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The epicardium, the mesothelial envelope of the vertebrate heart, is the source of multiple cardiac cell lineages during embryonic development and provides signals that are essential to myocardial growth and repair. Here we generate self-organizing human pluripotent stem cell-derived epicardioids that display retinoic acid-dependent morphological, molecular and functional patterning of the epicardium and myocardium typical of the left ventricular wall. By combining lineage tracing, single-cell transcriptomics and chromatin accessibility profiling, we describe the specification and differentiation process of different cell lineages in epicardioids and draw comparisons to human fetal development at the transcriptional and morphological levels. We then use epicardioids to investigate the functional cross-talk between cardiac cell types, gaining new insights into the role of IGF2/IGF1R and NRP2 signaling in human cardiogenesis. Finally, we show that epicardioids mimic the multicellular pathogenesis of congenital or stress-induced hypertrophy and fibrotic remodeling. As such, epicardioids offer a unique testing ground of epicardial activity in heart development, disease and regeneration.
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Coração , Pericárdio , Humanos , Pericárdio/metabolismo , Miocárdio , Diferenciação Celular/genética , Linhagem da Célula/genética , BiologiaRESUMO
Inter-organ communication is a major hallmark of health and is often orchestrated by hormones released by the anterior pituitary gland. Pituitary gonadotropes secrete follicle-stimulating hormone (FSH) and luteinizing hormone (LH) to regulate gonadal function and control fertility. Whether FSH and LH also act on organs other than the gonads is debated. Here, we find that gonadotrope depletion in adult female mice triggers profound hypogonadism, obesity, glucose intolerance, fatty liver, and bone loss. The absence of sex steroids precipitates these phenotypes, with the notable exception of fatty liver, which results from ovary-independent actions of FSH. We uncover paracrine FSH action on pituitary corticotropes as a mechanism to restrain the production of corticosterone and prevent hepatic steatosis. Our data demonstrate that functional communication of two distinct hormone-secreting cell populations in the pituitary regulates hepatic lipid metabolism.
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Fígado Gorduroso , Metabolismo dos Lipídeos , Camundongos , Feminino , Animais , Hormônio Foliculoestimulante/genética , Hormônio Foliculoestimulante/metabolismo , Hipófise/metabolismo , Hormônio Luteinizante/metabolismo , Fígado Gorduroso/metabolismoRESUMO
Cardiomyocytes (CMs) derived from human induced pluripotent stem cells (hiPSCs) represent an excellent in vitro model in cardiovascular research. Changes in their action potential (AP) dynamics convey information that is essential for disease modeling, drug screening and toxicity evaluation. High-throughput optical AP recordings utilizing intramolecular Förster resonance energy transfer (FRET) of the voltage-sensitive fluorescent protein (VSFP) have emerged as a substitute or complement to the resource-intensive patch clamp technique. Here, we functionally validated our recently generated voltage indicator hiPSC lines stably expressing CAG-promoter-driven VSFP in the AAVS1 safe harbor locus. By combining subtype-specific cardiomyocyte differentiation protocols, we established optical AP recordings in ventricular, atrial, and nodal CMs in 2D monolayers using fluorescence microscopy. Moreover, we achieved high-throughput optical AP measurements in single hiPSC-derived CMs in a 3D context. Overall, this system greatly expands the spectrum of possibilities for high-throughput, non-invasive and long-term AP analyses in cardiovascular research and drug discovery.
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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
Ca2+ sparks are instrumental to understand physiological and pathological Ca2+ signaling in the heart. High-speed two spatially dimensional (2D) confocal imaging (>120â¯Hz) enables acquisition of sparks with high-content information, however, owing to a wide variety of different acquisition modalities the question arises: how much they reflect the "true" Ca2+ spark properties. To address this issue, we compared a fast point and a 2D-array scanner equipped with a range of different detectors. As a quasi-standard biological sample, we employed Ca2+ sparks in permeabilized and intact mouse ventricular myocytes and utilized an unbiased, automatic Ca2+ spark analysis tool, iSpark. Data from the point scanner suffered from low pixel photon fluxes (PPF) concomitant with high Poissonian noise. Images from the 2D-array scanner displayed substantially increased PPF, lower Poissonian noise and almost 3-fold increased sign-to-noise ratios. Noteworthy, data from the 2D scanner suffered from considerable inter-pinhole crosstalk evident for the permeabilized cells. Spark properties, such as frequency, amplitude, decay time and spatial spread were distinctly different for any scanner/detector combination. Our study reveals that the apparent Ca2+ spark properties differ dependent on the particular recording modality and set-up employed, quantitatively.
