisms underlying anesthetic postconditioning are unclear.

Methods: Isolated perfused rat hearts were exposed to 40 min of ischemia followed by 1 h of reperfusion. Anesthetic postconditioning was induced by 15 min of 2.1 vol% isoflurane (1.5 minimum alveolar concentration) administered at the onset of reperfusion. In some experiments, atractyloside (10 microm), a mitochondrial permeability transition pore (mPTP) opener, and LY294002 (15 microm), a phosphatidylinositol 3-kinase inhibitor, were coadministered with isoflurane. Western blot analysis was used to determine phosphorylation of protein kinase B/Akt and its downstream target glycogen synthase kinase 3beta after 15 min of reperfusion. Myocardial tissue content of nicotinamide adenine dinucleotide served as a marker for mPTP opening. Accumulation of MitoTracker Red 580 (Molecular Probes, Invitrogen, Basel, Switzerland) was used to visualize mitochondrial function.

Results: Anesthetic postconditioning significantly improved functional recovery and decreased infarct size (36 +/- 1% in unprotected hearts vs. 3 +/- 2% in anesthetic postconditioning; P < 0.05). Isoflurane-mediated protection was abolished by atractyloside and LY294002. LY294002 inhibited isoflurane-induced phosphorylation of protein kinase B/Akt and glycogen synthase kinase 3beta and opened mPTP as determined by nicotinamide adenine dinucleotide measurements. Atractyloside, a direct opener of the mPTP, did not inhibit phosphorylation of protein kinase B/Akt and glycogen synthase kinase 3beta by isoflurane but reversed isoflurane-mediated cytoprotection. Microscopy showed accumulation of the mitochondrial tracker in isoflurane-protected functional mitochondria but no staining in mitochondria of unprotected hearts.

Conclusions: Anesthetic postconditioning by isoflurane effectively protects against reperfusion damage by preventing opening of the mPTP through inhibition of glycogen synthase kinase 3beta.
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http://dx.doi.org/10.1097/00000542-200511000-00013DOI Listing
November 2005

The M-band: an elastic web that crosslinks thick filaments in the center of the sarcomere.

Trends Cell Biol 2005 Sep;15(9):477-85

Institute of Cell Biology, Swiss Federal Institute of Technology, ETH-Hönggerberg, CH-8093 Zürich.

The sarcomere of striated muscle is an efficient molecular machine, characterized by perfect structural organization of contractile filaments. This order is ensured by the sarcomere cytoskeleton, an important element of which is the M-band, believed to maintain the thick filament lattice. We review here recent progress in understanding the M-band function and its structural organization. We explain how the M-band might reduce the intrinsic instability of thick filaments and help titin to maintain order in the sarcomeres. The M-band molecular structure has been clarified recently by biochemical and biophysical approaches that focused on the properties of the prominent M-band component myomesin. These have shown that antiparallel myomesin dimers might link the thick filaments in the M-band, a role analogous to that of alpha-actinin in the Z-disc. Furthermore, similar to titin, myomesin is a molecular spring with complex visco-elastic properties that can be modified by alternative splicing. M-band protein composition correlates with the expression of titin isoforms and appears to be a reliable marker for biomechanical conditions in contracting muscle. We propose that the M-band is in fact a dynamic structure that monitors the stress appearing in the thick filament lattice during contraction and quickly reorganizes to meet new physiological requirements.
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http://dx.doi.org/10.1016/j.tcb.2005.07.001DOI Listing
September 2005

Myomesin is a molecular spring with adaptable elasticity.

J Mol Biol 2005 Jun 7;349(2):367-79. Epub 2005 Apr 7.

Institute of Cell Biology, ETH Zürich-Hönggerberg, CH-8093 Zürich, Switzerland.

