RESUMO
BACKGROUND: Cation channels play an essential role in red blood cells (RBCs) ion homeostasis. One set of ion channels are the transient receptor potential channels of canonical type (TRPC channels). The abundance of these channels in primary erythroblasts, erythroid cell lines and RBCs was associated with an increase in intracellular Ca2+ upon stimulation with Erythropoietin (Epo). In contrast two independent studies on Epo-treated patients revealed diminished basal Ca2+ concentration or reduced phosphatidylserine exposure to the outer membrane leaflet. METHODS: To resolve the seemingly conflicting reports we challenged mature human and mouse RBCs of several genotypes with Epo and Prostaglandin E2 (PGE2) and recorded the intracellular Ca2+ content. Next Generation Sequencing was utilised to approach a molecular analysis of reticulocytes. RESULTS/CONCLUSIONS: Our results allow concluding that Epo and PGE2 regulation of the Ca2+ homeostasis is distinctly different between murine and human RBCs and that changes in intracellular Ca2+ upon Epo treatment is a primary rather than a compensatory effect. In human RBCs, Epo itself has no effect on Ca2+ fluxes but inhibits the PGE2-induced Ca2+ entry. In murine mature RBCs functional evidence indicates TRPC4/C5 mediated Ca2+ entry activated by Epo whereas PGE2 leads to a TRPC independent Ca2+ entry.
Assuntos
Cálcio/metabolismo , Dinoprostona/farmacologia , Eritrócitos/efeitos dos fármacos , Eritropoetina/farmacologia , Canais de Cátion TRPC/metabolismo , Animais , Cátions Bivalentes , Eritrócitos/citologia , Eritrócitos/metabolismo , Expressão Gênica , Humanos , Transporte de Íons/efeitos dos fármacos , Camundongos , Cultura Primária de Células , Especificidade da Espécie , Canais de Cátion TRPC/genéticaRESUMO
Genetically encoded Ca(2+) indicators constitute a powerful set of tools to investigate functional aspects of Ca(2+) signaling in isolated cardiomyocytes, cardiac tissue, and whole hearts. Here, we provide an overview of the concepts, experiences, state of the art, and ongoing developments in the use of genetically encoded Ca(2+) indicators for cardiac cells and heart tissue. This review is supplemented with in vivo viral gene transfer experiments and comparisons of available genetically encoded Ca(2+) indicators with each other and with the small molecule dye Fura-2. In the context of cardiac myocytes, we provide guidelines for selecting a genetically encoded Ca(2+) indicator. For future developments, we discuss improvements of a broad range of properties, including photophysical properties such as spectral spread and biocompatibility, as well as cellular and in vivo applications.
Assuntos
Sinalização do Cálcio/genética , Corantes Fluorescentes , Miócitos Cardíacos/química , Miócitos Cardíacos/fisiologia , Transgenes , Animais , Diagnóstico por Imagem/métodos , Técnicas de Transferência de Genes , Humanos , Miócitos Cardíacos/metabolismoRESUMO
Membrane potentials display the cellular status of non-excitable cells and mediate communication between excitable cells via action potentials. The use of genetically encoded biosensors employing fluorescent proteins allows a non-invasive biocompatible way to read out the membrane potential in cardiac myocytes and other cells of the circulation system. Although the approaches to design such biosensors date back to the time when the first fluorescent-protein based Förster Resonance Energy Transfer (FRET) sensors were constructed, it took 15 years before reliable sensors became readily available. Here, we review different developments of genetically encoded membrane potential sensors. Furthermore, it is shown how such sensors can be used in pharmacological screening applications as well as in circulation related basic biomedical research. Potentials and limitations will be discussed and perspectives of possible future developments will be provided.