Assuntos
Sinalização do Cálcio , Imageamento Tridimensional , Microscopia Confocal , Animais , Ventrículos do Coração/citologia , Camundongos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , SemicondutoresRESUMO
AIMS: After summarizing current concepts for the role of TRPC cation channels in cardiac cells and in processes triggered by mechanical stimuli arising e.g. during pressure overload, we analysed the role of TRPC1 and TRPC4 for background Ca2+ entry (BGCE) and for cardiac pressure overload induced transcriptional remodelling. METHODS AND RESULTS: Mn2+-quench analysis in cardiomyocytes from several Trpc-deficient mice revealed that both TRPC1 and TRPC4 are required for BGCE. Electrically-evoked cell shortening of cardiomyocytes from TRPC1/C4-DKO mice was reduced, whereas parameters of cardiac contractility and relaxation assessed in vivo were unaltered. As pathological cardiac remodelling in mice depends on their genetic background, and the development of cardiac remodelling was found to be reduced in TRPC1/C4-DKO mice on a mixed genetic background, we studied TRPC1/C4-DKO mice on a C57BL6/N genetic background. Cardiac hypertrophy was reduced in those mice after chronic isoproterenol infusion (-51.4%) or after one week of transverse aortic constriction (TAC; -73.0%). This last manoeuvre was preceded by changes in the pressure overload induced transcriptional program as analysed by RNA sequencing. Genes encoding specific collagens, the Mef2 target myomaxin and the gene encoding the mechanosensitive channel Piezo2 were up-regulated after TAC in wild type but not in TRPC1/C4-DKO hearts. CONCLUSIONS: Deletion of the TRPC1 and TRPC4 channel proteins protects against development of pathological cardiac hypertrophy independently of the genetic background. To determine if the TRPC1/C4-dependent changes in the pressure overload induced alterations in the transcriptional program causally contribute to cardio-protection needs to be elaborated in future studies.
Assuntos
Cálcio/metabolismo , Miócitos Cardíacos/metabolismo , Canais de Cátion TRPC/metabolismo , Remodelação Ventricular/fisiologia , Animais , Fenômenos Biomecânicos/fisiologia , Sinalização do Cálcio , Cardiomegalia/metabolismo , Regulação da Expressão Gênica , Humanos , Canais Iônicos/genética , Canais Iônicos/metabolismo , Masculino , Camundongos , Camundongos Knockout , Ativação Transcricional/fisiologiaRESUMO
The identification of spatiotemporally restricted Ca2+ signals, Ca2+ sparks, was instrumental for our understanding of cardiac Ca2+ homeostasis. High-speed 2D confocal imaging enables acquisition of such Ca2+ sparks with high-content information but their full appreciation is constrained by the lack of unbiased and easy-to-use analysis tools. We developed a software toolset for unbiased and automatic Ca2+ spark analysis for huge data sets of subcellular Ca2+ signals. iSpark was developed to be scanner and detector independent. In myocytes from hearts subjected to various degrees of hypertrophy we acquired >5.000.000 Ca2+ sparks from 14 mice. The iSpark-enabled analysis of this large Ca2+ spark data set showed that the highly organized distribution of Ca2+ sparks present in healthy cells disarrayed concomitant with the development of aberrant transverse tubules and disease severity. Thus, iSpark represents a versatile and universal tool for analyzing local Ca2+ signaling in healthy as well as diseased, aberrant local Ca2+ signal transduction. The results from the unbiased analysis of large data sets provide a deeper insight into possible mechanisms contributing to the onset and progression of cardiac diseases such as hypertrophy.
