RESUMO
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.
Assuntos
Cardiomiopatia Dilatada/metabolismo , Cardiomiopatia Dilatada/fisiopatologia , Miócitos Cardíacos/fisiologia , beta Catenina/genética , beta Catenina/metabolismo , Animais , Cardiomiopatia Dilatada/mortalidade , Fracionamento Celular , Linhagem Celular , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Técnicas de Introdução de Genes/métodos , Ventrículos do Coração/metabolismo , Ventrículos do Coração/patologia , Ventrículos do Coração/fisiopatologia , Integrases/genética , Camundongos , Camundongos Knockout , Miócitos Cardíacos/patologia , Fenótipo , Ratos , Transdução de Sinais/fisiologia , Taxa de Sobrevida , Transcrição Gênica/fisiologia , Proteínas Wnt/metabolismoRESUMO
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.
RESUMO
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.
Assuntos
Cardiomegalia/metabolismo , Glicólise , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Metabolismo dos Lipídeos , PPAR gama/metabolismo , Animais , Apoptose , Ácidos Graxos/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Camundongos , Camundongos Knockout , Miócitos Cardíacos/metabolismo , PPAR gama/genética , Monoéster Fosfórico Hidrolases/metabolismo , Regulação para Cima , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismoRESUMO
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.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Coração/embriologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Hipóxia/metabolismo , Miócitos Cardíacos/metabolismo , Transdução de Sinais , Animais , Conectina , Perda do Embrião/genética , Perda do Embrião/metabolismo , Desenvolvimento Embrionário , Regulação da Expressão Gênica no Desenvolvimento/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Fatores de Transcrição MEF2 , Camundongos , Camundongos Transgênicos , Proteínas Musculares/biossíntese , Proteínas Musculares/genética , Fatores de Regulação Miogênica/biossíntese , Fatores de Regulação Miogênica/genética , Proteínas Quinases/biossíntese , Proteínas Quinases/genética , Sarcômeros/genética , Sarcômeros/metabolismo , Transdução de Sinais/genética , Proteínas com Domínio T/biossíntese , Proteínas com Domínio T/genéticaRESUMO
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.
Assuntos
Aminoidrolases/genética , Células-Tronco Embrionárias/citologia , Desenvolvimento Muscular , Miócitos Cardíacos/citologia , Cadeias Leves de Miosina/genética , Aminoidrolases/metabolismo , Animais , Diferenciação Celular , Linhagem Celular , Células-Tronco Embrionárias/metabolismo , Vetores Genéticos , Proteínas de Fluorescência Verde , Humanos , Camundongos , Miócitos Cardíacos/metabolismo , Cadeias Leves de Miosina/metabolismo , Ratos , Proteínas Recombinantes de Fusão/metabolismo , TransfecçãoRESUMO
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.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Proteínas Musculares/biossíntese , Proteínas Musculares/fisiologia , Músculo Liso Vascular/metabolismo , Fosfoproteínas/genética , Animais , Feminino , Deleção de Genes , Humanos , Camundongos , Modelos Biológicos , Fibras Musculares Esqueléticas/metabolismo , Proteínas Musculares/genética , Miosinas/metabolismo , Fenótipo , Fosfoproteínas/fisiologia , Fosforilação , Condicionamento Físico AnimalRESUMO
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.
Assuntos
Citoesqueleto/química , Proteínas Musculares/química , Proteínas Musculares/metabolismo , Músculo Estriado/citologia , Proteínas Quinases/química , Processamento Alternativo , Sequência de Aminoácidos , Animais , Animais Recém-Nascidos , Sítios de Ligação , Conectina , Citoesqueleto/ultraestrutura , Dimerização , Embrião de Mamíferos , Epitopos/química , Éxons , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Glutationa Transferase/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Membro Posterior/metabolismo , Imuno-Histoquímica , Íntrons , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Proteínas Musculares/biossíntese , Proteínas Musculares/genética , Proteínas Musculares/ultraestrutura , Músculo Esquelético/metabolismo , Músculo Esquelético/ultraestrutura , Músculo Estriado/metabolismo , Músculo Estriado/ultraestrutura , Gravidez , Ligação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/ultraestrutura , Proteínas Quinases/ultraestrutura , Estrutura Terciária de Proteína , RNA Mensageiro/metabolismo , Sarcômeros/química , Sarcômeros/metabolismo , Sarcômeros/ultraestrutura , Homologia de Sequência de Aminoácidos , Somitos/metabolismoRESUMO
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.