Assuntos
Técnicas Biossensoriais , Potenciais da Membrana/genética , Miócitos Cardíacos/metabolismo , Potenciais de Ação/genética , Animais , Animais Geneticamente Modificados , Sistema Cardiovascular/metabolismo , Transferência Ressonante de Energia de Fluorescência , Expressão Gênica , Genes Reporter , Humanos , Proteínas Recombinantes de Fusão/genética , Pesquisa , Imagens com Corantes Sensíveis à VoltagemRESUMO
Cardiac excitation-contraction coupling (EC coupling) links the electrical excitation of the cell membrane to the mechanical contractile machinery of the heart. Calcium channels are major players of EC coupling and are regulated by voltage and Ca(2+)/calmodulin (CaM). CaM binds to the IQ motif located in the C terminus of the Ca(v)1.2 channel and induces Ca(2+)-dependent inactivation (CDI) and facilitation (CDF). Mutation of Ile to Glu (Ile1624Glu) in the IQ motif abolished regulation of the channel by CDI and CDF. Here, we addressed the physiological consequences of such a mutation in the heart. Murine hearts expressing the Ca(v)1.2(I1624E) mutation were generated in adult heterozygous mice through inactivation of the floxed WT Ca(v)1.2(L2) allele by tamoxifen-induced cardiac-specific activation of the MerCreMer Cre recombinase. Within 10 days after the first tamoxifen injection these mice developed dilated cardiomyopathy (DCM) accompanied by apoptosis of cardiac myocytes (CM) and fibrosis. In Ca(v)1.2(I1624E) hearts, the activity of phospho-CaM kinase II and phospho-MAPK was increased. CMs expressed reduced levels of Ca(v)1.2(I1624E) channel protein and I(Ca). The Ca(v)1.2(I1624E) channel showed "CDI" kinetics. Despite a lower sarcoplasmic reticulum Ca(2+) content, cellular contractility and global Ca(2+) transients remained unchanged because the EC coupling gain was up-regulated by an increased neuroendocrine activity. Treatment of mice with metoprolol and captopril reduced DCM in Ca(v)1.2(I1624E) hearts at day 10. We conclude that mutation of the IQ motif to IE leads to dilated cardiomyopathy and death.
Assuntos
Canais de Cálcio Tipo L/genética , Calmodulina/metabolismo , Cardiomiopatia Dilatada/genética , Cardiomiopatia Dilatada/mortalidade , Motivos de Aminoácidos/genética , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Animais , Antiarrítmicos/farmacologia , Sítios de Ligação/genética , Cálcio/metabolismo , Canais de Cálcio Tipo L/química , Canais de Cálcio Tipo L/metabolismo , Captopril/farmacologia , Cardiomiopatia Dilatada/tratamento farmacológico , Células Cultivadas , Insuficiência Cardíaca/tratamento farmacológico , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/mortalidade , Metoprolol/farmacologia , Camundongos , Camundongos Mutantes , Contração Miocárdica/fisiologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/fisiologia , Estrutura Terciária de Proteína/genética , Taxa de SobrevidaRESUMO
BACKGROUND: The recent discovery of a new myokine (irisin) potentially involved in health-related training effects has gained great attention, but evidence for a training-induced increase in irisin remains preliminary. Therefore, the present study aimed to determine whether irisin concentration is increased after regular exercise training in humans. METHODS: In a randomized controlled design, two guideline conforming training interventions were studied. Inclusion criteria were age 30 to 60 years, <1 hour/week regular activity, non-smoker, and absence of major diseases. 102 participants could be included in the analysis. Subjects in the training groups exercised 3 times per week for 26 weeks. The minimum compliance was defined at 70%. Aerobic endurance training (AET) consisted of 45 minutes of walking/running at 60% heart rate reserve. Strength endurance training (SET) consisted of 8 machine-based exercises (2 sets of 15 repetitions with 100% of the 20 repetition maximum). Serum irisin concentrations in frozen serum samples were determined in a single blinded measurement immediately after the end of the training study. Physical performance provided positive control for the overall efficacy of training. Differences between groups were tested for significance using analysis of variance. For post hoc comparisons with the control group, Dunnett's test was used. RESULTS: Maximum performance increased significantly in the training groups compared with controls (controls: ±0.0 ± 0.7 km/h; AET: 1.1 ± 0.6 km/h, P < 0.01; SET: +0.5 ± 0.7 km/h, P = 0.01). Changes in irisin did not differ between groups (controls: 101 ± 81 ng/ml; AET: 44 ± 93 ng/ml; SET: 60 ± 92 ng/ml; in both cases: P = 0.99 (one-tailed testing), 1-ß error probability = 0.7). The general upward trend was mainly accounted for by a negative association of irisin concentration with the storage duration of frozen serum samples (P < 0.01, ß = -0.33). After arithmetically eliminating this confounder, the differences between groups remained non-significant. CONCLUSIONS: A training-induced increase in circulating irisin could not be confirmed, calling into question its proposed involvement in health-related training effects. Because frozen samples are prone to irisin degradation over time, positive results from uncontrolled trials might exclusively reflect the longer storage of samples from initial tests.
