RESUMO
AIMS: We investigated newly generated immortalized heterozygous and homozygous R349P desmin knock-in myoblasts in conjunction with the corresponding desminopathy mice as models for desminopathies to analyse major protein quality control processes in response to the presence of R349P mutant desmin. METHODS: We used hetero- and homozygous R349P desmin knock-in mice for analyses and for crossbreeding with p53 knock-out mice to generate immortalized R349P desmin knock-in skeletal muscle myoblasts and myotubes. Skeletal muscle sections and cultured muscle cells were investigated by indirect immunofluorescence microscopy, proteasomal activity measurements and immunoblotting addressing autophagy rate, chaperone-assisted selective autophagy and heat shock protein levels. Muscle sections were further analysed by transmission and immunogold electron microscopy. RESULTS: We demonstrate that mutant desmin (i) increases proteasomal activity, (ii) stimulates macroautophagy, (iii) dysregulates the chaperone assisted selective autophagy and (iv) elevates the protein levels of αB-crystallin and Hsp27. Both αB-crystallin and Hsp27 as well as Hsp90 displayed translocation patterns from Z-discs as well as Z-I junctions, respectively, to the level of sarcomeric I-bands in dominant and recessive desminopathies. CONCLUSIONS: Our findings demonstrate that the presence of R349P mutant desmin causes a general imbalance in skeletal muscle protein homeostasis via aberrant activity of all major protein quality control systems. The augmented activity of these systems and the subcellular shift of essential heat shock proteins may deleteriously contribute to the previously observed increased turnover of desmin itself and desmin-binding partners, which triggers progressive dysfunction of the extrasarcomeric cytoskeleton and the myofibrillar apparatus in the course of the development of desminopathies.
Assuntos
Cardiomiopatias/genética , Cardiomiopatias/fisiopatologia , Desmina/genética , Músculo Esquelético/fisiopatologia , Distrofias Musculares/genética , Distrofias Musculares/fisiopatologia , Proteostase/genética , Animais , Autofagia/genética , Modelos Animais de Doenças , Camundongos , Músculo Esquelético/metabolismo , MutaçãoRESUMO
In this review, we present our current understanding of peripartum cardiomyopathy (PPCM) based on reports of the incidence, diagnosis and current treatment options. We summarise opinions on whether PPCM is triggered by vascular and/or hormonal causes and examine the influence of comorbidities such as preeclampsia. Two articles published in 2021 strongly support the hypothesis that PPCM may be a familial disease. Using large cohorts of PPCM patients, they summarised the available genomic DNA sequence data that are expressed in human cardiomyocytes. While PPCM is considered a disease predominately affecting the left ventricle, there are data to suggest that some cases also involve right ventricular failure. Finally, we conclude that there is sufficient evidence to warrant an RNAseq investigation and that this would be most informative if performed at the cardiomyocytes level rather than analysing genomic DNA from the peripheral circulation. Given the rarity of PPCM, the combined resources of international human heart tissue biobanks have assembled 30 ventricular tissue samples from PPCM patients, and we are actively seeking to enlarge this patient base by collaborating with human heart tissue banks and research laboratories who would like to join this endeavour.
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We report the cloning and functional characterization of myopodin, the second member of the synaptopodin gene family. Myopodin shows no significant homology to any known protein except synaptopodin. Northern blot analysis resulted in a 3.6-kb transcript for mouse skeletal and heart muscle. Western blots showed an 80-kD signal for skeletal and a 95-kD signal for heart muscle. Myopodin contains one PPXY motif and multiple PXXP motifs. Myopodin colocalizes with alpha-actinin and is found at the Z-disc as shown by immunogold electron microscopy. In myoblasts, myopodin shows preferential nuclear localization. During myotube differentiation, myopodin binds to stress fibers in a punctuated pattern before incorporation into the Z-disc. Myopodin can directly bind to actin and contains a novel actin binding site in the center of the protein. Myopodin has actin-bundling activity as shown by formation of latrunculin-A-sensitive cytosolic actin bundles and nuclear actin loops in transfected cells expressing green fluorescent protein-myopodin. Under stress conditions, myopodin accumulates in the nucleus and is depleted from the cytoplasm. Nuclear export of myopodin is sensitive to leptomycin B, despite the absence of a classical nuclear export sequence. We propose a dual role for myopodin as a structural protein also participating in signaling pathways between the Z-disc and the nucleus.