Assuntos
Proteínas Musculares/química , Fragmentos de Peptídeos/química , Sarcômeros/química , Sequência de Aminoácidos , Conectina , Cristalografia por Raios X , Citoesqueleto/química , Citoesqueleto/metabolismo , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Musculares/metabolismo , Fragmentos de Peptídeos/metabolismo , Sarcômeros/metabolismoRESUMO
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).
Assuntos
Fibras Musculares Esqueléticas/ultraestrutura , Proteínas Musculares/genética , Músculos Oculomotores/ultraestrutura , Sarcômeros/ultraestrutura , Animais , Conectina , Imuno-Histoquímica , Microscopia Confocal , Microscopia Eletrônica , Fibras Musculares Esqueléticas/metabolismo , Proteínas Musculares/metabolismo , Músculos Oculomotores/metabolismo , Reação em Cadeia da Polimerase , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Sarcômeros/metabolismoRESUMO
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.
Assuntos
Cardiomegalia/metabolismo , Divisão do Núcleo Celular , Citocinese , Coração/embriologia , Miócitos Cardíacos/fisiologia , Actomiosina/metabolismo , Amidas/farmacologia , Angiotensina II/metabolismo , Animais , Anti-Hipertensivos/farmacologia , Biomarcadores/análise , Calpaína/metabolismo , Cardiomegalia/induzido quimicamente , Proteínas Culina/metabolismo , Coração/crescimento & desenvolvimento , Hipertensão/patologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Miócitos Cardíacos/metabolismo , Miofibrilas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Piridinas/farmacologia , Ratos , Ratos Endogâmicos Dahl , Regulação para Cima , Proteínas rho de Ligação ao GTP/metabolismo , Quinases Associadas a rhoRESUMO
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.
Assuntos
Sistema de Condução Cardíaco/crescimento & desenvolvimento , Sistema de Condução Cardíaco/fisiologia , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia , Animais , Fascículo Atrioventricular/anormalidades , Fascículo Atrioventricular/crescimento & desenvolvimento , Fascículo Atrioventricular/fisiologia , Diferenciação Celular , Eletrofisiologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Sistema de Condução Cardíaco/anormalidades , Proteína Homeobox Nkx-2.5 , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Modelos Cardiovasculares , Ramos Subendocárdicos/anormalidades , Ramos Subendocárdicos/crescimento & desenvolvimento , Ramos Subendocárdicos/fisiologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fatores de Transcrição/deficiência , Função VentricularRESUMO
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.
Assuntos
Doxorrubicina/farmacologia , Contração Miocárdica/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Neuregulina-1/farmacologia , Estresse Oxidativo , Acetilcisteína/farmacologia , Animais , Antibióticos Antineoplásicos/farmacologia , Cálcio/metabolismo , Doxorrubicina/antagonistas & inibidores , Peróxido de Hidrogênio/farmacologia , Masculino , Miócitos Cardíacos/fisiologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Wistar , Receptor ErbB-2/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático , Transdução de Sinais , Função VentricularRESUMO
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.
Assuntos
Bioprótese , Células Endoteliais/transplante , Miócitos Cardíacos/transplante , Neovascularização Fisiológica , Transplante de Tecidos , Transplantes , Animais , Animais Recém-Nascidos , Células Cultivadas , Embrião de Galinha , Técnicas de Cocultura , Células Endoteliais/metabolismo , Células Endoteliais/ultraestrutura , Humanos , Miocárdio/metabolismo , Miocárdio/ultraestrutura , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/ultraestrutura , Ratos , Ratos Wistar , Engenharia Tecidual/métodos , Transplante de Tecidos/métodos , Transplante Heterólogo , Transplante HomólogoRESUMO
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.
Assuntos
Cardiomiopatia Dilatada/genética , Células Musculares/fisiologia , Infarto do Miocárdio/genética , Disfunção Ventricular Direita/genética , alfa Catenina/genética , Animais , Modelos Animais de Doenças , Eletrocardiografia , Camundongos , Camundongos Knockout , Células Musculares/patologia , Miocárdio/patologia , Proteínas/metabolismo , alfa Catenina/deficiênciaRESUMO
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.