Assuntos
Exercício Físico/fisiologia , Fibronectinas/sangue , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Soro/químicaRESUMO
In cardiac myocytes, cytochalasin D (CytoD) was reported to act as an actin disruptor and mechanical uncoupler. Using confocal and super-resolution STED microscopy, we show that CytoD preserves the actin filament architecture of adult rat ventricular myocytes in culture. Five hundred nanomolar CytoD was the optimal concentration to achieve both preservation of the T-tubular structure during culture periods of 3 days and conservation of major functional characteristics such as action potentials, calcium transients and, importantly, the contractile properties of single myocytes. Therefore, we conclude that the addition of CytoD to the culture of adult cardiac myocytes can indeed be used to generate a solid single-cell model that preserves both morphology and function of freshly isolated cells. Moreover, we reveal a putative link between cytoskeletal and T-tubular remodeling. In the absence of CytoD, we observed a loss of T-tubules that led to significant dyssynchronous Ca(2+)-induced Ca(2+) release (CICR), while in the presence of 0.5 µM CytoD, T-tubules and homogeneous CICR were majorly preserved. Such data suggested a possible link between the actin cytoskeleton, T-tubules and synchronous, reliable excitation-contraction-coupling. Thus, T-tubular re-organization in cell culture sheds some additional light onto similar processes found during many cardiac diseases and might link cytoskeletal alterations to changes in subcellular Ca(2+) signaling revealed under such pathophysiological conditions.
Assuntos
Citocalasina D/farmacologia , Miócitos Cardíacos/diagnóstico por imagem , Miócitos Cardíacos/metabolismo , Potenciais de Ação/efeitos dos fármacos , Animais , Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Citocalasina D/metabolismo , Ventrículos do Coração/citologia , Ventrículos do Coração/metabolismo , Masculino , Contração Miocárdica/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Ratos , Ratos Wistar , UltrassonografiaRESUMO
The major L-type voltage-gated calcium channels in heart consist of an α1C (Ca(V)1.2) subunit usually associated with an auxiliary ß subunit (Ca(V)ß2). In embryonic cardiomyocytes, both the complete and the cardiac myocyte-specific null mutant of Ca(V)ß2 resulted in reduction of L-type calcium currents by up to 75%, compromising heart function and causing defective remodeling of intra- and extra-embryonic blood vessels followed by embryonic death. Here we conditionally excised the Ca(V)ß2 gene (cacnb2) specifically in cardiac myocytes of adult mice (KO). Upon gene deletion, Ca(V)ß2 protein expression declined by >96% in isolated cardiac myocytes and by >74% in protein fractions from heart. These latter protein fractions include Ca(V)ß2 proteins expressed in cardiac fibroblasts. Surprisingly, mice did not show any obvious impairment, although cacnb2 excision was not compensated by expression of other Ca(V)ß proteins or changes of Ca(V)1.2 protein levels. Calcium currents were still dihydropyridine-sensitive, but current density at 0 mV was reduced by <29%. The voltage for half-maximal activation was slightly shifted to more depolarized potentials in KO cardiomyocytes when compared with control cells, but the difference was not significant. In summary, Ca(V)ß2 appears to be a much stronger modulator of L-type calcium currents in embryonic than in adult cardiomyocytes. Although essential for embryonic survival, Ca(V)ß2 down-regulation in cardiomyocytes is well tolerated by the adult mice.
Assuntos
Canais de Cálcio Tipo L/biossíntese , Regulação da Expressão Gênica , Proteínas Musculares/biossíntese , Miocárdio/enzimologia , Miócitos Cardíacos/metabolismo , Animais , Canais de Cálcio Tipo L/genética , Embrião de Mamíferos/enzimologia , Embrião de Mamíferos/patologia , Fibroblastos/enzimologia , Fibroblastos/patologia , Camundongos , Camundongos Knockout , Proteínas Musculares/genética , Miocárdio/patologia , Miócitos Cardíacos/patologia , Especificidade de Órgãos/genéticaRESUMO
AIMS: In ventricular myocytes, transverse-tubules (T-tubules) are instrumental for excitation-contraction (EC)coupling and their disarray is a hallmark of cardiac diseases. BIN1 is a key contributor to their biogenesis. Our study set out to investigate the role of human BIN1 splice variants in the maintenance and regeneration of EC-coupling in rat adult ventricular myocytes and human-induced pluripotent stem cell-derived cardiac myocytes (hiPS-CMs). METHODS AND RESULTS: In heart samples from healthy human donors expression patterns of five BIN1 splice variants were identified. Following viral transduction of human BIN1 splice variants in cellular models of T-tubular disarray, we employed high-speed confocal calcium imaging and CaCLEAN analysis to identify functional EC-coupling sites (couplons) and T-tubular architecture. Adult rat ventricular myocytes were used to investigate the regeneration after loss and maintenance of EC-coupling while we studied the enhancement of EC-coupling in hiPS-CMs. All five human BIN1 splice variants induced de-novo generation of T-tubules in both cell types. Isoforms with the phosphoinositide-binding motif (PI) were most potent in maintenance and regeneration of T-tubules and functional EC-coupling in adult rat myocytes. In hiPSC-CMs, BIN1 variants with PI-motif-induced de novo generation of T-tubules, functional couplons and enhanced calcium handling. CONCLUSION: BIN1 is essential for the maintenance, regeneration, and de novo generation of functional T-tubules. Isoforms with PI-motifs appeared as particulalrly potent. These T-tubules trigger the development of functional couplons resulting in enhanced calcium handling.