Assuntos
Núcleo Celular/metabolismo , Citoplasma/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas Musculares/metabolismo , Actinas/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Diferenciação Celular , Linhagem Celular , Expressão Gênica , Proteínas de Fluorescência Verde , Resposta ao Choque Térmico , Calefação , Humanos , Proteínas Luminescentes/genética , Camundongos , Proteínas dos Microfilamentos/classificação , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/fisiologia , Dados de Sequência Molecular , Proteínas Musculares/classificação , Proteínas Musculares/genética , Proteínas Musculares/fisiologia , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Músculo Liso/citologia , Músculo Liso/metabolismo , Mutagênese , Miocárdio/metabolismo , Sinais de Localização Nuclear/genética , Sinais de Localização Nuclear/metabolismo , Transporte Proteico , RNA Mensageiro , Proteínas Recombinantes de Fusão/genética , Homologia de Sequência de Aminoácidos , Estresse Fisiológico , Tiazóis/farmacologia , TiazolidinasRESUMO
Kettin is a high molecular mass protein of insect muscle that in the sarcomeres binds to actin and alpha-actinin. To investigate kettin's functional role, we combined immunolabeling experiments with mechanical and biochemical studies on indirect flight muscle (IFM) myofibrils of Drosophila melanogaster. Micrographs of stretched IFM sarcomeres labeled with kettin antibodies revealed staining of the Z-disc periphery. After extraction of the kettin-associated actin, the A-band edges were also stained. In contrast, the staining pattern of projectin, another IFM-I-band protein, was not altered by actin removal. Force measurements were performed on single IFM myofibrils to establish the passive length-tension relationship and record passive stiffness. Stiffness decreased within seconds during gelsolin incubation and to a similar degree upon kettin digestion with mu-calpain. Immunoblotting demonstrated the presence of kettin isoforms in normal Drosophila IFM myofibrils and in myofibrils from an actin-null mutant. Dotblot analysis revealed binding of COOH-terminal kettin domains to myosin. We conclude that kettin is attached not only to actin but also to the end of the thick filament. Kettin along with projectin may constitute the elastic filament system of insect IFM and determine the muscle's high stiffness necessary for stretch activation. Possibly, the two proteins modulate myofibrillar stiffness by expressing different size isoforms.
Assuntos
Proteínas de Drosophila , Drosophila melanogaster/fisiologia , Proteínas de Insetos/metabolismo , Proteínas Musculares/metabolismo , Miofibrilas/fisiologia , Sarcômeros/metabolismo , Actinas/metabolismo , Animais , Fenômenos Biomecânicos , Calpaína/farmacologia , Conectina , Voo Animal , Gelsolina/farmacologia , Immunoblotting , Microscopia de Fluorescência , Ligação Proteica , Isoformas de Proteínas , Sarcômeros/efeitos dos fármacos , Sarcômeros/ultraestruturaRESUMO
In cardiac muscle, the giant protein titin exists in different length isoforms expressed in the molecule's I-band region. Both isoforms, termed N2-A and N2-B, comprise stretches of Ig-like modules separated by the PEVK domain. Central I-band titin also contains isoform-specific Ig-motifs and nonmodular sequences, notably a longer insertion in N2-B. We investigated the elastic behavior of the I-band isoforms by using single-myofibril mechanics, immunofluorescence microscopy, and immunoelectron microscopy of rabbit cardiac sarcomeres stained with sequence-assigned antibodies. Moreover, we overexpressed constructs from the N2-B region in chick cardiac cells to search for possible structural properties of this cardiac-specific segment. We found that cardiac titin contains three distinct elastic elements: poly-Ig regions, the PEVK domain, and the N2-B sequence insertion, which extends approximately 60 nm at high physiological stretch. Recruitment of all three elements allows cardiac titin to extend fully reversibly at physiological sarcomere lengths, without the need to unfold Ig domains. Overexpressing the entire N2-B region or its NH(2) terminus in cardiac myocytes greatly disrupted thin filament, but not thick filament structure. Our results strongly suggest that the NH(2)-terminal N2-B domains are necessary to stabilize thin filament integrity. N2-B-titin emerges as a unique region critical for both reversible extensibility and structural maintenance of cardiac myofibrils.