Assuntos
Proteínas de Ligação ao GTP/fisiologia , Miócitos Cardíacos/metabolismo , Actinina/análise , Actinina/metabolismo , Amidas/farmacologia , Animais , Proteínas de Ciclo Celular/análise , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Citocalasina D/farmacologia , Citocinese/efeitos dos fármacos , Citocinese/fisiologia , Proteínas do Citoesqueleto/análise , Proteínas do Citoesqueleto/metabolismo , Inibidores Enzimáticos/farmacologia , GTP Fosfo-Hidrolases/análise , GTP Fosfo-Hidrolases/metabolismo , Proteínas de Ligação ao GTP/análise , Proteínas de Ligação ao GTP/metabolismo , Coração/embriologia , Coração/crescimento & desenvolvimento , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Camundongos , Microtúbulos/química , Microtúbulos/metabolismo , Modelos Biológicos , Miocárdio/química , Miocárdio/citologia , Miocárdio/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Miosina não Muscular Tipo IIB/análise , Miosina não Muscular Tipo IIB/metabolismo , Piridinas/farmacologia , Ratos , Proteínas de Schizosaccharomyces pombe , Septinas , Fuso Acromático/química , Fuso Acromático/metabolismo , Fatores de Tempo , Fatores de TranscriçãoRESUMO
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.
Assuntos
Anestésicos Inalatórios/farmacologia , Cardiotônicos , Isoflurano/farmacologia , Proteína Oncogênica v-akt/fisiologia , Proteínas Proto-Oncogênicas c-akt/fisiologia , Transdução de Sinais/efeitos dos fármacos , Remodelação Ventricular/efeitos dos fármacos , Animais , Western Blotting , Hemodinâmica/efeitos dos fármacos , Técnicas In Vitro , Masculino , Miocárdio/ultraestrutura , RNA Mensageiro/biossíntese , RNA Mensageiro/isolamento & purificação , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
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.
Assuntos
Coração/embriologia , Junções Aderentes/fisiologia , Junções Aderentes/ultraestrutura , Animais , Diferenciação Celular/fisiologia , Tamanho Celular , Conexina 43/metabolismo , Crioultramicrotomia , Desmossomos/metabolismo , Matriz Extracelular/metabolismo , Matriz Extracelular/ultraestrutura , Junções Comunicantes/metabolismo , Coração/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Microscopia de Fluorescência/métodos , Miofibrilas/metabolismo , Transporte ProteicoRESUMO
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.
Assuntos
Anestésicos Inalatórios/farmacologia , Inibidores Enzimáticos/farmacologia , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Isoflurano/farmacologia , Mitocôndrias Cardíacas/efeitos dos fármacos , Anestésicos Inalatórios/antagonistas & inibidores , Animais , Western Blotting , Cromonas/farmacologia , Corantes , Condicionamento Psicológico/efeitos dos fármacos , Glicogênio Sintase Quinase 3 beta , Hemodinâmica/efeitos dos fármacos , Técnicas In Vitro , Isoflurano/antagonistas & inibidores , Masculino , Mitocôndrias Cardíacas/metabolismo , Morfolinas/farmacologia , Infarto do Miocárdio/enzimologia , Infarto do Miocárdio/patologia , Infarto do Miocárdio/prevenção & controle , Miocárdio/metabolismo , NAD/metabolismo , Compostos Orgânicos , Permeabilidade/efeitos dos fármacos , Fosforilação , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacosRESUMO
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.
Assuntos
Miosinas/fisiologia , Sarcômeros/fisiologia , Animais , Conectina , Elasticidade , Humanos , Microscopia Eletrônica , Modelos Biológicos , Contração Muscular/fisiologia , Proteínas Musculares/fisiologia , Músculo Esquelético/fisiologia , Músculo Esquelético/ultraestrutura , Miofibrilas/fisiologia , Miofibrilas/ultraestrutura , Isoformas de Proteínas/fisiologia , Proteínas Quinases/fisiologia , Sarcômeros/ultraestrutura , Estresse MecânicoRESUMO
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.