Assuntos
Células-Tronco Pluripotentes Induzidas , Miócitos Cardíacos , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Miócitos Cardíacos/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Isoformas de Proteínas/metabolismo , Ratos , Regeneração , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
BACKGROUND/AIMS: QT-interval screens are increasingly important for cardiac safety on all new medications. So far, investigations rely on animal experiments or cell-based screens solely probing for conductance alterations in heterologously expressed hERG-channels in cell lines allowing for a high degree of automation. Adult cardiomyocytes can not be handled by automated patch-clamp setups. Therefore optical screening of primary isolated ventricular myocytes is regarded as an alternative. Several optical voltage sensors have been reported for ratiometric measurements, but they all influenced the naïve action potential. The aim of the present study was to explore the recording conditions and define settings that allow optical QT-interval screens. METHODS: Based on an improved optical design, individual action potentials could be recorded with an exceptional signal-to-noise-ratio. The sensors were validated using the patch-clamp technique, confocal microscopy and fluorescence lifetime imaging in combination with global unmixing procedures. RESULTS: We show that the small molecule dye di-8-ANEPPS and the novel genetically encoded sensor Mermaid provide quantitative action potential information. When applying such sensors we identified distinctly different pharmacological profiles of action potentials for adult and neonatal rat cardiomyocytes. CONCLUSION: Optical methods can be used for QT-interval investigations based on cellular action potentials using either the small molecule dye di-8-ANEPPS or the genetically encoded sensor Mermaid. Adult cardiomyocytes are superior to neonatal cardiomyocytes for such pharmacological investigations. Optical QT-screens may replace intricate animal experiments.
Assuntos
Potenciais de Ação/fisiologia , Miócitos Cardíacos/fisiologia , Animais , Animais Recém-Nascidos , Eletrocardiografia , Corantes Fluorescentes/química , Corantes Fluorescentes/farmacologia , Masculino , Microscopia Confocal , Técnicas de Patch-Clamp , Compostos de Piridínio/química , Compostos de Piridínio/farmacologia , Ratos , Ratos WistarRESUMO
(1) Background: It is known that sickle cells contain a higher amount of Ca2+ compared to healthy red blood cells (RBCs). The increased Ca2+ is associated with the most severe symptom of sickle cell disease (SCD), the vaso-occlusive crisis (VOC). The Ca2+ entry pathway received the name of Psickle but its molecular identity remains only partly resolved. We aimed to map the involved Ca2+ signaling to provide putative pharmacological targets for treatment. (2) Methods: The main technique applied was Ca2+ imaging of RBCs from healthy donors, SCD patients and a number of transgenic mouse models in comparison to wild-type mice. Life-cell Ca2+ imaging was applied to monitor responses to pharmacological targeting of the elements of signaling cascades. Infection as a trigger of VOC was imitated by stimulation of RBCs with lysophosphatidic acid (LPA). These measurements were complemented with biochemical assays. (3) Results: Ca2+ entry into SCD RBCs in response to LPA stimulation exceeded that of healthy donors. LPA receptor 4 levels were increased in SCD RBCs. Their activation was followed by the activation of Gi protein, which in turn triggered opening of TRPC6 and CaV2.1 channels via a protein kinase Cα and a MAP kinase pathway, respectively. (4) Conclusions: We found a new Ca2+ signaling cascade that is increased in SCD patients and identified new pharmacological targets that might be promising in addressing the most severe symptom of SCD, the VOC.