Assuntos
Citoesqueleto de Actina/metabolismo , Proteínas Musculares/metabolismo , Miocárdio/metabolismo , Miofibrilas/metabolismo , Proteínas Quinases/metabolismo , Citoesqueleto de Actina/ultraestrutura , Actinas/metabolismo , Animais , Anticorpos/imunologia , Células Cultivadas , Galinhas , Conectina , Elasticidade , Epitopos/imunologia , Microscopia Imunoeletrônica , Modelos Biológicos , Proteínas Motores Moleculares/metabolismo , Proteínas Musculares/química , Proteínas Musculares/genética , Miocárdio/citologia , Miocárdio/ultraestrutura , Miofibrilas/ultraestrutura , Miosinas/metabolismo , Dobramento de Proteína , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Quinases/química , Proteínas Quinases/genética , Coelhos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Sarcômeros/metabolismo , Sarcômeros/ultraestrutura , TransfecçãoRESUMO
In the original version of this article, the name of one of the authors is not correct. The correct name should be W. A. Linke, which is shown correctly in the authorgroup section above.
RESUMO
The Sydney Heart Bank (SHB) is one of the largest human heart tissue banks in existence. Its mission is to provide high-quality human heart tissue for research into the molecular basis of human heart failure by working collaboratively with experts in this field. We argue that, by comparing tissues from failing human hearts with age-matched non-failing healthy donor hearts, the results will be more relevant than research using animal models, particularly if their physiology is very different from humans. Tissue from heart surgery must generally be used soon after collection or it significantly deteriorates. Freezing is an option but it raises concerns that freezing causes substantial damage at the cellular and molecular level. The SHB contains failing samples from heart transplant patients and others who provided informed consent for the use of their tissue for research. All samples are cryopreserved in liquid nitrogen within 40 min of their removal from the patient, and in less than 5-10 min in the case of coronary arteries and left ventricle samples. To date, the SHB has collected tissue from about 450 failing hearts (>15,000 samples) from patients with a wide range of etiologies as well as increasing numbers of cardiomyectomy samples from patients with hypertrophic cardiomyopathy. The Bank also has hearts from over 120 healthy organ donors whose hearts, for a variety of reasons (mainly tissue-type incompatibility with waiting heart transplant recipients), could not be used for transplantation. Donor hearts were collected by the St Vincent's Hospital Heart and Lung transplantation team from local hospitals or within a 4-h jet flight from Sydney. They were flushed with chilled cardioplegic solution and transported to Sydney where they were quickly cryopreserved in small samples. Failing and/or donor samples have been used by more than 60 research teams around the world, and have resulted in more than 100 research papers. The tissues most commonly requested are from donor left ventricles, but right ventricles, atria, interventricular system, and coronary arteries vessels have also been reported. All tissues are stored for long-term use in liquid N or vapor (170-180 °C), and are shipped under nitrogen vapor to avoid degradation of sensitive molecules such as RNAs and giant proteins. We present evidence that the availability of these human heart samples has contributed to a reduction in the use of animal models of human heart failure.