The M-band is a transverse structure in the center of the sarcomere, which is thought to stabilize the thick filament lattice. It was shown recently that the constitutive vertebrate M-band component myomesin can form antiparallel dimers, which might cross-link the neighboring thick filaments. Myomesin consists mainly of immunoglobulin-like (Ig) and fibronectin type III (Fn) domains, while several muscle types express the EH-myomesin splice isoform, generated by the inclusion of the unique EH-segment of about 100 amino acid residues (aa) in the center of the molecule. Here we use atomic force microscopy (AFM), transmission electron microscopy (TEM) and circular dichroism (CD) spectroscopy for the biophysical characterization of myomesin. The AFM identifies the "mechanical fingerprints" of the modules constituting the myomesin molecule. Stretching of homomeric polyproteins, constructed of Ig and Fn domains of human myomesin, produces a typical saw-tooth pattern in the force-extension curve. The domains readily refold after relaxation. In contrast, stretching of a heterogeneous polyprotein, containing several repeats of the My6-EH fragment reveals a long initial plateau corresponding to the sum of EH-segment contour lengths, followed by several My6 unfolding peaks. According to this, the EH-segment is characterized as an entropic chain with a persistence length of about 0.3nm. In TEM pictures, the EH-domain appears as a gap in the molecule, indicating a random coil conformation similar to the PEVK region of titin. CD spectroscopy measurements support this result, demonstrating a mostly non-folded conformation for the EH-segment. We suggest that similarly to titin, myomesin is a molecular spring, whose elasticity is modulated by alternative splicing. The Ig and Fn domains might function as reversible "shock absorbers" by sequential unfolding in the case of extremely high or long sustained stretching forces. These complex visco-elastic properties of myomesin might be crucial for the stability of the sarcomere.
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http://dx.doi.org/10.1016/j.jmb.2005.03.055DOI Listing
June 2005

Bacterial cell penetration by beta3-oligohomoarginines: indications for passive transfer through the lipid bilayer.

Chembiochem 2005 Jun;6(6):982-5

Swiss Federal Institute of Environmental Science and Technology (EAWAG), Uberlandstrasse 133, 8600 Dübendorf, Switzerland.

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http://dx.doi.org/10.1002/cbic.200400394DOI Listing
June 2005

Distinct functions of junD in cardiac hypertrophy and heart failure.

Genes Dev 2005 Jan;19(2):208-13

Institute of Molecular Pathology, A-1030 Vienna, Austria.

Cardiac hypertrophic stimuli induce both adaptive and maladaptive growth response pathways in heart. Here we show that mice lacking junD develop less adaptive hypertrophy in heart after mechanical pressure overload, while cardiomyocyte-specific expression of junD in mice results in spontaneous ventricular dilation and decreased contractility. In contrast, fra-1 conditional knock-out mice have a normal hypertrophic response, whereas hearts from fra-1 transgenic mice decompensate prematurely. Moreover, fra-1 transgenic mice simultaneously lacking junD reveal a spontaneous dilated cardiomyopathy associated with increased cardiomyocyte apoptosis and a primary mitochondrial defect. These data suggest that junD promotes both adaptive-protective and maladaptive hypertrophy in heart, depending on its expression levels.
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http://dx.doi.org/10.1101/gad.327005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC545879PMC
January 2005

Dimerisation of myomesin: implications for the structure of the sarcomeric M-band.

J Mol Biol 2005 Jan;345(2):289-98

Institute of Cell Biology, ETH Zurich-Honggerberg, CH-8093 Zurich, Switzerland.

The sarcomeric M-band is thought to provide a link between the thick and the elastic filament systems. So far, relatively little is known about its structural components and their three-dimensional organisation. Myomesin seems to be an essential component of the M-band, since it is expressed in all types of vertebrate striated muscle fibres investigated and can be found in its mature localisation pattern as soon as the first myofibrils are assembled. Previous work has shown that the N-terminal and central part of myomesin harbour binding sites for myosin, titin and muscle creatine kinase. Intrigued by the highly conserved domain layout of the C-terminal half, we screened for new interaction partners by yeast two-hybrid analysis. This revealed a strong interaction of myomesin with itself. This finding was confirmed by several biochemical assays. Our data suggest that myomesin can form antiparallel dimers via a binding site residing in its C-terminal domain 13. We suggest that, similar to alpha-actinin in the Z-disc, the myomesin dimers cross-link the contractile filaments in the M-band. The new and the already previously identified myomesin interaction sites are integrated into the first three-dimensional model of the sarcomeric M-band on a molecular basis.
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http://dx.doi.org/10.1016/j.jmb.2004.10.040DOI Listing
January 2005

The molecular composition of the sarcomeric M-band correlates with muscle fiber type.

Eur J Cell Biol 2004 Jul;83(5):193-204

Institute of Cell Biology, ETH-Zurich Hoenggerberg, Zurich, Switzerland.