Assuntos
Anemia Falciforme/sangue , Sinalização do Cálcio , Eritrócitos/metabolismo , Lisofosfolipídeos/metabolismo , Animais , Cálcio/metabolismo , Canais de Cálcio Tipo N/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Células HeLa , Humanos , Sistema de Sinalização das MAP Quinases , Camundongos , Modelos Biológicos , Proteína Quinase C/metabolismo , Canal de Cátion TRPC6/metabolismo , Doadores de TecidosRESUMO
It is well known that for cardiomyocytes, isolation and culturing induce largely unknown remodelling processes. We analysed changes in the structure of cell compartments with optical techniques such as confocal microscopy and fluorescence redistribution after photobleaching employing adenoviral-mediated transduction of targeted fluorescent proteins and small molecule dyes. We identified characteristic remodelling processes: the T-tubular membrane system was gradually lost by a process referred to as "sequential pinching off", in an outward direction. Mitochondria fell in one of three classes, very small (0.9 microm length), medium long (1.8 microm) or extended shape (3.6 microm) organelles. Over the culturing time mitochondria gradually fused. Bleaching of individual mitochondria revealed association between apparently separated mitochondria by "tunnelling" via sub-resolution organelle-tubes. This tunnelling process was increasing over the culturing time. A gradual loss of the cross-striation arrangement in the endoplasmic/sarcoplasmic reticulum was visualised. Analysis of large populations of Ca(2+) sparks by video-rate confocal 2D-scanning revealed significant albeit small changes of these elementary SR-Ca(2+) release events in adult cardiomyocytes that could be related to changes in SR-Ca(2+) content rather than resting Ca(2+) concentration. In conclusion, primary isolated cardiomyocytes from adult hearts undergo a well-defined, but reproducible subcellular remodelling during optimised long term culture.
Assuntos
Cálcio/metabolismo , Ventrículos do Coração/metabolismo , Miócitos Cardíacos/metabolismo , Organelas/metabolismo , Animais , Técnicas de Cultura de Células , Permeabilidade da Membrana Celular , Retículo Endoplasmático/metabolismo , Ventrículos do Coração/citologia , Microscopia Confocal , Mitocôndrias Cardíacas/metabolismo , Miócitos Cardíacos/citologia , RatosRESUMO
Very young red blood cells, namely reticulocytes, can be quite easily recognized and labeled by cluster of differentiation antibodies (CD71, transferrin receptor) or by staining remnant RNA with thiazol orange. In contrast, age specific erythrocyte labeling is more difficult in later periods of their life time. While erythrocytes contain band 4.1 protein, a molecular clock, so far it has not been possible to read this clock on individual cells. One concept to track erythrocytes during their life time is to mark them when they are young, either directly in vivo or ex vivo followed by a transfusion. Several methods like biotinylation, use of isotopes or fluorescent labeling have proved to be useful experimental approaches but also have several inherent disadvantages. Genetic engineering of mice provides additional options to express fluorescent proteins in erythrocytes. To allow co-staining with popular green fluorescent dyes like Fluo-4 or other fluorescein-based dyes, we bred a mouse line expressing a tandem red fluorescent protein (tdRFP). Within this Brief Research Report, we provide the initial characterisation of this mouse line and show application examples ranging from transfusion experiments and intravital microscopy to multicolour flow cytometry and confocal imaging. We provide a versatile new tool for erythrocyte research and discuss a range of experimental opportunities to study membrane processes and other aspects of erythrocyte development and aging with help of these animals.
RESUMO
AIMS: Signalling via Gq-coupled receptors is of profound importance in many cardiac diseases such as hypertrophy and arrhythmia. Nevertheless, owing to their widespread expression and the inability to selectively stimulate such receptors in vivo, their relevance for cardiac function is not well understood. We here use DREADD technology to understand the role of Gq-coupled signalling in vivo in cardiac function. METHODS AND RESULTS: We generated a novel transgenic mouse line that expresses a Gq-coupled DREADD (Dq) in striated muscle under the control of the muscle creatine kinase promotor. In vivo injection of the DREADD agonist clozapine-N-oxide (CNO) resulted in a dose-dependent, rapid mortality of the animals. In vivo electrocardiogram data revealed severe cardiac arrhythmias including lack of P waves, atrioventricular block, and ventricular tachycardia. Following Dq activation, electrophysiological malfunction of the heart could be recapitulated in the isolated heart ex vivo. Individual ventricular and atrial myocytes displayed a positive inotropic response and arrhythmogenic events in the absence of altered action potentials. Ventricular tissue sections revealed a strong co-localization of Dq with the principal cardiac connexin CX43. Western blot analysis with phosphor-specific antibodies revealed strong phosphorylation of a PKC-dependent CX43 phosphorylation site following CNO application in vivo. CONCLUSION: Activation of Gq-coupled signalling has a major impact on impulse generation, impulse propagation, and coordinated impulse delivery in the heart. Thus, Gq-coupled signalling does not only modulate the myocytes' Ca2+ handling but also directly alters the heart's electrophysiological properties such as intercellular communication. This study greatly advances our understanding of the plethora of modulatory influences of Gq signalling on the heart in vivo.