RESUMO
In the pathogenesis of dilated cardiomyopathy, cytoskeletal proteins play an important role. In this study, we analyzed titin expression in left ventricles of 19 control human donors and 9 severely diseased (nonischemic) dilated cardiomyopathy (DCM) transplant-patients, using gel-electrophoresis, immunoblotting, and quantitative RT-PCR. Both human-heart groups coexpressed smaller (approximately 3 MDa) N2B-isoform and longer (3.20 to 3.35 MDa) N2BA-isoforms, but the average N2BA:N2B-protein ratio was shifted from approximately 30:70 in controls to 42:58 in DCM hearts, due mainly to increased expression of N2BA-isoforms >3.30 MDa. Titin per unit tissue was decreased in some DCM hearts. The titin-binding protein obscurin also underwent isoform-shifting in DCM. Quantitative RT-PCR revealed a 47% reduction in total-titin mRNA levels in DCM compared with control hearts, but no differences in N2B, all-N2BA, and individual-N2BA transcripts. The reduction in total-titin transcripts followed from a decreased area occupied by myocytes and increased connective tissue in DCM hearts, as detected by histological analysis. Force measurements on isolated cardiomyofibrils showed that sarcomeric passive tension was reduced on average by 25% to 30% in DCM, a reduction readily predictable with a model of wormlike-chain titin elasticity. Passive-tension measurements on human-heart fiber bundles, before and after titin proteolysis, revealed a much-reduced relative contribution of titin to total passive stiffness in DCM. Results suggested that the titin-isoform shift in DCM depresses the proportion of titin-based stiffness by approximately 10%. We conclude that a lower-than-normal proportion of titin-based stiffness in end-stage failing hearts results partly from loss of titin and increased fibrosis, partly from titin-isoform shift. The titin-isoform shift may be beneficial for myocardial diastolic function, but could impair the contractile performance in systole.
Assuntos
Cardiomiopatia Dilatada/patologia , Regulação da Expressão Gênica/fisiologia , Proteínas Musculares/fisiologia , Proteínas Quinases/fisiologia , Animais , Fenômenos Biomecânicos , Western Blotting , Cardiomiopatia Dilatada/genética , Cardiomiopatia Dilatada/metabolismo , Conectina , Fibrose , Fatores de Troca do Nucleotídeo Guanina/biossíntese , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/fisiologia , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/patologia , Ventrículos do Coração/química , Ventrículos do Coração/patologia , Humanos , Modelos Biológicos , Peso Molecular , Proteínas Musculares/biossíntese , Proteínas Musculares/química , Proteínas Musculares/genética , Miocárdio/patologia , Miofibrilas/fisiologia , Maleabilidade , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiologia , Proteínas Quinases/biossíntese , Proteínas Quinases/química , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Troca de Nucleotídeo Guanina Rho , Sus scrofaRESUMO
The giant muscle protein titin contains a unique sequence, the PEVK domain, the elastic properties of which contribute to the mechanical behavior of relaxed cardiomyocytes. Here, human N2-B-cardiac PEVK was expressed in Escherichia coli and tested-along with recombinant cardiac titin constructs containing immunoglobulin-like or fibronectin-like domains-for a possible interaction with actin filaments. In the actomyosin in vitro motility assay, only the PEVK construct inhibited actin filament sliding over myosin. The slowdown occurred in a concentration-dependent manner and was accompanied by an increase in the number of stationary actin filaments. High [Ca(2+)] reversed the PEVK effect. PEVK concentrations >/=10 microgram/mL caused actin bundling. Actin-PEVK association was found also in actin fluorescence binding assays without myosin at physiological ionic strength. In cosedimentation assays, PEVK-titin interacted weakly with actin at 0 degrees C, but more strongly at 30 degrees C, suggesting involvement of hydrophobic interactions. To probe the interaction in a more physiological environment, nonactivated cardiac myofibrils were stretched quickly, and force was measured during the subsequent hold period. The observed force decline could be fit with a three-order exponential-decay function, which revealed an initial rapid-decay component (time constant, 4 to 5 ms) making up 30% to 50% of the whole decay amplitude. The rapid, viscous decay component, but not the slower decay components, decreased greatly and immediately on actin extraction with Ca(2+)-independent gelsolin fragment, both at physiological sarcomere lengths and beyond actin-myosin overlap. Steady-state passive force dropped only after longer exposure to gelsolin. We conclude that interaction between PEVK-titin and actin occurs in the sarcomere and may cause viscous drag during diastolic stretch of cardiac myofibrils. The interaction could also oppose shortening during contraction.