The M-band is the transverse structure that cross-links the thick filaments in the center and provides a perfect alignment of the A-band in the activated sarcomere. The molecular composition of the M-bands in adult mouse skeletal muscle is fiber-type dependent. All M-bands in fast fibers contain M-protein while M-bands in slow fibers contain a significant proportion of the EH-myomesin isoform, previously detected only in embryonic heart muscle. This fiber-type specificity develops during the first postnatal weeks. However, the ratio between the amounts of myosin and of myomesin, taken as sum of both isoforms, remains nearly constant in all studied muscles. Ultrastructural analysis demonstrates that some of the soleus fibers show a diffuse appearance of the M-band, resembling the situation in the embryonic heart. A model is proposed to explain the functional consequence of differential M-band composition for the physiological and morphological properties of sarcomeres in different muscle types.
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http://dx.doi.org/10.1078/0171-9335-00383DOI Listing
July 2004

Sequential myofibrillar breakdown accompanies mitotic division of mammalian cardiomyocytes.

J Cell Sci 2004 Jul;117(Pt 15):3295-306

Institute of Cell Biology, Swiss Federal Institute of Technology, ETH Hönggerberg, 8093 Zurich.

The contractile tissue of the heart is composed of individual cardiomyocytes. During mammalian embryonic development, heart growth is achieved by cell division while at the same time the heart is already exerting its essential pumping activity. There is still some debate whether the proliferative activity is carried out by a less differentiated, stem cell-like type of cardiomyocytes or whether embryonic cardiomyocytes are able to perform both of these completely different dynamic tasks, contraction and cell division. Our analysis of triple-stained specimen of cultured embryonic cardiomyocytes and of whole mount preparations of embryonic mouse hearts by confocal microscopy revealed that differentiated cardiomyocytes are indeed able to proliferate. However, to go through cell division, a disassembly of the contractile elements, the myofibrils, has to take place. This disassembly occurs in two steps with Z-disk and thin (actin)-filament-associated proteins getting disassembled before disassembly of the M-bands and the thick (myosin) filaments happens. After cytokinesis reassembly of the myofibrillar proteins to their mature cross-striated pattern can be seen. Another interesting observation was that the cell-cell contacts remain seemingly intact during division, probably reflecting the requirement of intact integration sites of the individual cells in the contractile tissue. Our results suggest that embryonic cardiomyocytes have developed an interesting strategy to deal with their major cytoskeletal elements, the myofibrils, during mitosis. The complex disassembly-reassembly process might also provide a mechanistic explanation, why cardiomyocytes cede to divide postnatally.
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http://dx.doi.org/10.1242/jcs.01159DOI Listing
July 2004

Myofibrillogenesis in the developing chicken heart: role of actin isoforms and of the pointed end actin capping protein tropomodulin during thin filament assembly.

Dev Dyn 2004 Apr;229(4):745-55

Institute of Cell Biology, ETH-Zürich Hönggerberg, Zürich, Switzerland.

Recently, important differences between myofibrillogenesis in cultured cardiomyocytes vs. the three-dimensional setting in situ could be determined. We investigated thin filament assembly in situ by confocal microscopy of whole-mount preparations of immunostained embryonic chicken hearts. Of interest, a distinct localisation of different actin isoforms was observed in immature thin filaments. Cardiac alpha-actin is restricted to filaments with a length comparable to mature thin filaments as soon as the first contractions occur, while vascular alpha-actin makes up filaments that extend toward the M-band. The pointed-end actin filament capping protein tropomodulin can be found initially in close association with the plasma membrane, but attains its mature localisation pattern at the ends of the thin filaments only comparatively late during myofibrillogenesis. Thus tropomodulin acts as a length stabilising element of actin filaments also in developing cardiomyocytes in situ, but plays an additional role together with membrane-associated actin filaments in the earliest steps of myofibril assembly.
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http://dx.doi.org/10.1002/dvdy.10482DOI Listing
April 2004

Design of artificial myocardial microtissues.

Tissue Eng 2004 Jan-Feb;10(1-2):201-14

Institute of Biotechnology, Swiss Federal Institute of Technology, Zurich, Switzerland.