Assuntos
Potenciais de Ação , Arritmias Cardíacas/metabolismo , Sinalização do Cálcio , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Frequência Cardíaca , Miocárdio/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animais , Arritmias Cardíacas/genética , Arritmias Cardíacas/fisiopatologia , Clozapina/análogos & derivados , Clozapina/farmacologia , Conexina 43/metabolismo , Creatina Quinase Forma MM/genética , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Preparação de Coração Isolado , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fosforilação , Regiões Promotoras Genéticas , Proteína Quinase C/metabolismo , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/genéticaRESUMO
For studying heart pathologies on the cellular level, cultured adult cardiac myocytes represent an important approach. We aimed to explore a novel adult rat ventricular myocyte culture system with minimised dedifferentiation allowing extended experimental manipulation of the cells such as expression of exogenous proteins. Various culture conditions were investigated including medium supplement, substrate coating and electrical pacing for one week. Adult myocytes were probed for (i) viability, (ii) morphology, (iii) frequency dependence of contractions, (iv) Ca(2+) transients, and (v) their tolerance towards adenovirus-mediated expression of the Ca(2+) sensor "inverse pericam". Conventionally, in either serum supplemented or serum-free medium, myocytes dedifferentiated into flat cells within 3 days or cell physiology and morphology were impaired, respectively. In contrast, myocytes cultured in medium supplemented with an insulin-transferrin-selenite mixture on substrates coated with extracellular matrix proteins showed an increased cell attachment and a conserved cross-striation. Moreover, these myocytes displayed optimised preservation of their contractile behaviour and Ca(2+) signalling even under conditions of continuous electrical pacing. Sustained expression of inverse pericam did not alter myocyte function and allowed long lasting high speed Ca(2+) imaging of electrically driven adult myocytes. Our single-cell model thus provides a new advance for high-content screening of these highly specialised cells.
Assuntos
Cálcio/análise , Técnicas de Cultura de Células , Corantes Fluorescentes/análise , Miócitos Cardíacos/citologia , Animais , Sinalização do Cálcio , Sobrevivência Celular , Células Cultivadas , Estimulação Elétrica , Corantes Fluorescentes/metabolismo , Ventrículos do Coração/citologia , Proteínas Luminescentes/análise , Proteínas Luminescentes/metabolismo , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/ultraestrutura , RatosRESUMO
A gain-of-function mutation in the Ca2+-activated transient receptor potential melastatin member 4 (TRPM4A432T) is linked to life-threatening cardiac conduction disturbance, but the underlying mechanism is unclear. For deeper insights, we used photolysis of caged Ca2+, quantitative Ca2+, and electrophysiological measurements. TRPM4A432T's 2-fold larger membrane current was associated with 50% decreased plasma membrane expression. Kinetic analysis unveiled 4-fold slower deactivation that was responsible for the augmented membrane current progressively rising during repetitive human cardiac action potentials. Rational mutagenesis of TRPM4 at position 432 revealed that the bulkiness of the amino acid was key to TRPM4A432T's aberrant gating. Charged amino acids rendered the channel non-functional. The slow deactivation caused by an amino acid substitution at position 432 from alanine to the bulkier threonine represents a key contributor to the gain of function in TRPM4A432T. Thus, our results add a mechanism in the etiology of TRP channel-linked human cardiac channelopathies.