Assuntos
Citoesqueleto de Actina/metabolismo , Proteínas Musculares/metabolismo , Miocárdio/metabolismo , Miofibrilas/metabolismo , Proteínas Quinases/metabolismo , Motivos de Aminoácidos/fisiologia , Animais , Ligação Competitiva/fisiologia , Bioensaio , Galinhas , Conectina , Humanos , Técnicas In Vitro , Substâncias Macromoleculares , Proteínas Musculares/genética , Contração Miocárdica/fisiologia , Ligação Proteica/fisiologia , Proteínas Quinases/genética , Estrutura Terciária de Proteína/fisiologia , Coelhos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sarcômeros/fisiologia , Estresse Mecânico , Temperatura , ViscosidadeRESUMO
Vertebrate striated muscle behaves elastically when stretched and this property is thought to reside primarily within the giant filamentous protein, titin (connectin). The elastic portion of titin comprises two distinct structural motifs, immunoglobulin (Ig) domains and the PEVK titin, which is a novel motif family rich in proline, glutamate, valine and lysine residues. The respective contributions of the titin Ig and the PEVK sequences to the elastic properties of the molecule have been unknown so far. We have measured both the passive tension in single, isolated myofibrils from cardiac and skeletal muscle and the stretch-induced translational movement of I-band titin antibody epitopes following immunofluorescent labelling of sites adjacent to the PEVK and Ig domain regions. We found that with myofibril stretch, I-band titin does not extend homogeneously. The Ig domain region lengthened predominantly during small stretch, but such lengthening did not result in measurable passive tension and might be explained by straightening, rather than by unfolding, of the Ig repeats. At moderate to extreme stretch, the main extensible region was found to be the PEVK segment whose unravelling was correlated with a steady passive tension increase. In turn, PEVK domain transition from a linearly extended to a folded state appears to be principally responsible for the elasticity of muscle fibers. Thus, the length of the PEVK sequence may determine the tissue-specificity of muscle stiffness, whereas the expression of different Ig domain motif lengths may set the characteristic slack sarcomere length of a muscle type.
Assuntos
Proteínas Musculares/química , Músculo Esquelético/química , Miofibrilas/química , Proteínas Quinases/química , Sequência de Aminoácidos , Animais , Anticorpos/imunologia , Conectina , Elasticidade , Epitopos/imunologia , Imunofluorescência , Imunoglobulinas , Microscopia de Fluorescência , Microscopia de Contraste de Fase , Dados de Sequência Molecular , Contração Muscular , Proteínas Musculares/imunologia , Proteínas Musculares/fisiologia , Músculo Esquelético/fisiologia , Miocárdio/química , Miofibrilas/fisiologia , Conformação Proteica , Proteínas Quinases/imunologia , Proteínas Quinases/fisiologia , Ratos , Sarcômeros/metabolismoRESUMO
Titins are giant filamentous proteins which connect Z-discs and M-lines in the sarcomeres of vertebrate striated muscles. Comparison of the N-terminal region of titin (Z-disc region) from different skeletal and cardiac muscles reveals a 900-residue segment which is expressed in different length variants, dependent on tissue type. When searching for ligands of this differentially expressed domain by a yeast-two hybrid approach, we detected binding to alpha-actinin. The isolated alpha-actinin cDNAs were derived from the C-terminal region of the alpha-actinin isoform (alpha-actinin-2) encoded by the ACTN2 gene. Therefore, the two antiparallel subunits of an alpha-actinin-2 homodimer will attach to actin at their respective C termini, whereas they will bind to the Z-disc titin at their N termini. This may thus explain how alpha-actinins can cross-link antiparallel titin and thin filaments from opposing sarcomeres. The alpha-actinin-2 binding site of the Z-disc titin is located within a sequence of 45-residue repeats, referred to as Z-repeat region. Both the N-terminal and C-terminal Z-repeats have alpha-actinin binding properties and are expressed in all striated muscles. By contrast, the more central Z-repeats are expressed in slow and fast skeletal muscles, as well as embryonic and adult cardiac muscles, in different copy numbers. Such alternative splicing of the Z-disc titin appears to be important for the tissue and fibre type diversity of the Z-disc lattice.