Cultivation technologies promoting organization of mammalian cells in three dimensions are essential for gene-function analyses as well as drug testing and represent the first step toward the design of tissue replacements and bioartificial organs. Embedded in a three-dimensional environment, cells are expected to develop tissue-like higher order intercellular structures (cell-cell contacts, extracellular matrix) that orchestrate cellular functions including proliferation, differentiation, apoptosis, and angiogenesis with unmatched quality. We have refined the hanging drop cultivation technology to pioneer beating heart microtissues derived from pure primary rat and mouse cardiomyocyte cultures as well as mixed populations reflecting the cell type composition of rodent hearts. Phenotypic characterization combined with detailed analysis of muscle-specific cell traits, extracellular matrix components, as well as endogenous vascular endothelial growth factor (VEGF) expression profiles of heart microtissues revealed (1). a linear cell number-microtissue size correlation, (2). intermicrotissue superstructures, (3). retention of key cardiomyocyte-specific cell qualities, (4). a sophisticated extracellular matrix, and (5). a high degree of self-organization exemplified by the tendency of muscle structures to assemble at the periphery of these myocardial spheroids. Furthermore (6). myocardial spheroids support endogenous VEGF expression in a size-dependent manner that will likely promote vascularization of heart microtissues produced from defined cell mixtures as well as support connection to the host vascular system after implantation. As cardiomyocytes are known to be refractory to current transfection technologies we have designed lentivirus-based transduction strategies to lead the way for genetic engineering of myocardial microtissues in a clinical setting.
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http://dx.doi.org/10.1089/107632704322791853DOI Listing
September 2004

M-band: a safeguard for sarcomere stability?

J Muscle Res Cell Motil 2003 ;24(2-3):191-203

Institute of Cell Biology, ETH-Zurich Hoenggerberg, CH-8093 Zurich, Switzerland.

The sarcomere of striated muscle is a very efficient machine transforming chemical energy into movement. However, a wrong distribution of the generated forces may lead to self-destruction of the engine itself. A well-known example for this is eccentric contraction (elongation of the sarcomere in the activated state), which damages sarcomeric structure and leads to a reduced muscle performance. The goal of this review is to discuss the involvement of different cytoskeletal systems, in particular the M-band filaments, in the mechanisms that provide stability during sarcomeric contraction. The M-band is the transverse structure in the center of the sarcomeric A-band, which is responsible both for the regular packing of thick filaments and for the uniform distribution of the tension over the myosin filament lattice in the activated sarcomere. Although some proteins from the Ig-superfamily, like myomesin and M-protein, are the major candidates for the role of M-band bridges, the exact molecular organisation of the M-band is not clear. However, the protein composition of the M-band seems to modulate the mechanical characteristics of the thick filament lattice, in particular its stiffness, adjusting it to the specific demands in different muscle types. The special M-band design in slow fibers might be part of structural adaptations, favouring sarcomere stability for a continuous contractile activity over a broad working range. In conclusion, we discuss why the interference with M-band structure might have fatal consequences for the integrity of the working sarcomere.
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http://dx.doi.org/10.1023/a:1026094924677DOI Listing
June 2004

Identification, characterization, and expression of a novel alpha-tropomyosin isoform in cardiac tissues in developing chicken.

J Cell Biochem 2003 Jun;89(3):427-39

Department of Cell and Developmental Biology, Syracuse, New York, USA.

Tropomyosins are present in various muscle (skeletal, cardiac, and smooth) and non-muscle cells with different isoforms characteristic of specific cell types. We describe here a novel smooth/striated chimeric isoform that was expressed in developing chick heart in addition to the classically described TM-4 type. This novel alpha-Tm tropomyosin isoform, designated as alpha-Tm-2, contains exon 2a (in place of exon 2b). The known striated muscle isoform (alpha-Tm-1) was also expressed in embryonic hearts along with the striated muscle isoform of TM-4. In adult heart, TM-4 was expressed, however, expression of both alpha-Tm-1 and alpha-Tm-2 isoforms was drastically reduced or downregulated. Interestingly, we were unable to detect the expression of alpha-Tm-2 in embryonic and adult skeletal muscle, however, the alpha-Tm-1 isoform is expressed in embryonic and adult skeletal muscle. Examination of other possible isoforms of the alpha-TM gene, i.e., alpha-smooth muscle tropomyosin (alpha-Sm), alpha-Fibroblast-1 (alpha-F1), and alpha-Fibroblast-2 (alpha-F2) revealed expression in embryonic hearts and a significant reduction of each of these isoforms in adult heart. In order to elucidate the role of the newly discovered tropomyosin isoform in chicken, we ectopically expressed the GFP fusion protein of alpha-Tm-1 and alpha-Tm-2 separately into cardiomyocytes isolated from neonatal rats. Each isoform was incorporated into organized myofibrils. Our results suggest that the alpha-TM gene may undergo both positive and negative transcriptional control in chicken hearts during development.
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http://dx.doi.org/10.1002/jcb.10504DOI Listing
June 2003