Assuntos
Mutação com Ganho de Função/genética , Estudos de Associação Genética , Sistema de Condução Cardíaco/metabolismo , Sistema de Condução Cardíaco/patologia , Canais de Cátion TRPM/genética , Potenciais de Ação , Aminoácidos/química , Cálcio/metabolismo , Membrana Celular/metabolismo , Glicosilação , Células HEK293 , Humanos , Ativação do Canal Iônico , Cinética , Modelos Moleculares , Mutação/genética , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosforilação , Domínios Proteicos , Proteína Quinase C/metabolismo , Canais de Cátion TRPM/sangue , Canais de Cátion TRPM/químicaRESUMO
The eukaryotic cell cycle is regulated by a network of different protein kinases and phosphatases which are by various mechanisms linked to the growth suppressor p53. Cell cycle regulation is quite similar from yeast to man. Although there is no endogenous p53 in yeast expression of human p53 led to growth arrest of yeast cells which can be suppressed by simultaneous overexpression of cdc25C, a phosphatase regulating entry into mitosis. Herein, we show that overexpression of cdc25C in mammalian cells is insufficient in suppressing a p53 induced growth arrest. We further show that p53 is co-immunoprecipitated with cdc25C and p53 inhibits the cdc25C phosphatase activity in a dose-dependent manner. Thus, our data show that p53 like other binding partners of cdc25C, regulates entry into mitosis by binding to cdc25C.
Assuntos
Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Regulação Neoplásica da Expressão Gênica , Genes p53 , Proteína Supressora de Tumor p53/metabolismo , Fosfatases cdc25/genética , Fosfatases cdc25/metabolismo , Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Relação Dose-Resposta a Droga , Humanos , Imunoprecipitação , Mitose , Modelos Biológicos , Monoéster Fosfórico Hidrolases/metabolismo , Schizosaccharomyces/metabolismoRESUMO
The precise role of hormones binding to Gαq protein-coupled receptors (H-GαqPCRs) in chronic heart diseases remains poorly understood. To address this, we used a model of cultured adult rat ventricular myocytes stimulated with endothelin-1 (ET-1) or phenylephrine (PE) over a period of 8 days in vitro (DIV). Chronically treated cells showed an increased number of arrhythmogenic Ca(2+) transients when electrically paced at 0.5 Hz. While their post-rest behaviour was preserved, from DIV6 onwards the amplitude of caffeine-evoked Ca(2+) transients was increased in hormone-treated cells, suggesting an elevated sarcoplasmic reticulum Ca(2+) load. The duration of electrically evoked global Ca(2+) transients gradually increased over the culturing time indicating decreased activity of processes removing cytosolic Ca(2+). In treated cells, spontaneous Ca(2+) sparks displayed smaller amplitudes from DIV6 onwards, and a slower decay period for PE (from DIV3) and for ET-1 (from DIV6). This cellular functional remodelling was associated with changes in gene expression: chronic ET-1 treatment decreased PKCγ transcripts, whereas PE increased PKCγ and SERCA2a transcripts as probed by qPCR. Western blot analysis confirmed the upregulation of PKCγ with PE. To study ET-1 receptor desensitization in vivo, osmotic minipumps containing either NaCl or ET-1 were implanted in mice and Ca(2+) signalling was studied in acutely isolated ventricular myocytes after 2 weeks of chronic treatment. Interestingly, while cellular responses to isoproterenol stimulation were preserved in ET-1 treated animals, the inotropic response of myocytes to ET-1 stimulation was abrogated. We therefore conclude that chronic stimulation of cardiac myocytes by H-GαqPCRs induces cellular remodelling of Ca(2+) cycling with altered PKCγ expression and promotion of arrhythmogenic cellular responses.
Assuntos
Cálcio/metabolismo , Endotelina-1/administração & dosagem , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Animais , Células Cultivadas , Bombas de Infusão Implantáveis , Masculino , Ratos , Ratos WistarRESUMO
BACKGROUND: Although both Gαq- and Gα11-protein signaling are believed to be involved in the regulation of cardiac hypertrophy, their detailed contribution to myocardial function remains elusive. METHODS AND RESULTS: We studied remodeling processes in healthy transgenic mice with genetically altered Gαq/Gα11-expression, in particular a global Gα11-knockout and a novel inducible cardiac specific Gαq-knockout, as well as a combined double knockout (dKO) mouse line. Echocardiography and telemetric ECG recordings revealed that compared with wild type mice, hearts of dKO mice showed an increased ejection fraction and a decreased heart rate, irrespective of age resulting in a maintained cardiac output. We attributed these findings to the lack of Gα11, which the absence was associated with a decreased afterload. Histological analysis of the extracellular matrix in the heart depicted a diminished presence of collagen in aging hearts of dKO mice compared to wild-type mice. The results of a transcriptome analysis on isolated ventricular cardiac myocytes revealed alterations of the activity of genes involved in the Gαq/Gα11-dependent regulation of the extracellular matrix, such as the matricellular protein Cyr61. CONCLUSIONS: From our data we conclude that Gαq/Gα11 signaling pathways play a pivotal role in maintaining gene activity patterns. For the heart we revealed their importance in modulating the properties of the extracellular matrix, a mechanism that might be an important contributor and mechanistic basis for the development of pressure-overload induced cardiac hypertrophy.