Assuntos
Actinina/metabolismo , Expressão Gênica , Proteínas Musculares/genética , Músculo Esquelético/fisiologia , Proteínas Quinases/genética , Sequência de Aminoácidos , Animais , Conectina , Humanos , Dados de Sequência Molecular , Proteínas Musculares/metabolismo , Proteínas Quinases/metabolismo , Coelhos , Sequências Repetitivas de Ácido Nucleico , Homologia de Sequência de Aminoácidos , VertebradosRESUMO
Myasthenia gravis (MG) patients develop autoantibodies primarily against the acetylcholine receptor in the motor endplate, but also against intracellular striated muscle proteins, notably titin, the giant elastic protein of the myofibrillar cytoskeleton. Titin antibodies have previously been shown to be directed against a single epitope on the molecule, located at the A-band/I-band junction and referred to as the main immunogenic region (MIR) of titin. By using immunofluorescence microscopy on stretched single myofibrils, we now report that approximately 40% of the sera from 18 MG/thymoma patients and 8 late-onset MG patients with thymus atrophy contain antibodies that bind to a more central I-band titin region. This region consists of homologous immunoglobulin domains and is known to be differentially spliced dependent on muscle type. All patients with I-band titin antibodies also had antibodies against the MIR. Although a statistically significant correlation between the occurrence of I-band titin antibodies and MG severity was not apparent, the results could hint at an initial immunoreactivity to titin's MIR, followed by reactivity along the titin molecule in the course of the disease.
Assuntos
Autoanticorpos/imunologia , Doenças Autoimunes/imunologia , Proteínas do Citoesqueleto/imunologia , Epitopos/imunologia , Proteínas Musculares/imunologia , Miastenia Gravis/imunologia , Proteínas Quinases/imunologia , Adulto , Idoso , Doenças Autoimunes/etiologia , Conectina , Feminino , Humanos , Masculino , Microscopia de Fluorescência , Microscopia Imunoeletrônica , Pessoa de Meia-Idade , Miastenia Gravis/etiologia , Reação em Cadeia da Polimerase , Receptores Colinérgicos/imunologia , Índice de Gravidade de Doença , Timoma/complicações , Timoma/imunologia , Timo/patologia , Neoplasias do Timo/complicações , Neoplasias do Timo/imunologiaRESUMO
Titin, the giant protein of striated muscle, provides a continuous link between the Z-disk and the M-line of a sarcomere. The elastic I-band section of titin comprises two main structural elements, stretches of immunoglobulin-like domains and a unique sequence, the PEVK segment. Both elements contribute to the extensibility and passive force development of nonactivated muscle. Extensibility of the titin segments in skeletal muscle has been determined by immunofluorescence/immunoelectron microscopy of sarcomeres stained with sequence-assigned titin antibodies. The force developed upon stretch of titin has been measured on isolated molecules or recombinant titin fragments with the help of optical tweezers and the atomic force microscope. Force has also been measured in single isolated myofibrils. The force-extension relation of titin could be readily fitted with models of biopolymer elasticity. For physiologically relevant extensions, the elasticity of the titin segments was largely explainable by an entropic-spring mechanism. The modelling explains why during stretch of titin, the Ig-domain regions (with folded modules) extend before the PEVK domain. In cardiac muscle, I-band titin is expressed in different isoforms, termed N2-A and N2-B. The N2-A isoform resembles that of skeletal muscle, whereas N2-B titin is shorter and is distinguished by cardiac-specific Ig-motifs and nonmodular sequences within the central I-band section. Examination of N2-B titin extensibility revealed that this isoform extends by recruiting three distinct elastic elements: poly-Ig regions and the PEVK domain at lower stretch and, in addition, a unique 572-residue sequence insertion at higher physiological stretch. Extension of all three elements allows cardiac titin to stretch fully reversibly at physiological sarcomere lengths, without the need to unfold individual Ig domains. However, unfolding of a very small number of Ig domains remains a possibility.
Assuntos
Proteínas Musculares/fisiologia , Músculo Esquelético/fisiologia , Proteínas Quinases/fisiologia , Animais , Conectina , Elasticidade , Humanos , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Proteínas Quinases/metabolismo , VertebradosRESUMO
Impaired cellular coupling is thought to be a very important factor for the genesis of cardiac arrhythmia. Cellular coupling is mediated by gap junctions. However, there are no therapeutic agents or experimental substances yet that increase cellular coupling. In addition, it has been shown that most antiarrhythmic drugs available now possess serious adverse effects. Thus, there is an urgent need for new antiarrhythmic agents. Previous studies using epicardial mapping in isolated rabbit hearts provided indirect evidence supporting the hypothesis that a newly synthesised antiarrhythmic peptide (Gly-Ala-Gly-4Hyp-Pro-Tyr-CONH2 = AAP10) might act via an increase in cellular, i.e., gap junctional coupling. The aim of the present study was to test this hypothesis. Measurement of the stimulus-response interval in papillary muscle showed a decrease of about 10% after application of 1 microM AAP10. These results are compatible with the hypothesis of AAP10 acting on gap junctions. In order to prove this hypothesis, gap junction conductance was measured directly by performing double-cell voltage-clamp experiments in isolated pairs of guinea-pig myocytes. During a 10 min control period gap junction conductance slowly decreased with a rate of -2.5 +/- 2.0 nS/min. After application of 10 nM AAP10 this behaviour reversed and gap junction conductance now increased with +1.0 +/- 0.7 nS/min. Upon washout of AAP10 gap junction conductance again decreased with a rate similar to that under control conditions. Another important finding was that we could not detect any other actions of AAP10 on cardiac myocytes. All parameters of the transmembrane action potential remained unchanged and, similarly, no changes in the IV relationship of single cardiac myocytes treated with 10 nM AAP10 could be observed. We conclude that AAP10 increases gap junction conductance, i.e., cellular coupling in the heart. This finding might be the first step towards the development of a new class of antiarrhythmic agents.
Assuntos
Antiarrítmicos/farmacologia , Junções Comunicantes/efeitos dos fármacos , Oligopeptídeos/farmacologia , Animais , Junções Comunicantes/fisiologia , Cobaias , Técnicas In Vitro , MasculinoRESUMO
Disturbances in gap junction distribution and a decrease in the connexin43 content of the heart were shown to occur after myocardial infarction and in ischemic heart disease, respectively. These changes are now thought to play an important role in the genesis of arrhythmias associated with these diseases. It is thought that agents that can increase cellular coupling might be beneficial in these situations. Recently, we presented data showing that the synthetic peptide AAP10 acts antiarrhythmically in a model of regional ischemia. The data suggested that AAP10 might act via an increase in cellular coupling. The goal of this study was to establish whether AAP10 can interact with cardiac gap junctions. Measurements of the stimulus-response-interval (SRI) in guinea pig papillary muscle showed that high concentrations of AAP10 (1 microM) can decrease the SRI by about 10% under normoxic conditions. At lower concentrations (10 nM) AAP10 had no effect on SRI under normoxic conditions but prevented the increase in the SRI induced by perfusion with hypoxic, glucose-free Tyrode's solution. Double-cell voltage-clamp experiments confirmed that AAP10 can interact with cardiac gap junctions. 10 nM AAP10 could either diminish or reverse the run-down of gap junction conductance normally observed in pairs of guinea pig ventricular myocytes. During control gap junction conductance decreased with a rate of -2.5 +/- 2.0 nS/min. After application of 10 nM AAP10 gap junction conductance increased with a rate of +1.0 +/- 0.7 nS/min (p < 0.01). After washout of AAP10 gap junction conductance decreased again with a rate not significantly different from control. Our results show that AAP10 does interact with gap junctions. Because no other effects of AAP10 on other electrophysiological parameters could be found, this action on gap junctions might be the basis of AAP10's antiarrhythmic effect seen in previous studies.