Assuntos
Cardiomegalia/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/deficiência , Frequência Cardíaca/fisiologia , Remodelação Ventricular/fisiologia , Animais , Cardiomegalia/genética , Cardiomegalia/patologia , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Miócitos Cardíacos/fisiologia , Transdução de Sinais/fisiologia , Volume Sistólico/fisiologiaRESUMO
PURPOSE: The aim of this study was to compare the proliferation and attachment behavior of fibroblasts and epithelial cells on differently structured abutment materials. MATERIALS AND METHODS: Three different surface topographies were prepared on zirconia and titanium alloy specimens and defined as follows: machined (as delivered without further surface modification), smooth (polished), and rough (sandblasted). Energy-dispersive X-ray spectroscopy, topographical analysis, and water contact angle measurements were used to analyze the surface properties. Fibroblasts (HGF1) and epithelial cells (HNEpC) grown on the specimens were investigated 24 hours and 72 hours after seeding and counted using fluorescence imaging. To investigate adhesion, the abundance and arrangement of the focal adhesion protein vinculin were evaluated by immunocytochemistry. RESULTS: Similar surface topographies were created on both materials. Fibroblasts exhibited significant higher proliferation rates on comparable surface topographies of zirconia compared with the titanium alloy. The proliferation of fibroblasts and epithelial cells was optimal on different substrate/topography combinations. Cell spreading was generally higher on polished and machined surfaces than on sandblasted surfaces. Rough surfaces provided favorable properties in terms of cellular adhesion of fibroblasts but not of epithelial cells. CONCLUSIONS: Our data support complex soft tissue cell-substrate interactions: the fibroblast and epithelial cell response is influenced by both the material and surface topography.
Assuntos
Dente Suporte , Implantes Dentários , Células Epiteliais/fisiologia , Fibroblastos/fisiologia , Western Blotting , Adesão Celular , Proliferação de Células , Células Cultivadas , Humanos , Imuno-Histoquímica , Teste de Materiais , Microscopia Eletrônica de Varredura , Espectrometria por Raios X , Propriedades de Superfície , Titânio , ZircônioRESUMO
PURPOSE: This study aimed to investigate the effects of a 6-month preventive resistance training program on resting metabolic rate (RMR) and its associations with fat-free mass (FFM) and the newly described myokine irisin as two potential mechanistic links between exercise training and RMR. METHODS: In a randomized controlled trial, 74 sedentary healthy male and female participants either completed 6 months of high-repetition resistance training 3 d·wk in accordance with the American College of Sports Medicine recommendations (RT: n = 37; 47 ± 7 yr; body mass index, 25.0 ± 3.4 kg·m) or served as controls (CO: n = 37; 50 ± 7 yr; body mass index, 24.2 ± 3.2 kg·m). Strength (one-repetition maximum), RMR (indirect calorimetry), body fat (caliper method), and serum irisin concentration (enzyme-linked immunosorbent assay) were measured before and after 6 months of training. RESULTS: Training led to an increase in strength (one-repetition maximum leg press, 16% ± 7%; P < 0.001). RMR increased in RT (1671 ± 356 vs 1843 ± 385 kcal·d, P < 0.001) but not in CO (1587 ± 285 vs 1602 ± 294 kcal·d, P = 0.97; group-time interaction, P < 0.01). Body weight (RT, -0.5 ± 2.4 kg; CO, 0.1 ± 2.3 kg), body fat percentage (RT, -1.1% ± 2.5%; CO, -0.7% ± 2.9%), and FFM (RT, 0.4 ± 2.1 kg; CO, 0.6 ± 1.9 kg) did not develop differently between groups (group-time interaction: P = 0.29, P = 0.54, and P = 0.59, respectively). Serum irisin concentration increased in CO (70.8 ± 83.4 ng·mL, P < 0.001) but not in RT (22.4 ± 92.6 ng·mL, P = 0.67; group-time interaction, P < 0.01). The change in RMR was not associated with the change in FFM (r = -0.11, P = 0.36) or irisin (r = -0.004, P = 0.97). CONCLUSIONS: Preventive resistance training elicits an increase in RMR. However, in contrast to currently discussed hypotheses, this increase does not seem to be mediated by training-induced changes in FFM or circulating irisin concentration, which casts doubt in the meaning of irisin for human energy balance.