Assuntos
Antiarrítmicos/farmacologia , Junções Comunicantes/efeitos dos fármacos , Oligopeptídeos/farmacologia , Potenciais de Ação/efeitos dos fármacos , Animais , Cobaias , Técnicas In Vitro , Masculino , Isquemia Miocárdica/fisiopatologia , Músculos Papilares/efeitos dos fármacos , Técnicas de Patch-Clamp , Tempo de Reação/efeitos dos fármacosRESUMO
Skeletal-muscle titin contains in its I-band section two main elastic elements, stretches of Ig-like domains and the PEVK segment. Both elements contribute to the extensibility and passive force development of relaxed skeletal muscle fibers during stretch. To explore the nature of elasticity of the segments, their force-extension relation was determined with immunofluorescence and immunoelectron microscopy, combined with isolated myofibril mechanics. The results were then fitted with recent models of biopolymer elasticity. Whereas an entropic-spring mechanism may account for the elasticity of the Ig-domain segments, PEVK-titin elasticity appears to have both entropic and enthalpic origins. The modeling explains why the two elements extend sequentially upon stretch: elongation of the Ig-domain regions (with folded modules) is followed by unraveling of the PEVK domain. I-band titin in cardiac muscle is expressed in two main isoforms, N2-A and N2-B. The N2-A isoform is similar to that found in skeletal muscle, whereas the N2-B titin is distinguished by cardiac-specific Ig-motifs and nonmodular sequences within the central I-band section. By examining the extensibility of N2-B titin, it was found that this isoform extends by recruiting three distinct elastic elements: poly-Ig regions and the PEVK domain at low to modest stretch, and in addition, a unique 572-residue sequence insertion at higher physiological stretch. Extension of all three elements allows cardiac titin to stretch fully reversibly at physiological sarcomere lengths, without the need to unfold individual Ig domains.
Assuntos
Proteínas Musculares/química , Proteínas Musculares/fisiologia , Músculo Esquelético/fisiologia , Miofibrilas/fisiologia , Proteínas Quinases/química , Proteínas Quinases/fisiologia , Sarcômeros/fisiologia , Animais , Conectina , Elasticidade , Coração/fisiologia , Músculo Esquelético/ultraestrutura , Miocárdio/ultraestrutura , Isoformas de Proteínas/química , Isoformas de Proteínas/fisiologia , TermodinâmicaRESUMO
"The first part of the...paper [compares]....mortality trends of various age groups in twelve selected industrialized countries..., covering the period from the beginning of the 50s until 1980/81.... Essential changes regarding the age-group specific mortality trends will be [outlined]....An estimation of trends of age-group specific mortality rates in the selected countries will be presented in the second part of this paper.... The trend and the levels of the estimated mortality rates as well as the comparison between the selected countries will be described." Rates are presented separately for males and females.
Assuntos
Fatores Etários , Países Desenvolvidos , Mortalidade , Dinâmica Populacional , Projetos de Pesquisa , Fatores Sexuais , Estatística como Assunto , Demografia , População , Características da População , PesquisaRESUMO
PIP: Mathematical equations are used to analyze changes in fertility for West Gertmany during the years 1964 to 1981. Tables depict the changes according to various demographic indexes (Monaks-Wert, Saisonindex, Monatlicher Wochentags-Indes). Monthly changes are charted for the years 1979-1981. The statistical methods used to derive the indexes are explained.^ieng