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Sinalização do Cálcio , Processamento de Imagem Assistida por Computador , Miócitos Cardíacos/metabolismo , Software , Animais , Camundongos , Microscopia de Fluorescência , Miócitos Cardíacos/citologiaRESUMO
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
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
In beating cardiomyocytes, synchronized localized Ca2+ transients from thousands of active excitation-contraction coupling sites (ECC couplons) comprising plasma and sarcoplasmic reticulum membrane calcium channels are important determinants of the heart's performance. Nevertheless, our knowledge about the properties of ECC couplons is limited by the lack of appropriate experimental and analysis strategies. We designed CaCLEAN to untangle the fundamental characteristics of ECC couplons by combining the astronomer's CLEAN algorithm with known properties of calcium diffusion. CaCLEAN empowers the investigation of fundamental properties of ECC couplons in beating cardiomyocytes without pharmacological interventions. Upon examining individual ECC couplons at the nanoscopic level, we reveal their roles in the negative amplitude-frequency relationship and in ß-adrenergic stimulation, including decreasing and increasing firing reliability, respectively. CaCLEAN combined with 3D confocal imaging of beating cardiomyocytes provides a functional 3D map of active ECC couplons (on average, 17,000 per myocyte). CaCLEAN will further enlighten the ECC-couplon-remodelling processes that underlie cardiac diseases.
Assuntos
Acoplamento Excitação-Contração , Coração/fisiologia , Imageamento Tridimensional/métodos , Algoritmos , Animais , RatosAssuntos
Arritmias Cardíacas/fisiopatologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Miócitos Cardíacos/fisiologia , Imagens com Corantes Sensíveis à Voltagem/métodos , Potenciais de Ação/fisiologia , Antiarrítmicos/farmacologia , Estudos de Casos e Controles , Cisaprida/farmacologia , Avaliação de Medicamentos/métodos , Humanos , Ivabradina/farmacologia , Síndrome do QT Longo/fisiopatologia , Proteínas Luminescentes/metabolismo , Modelos Cardiovasculares , Miócitos Cardíacos/metabolismo , Bloqueadores dos Canais de Potássio/farmacologiaRESUMO
Cardiovascular disease-related deaths frequently arise from arrhythmias, but treatment options are limited due to perilous side effects of commonly used antiarrhythmic drugs. Cardiac rhythmicity strongly depends on cardiomyocyte Ca2+ handling and prevalent cardiac diseases are causally associated with perturbations in intracellular Ca2+ handling. Therefore, intracellular Ca2+ transporters are lead candidate structures for novel and safer antiarrhythmic therapies. Mitochondria and mitochondrial Ca2+ transport proteins are important regulators of cardiac Ca2+ handling. Here we evaluated the potential of pharmacological activation of mitochondrial Ca2+ uptake for the treatment of cardiac arrhythmia. To this aim,we tested substances that enhance mitochondrial Ca2+ uptake for their ability to suppress arrhythmia in a murine model for ryanodine receptor 2 (RyR2)-mediated catecholaminergic polymorphic ventricular tachycardia (CPVT) in vitro and in vivo and in induced pluripotent stem cell-derived cardiomyocytes from a CPVT patient. In freshly isolated cardiomyocytes of RyR2R4496C/WT mice efsevin, a synthetic agonist of the voltage-dependent anion channel 2 (VDAC2) in the outer mitochondrial membrane, prevented the formation of diastolic Ca2+ waves and spontaneous action potentials. The antiarrhythmic effect of efsevin was abolished by blockade of the mitochondrial Ca2+ uniporter (MCU), but could be reproduced using the natural MCU activator kaempferol. Both mitochondrial Ca2+ uptake enhancers (MiCUps), efsevin and kaempferol, significantly reduced episodes of stress-induced ventricular tachycardia in RyR2R4496C/WT mice in vivo and abolished diastolic, arrhythmogenic Ca2+ events in human iPSC-derived cardiomyocytes.
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Patients with hypertension and hyperaldosteronism show an increased risk of stroke compared with patients with essential hypertension. Aim of the study was to assess the effects of aldosterone on left atrial function in rats as a potential contributor to thromboembolism. Osmotic mini-pumps delivering 1.5 µg aldosterone/h were implanted in rats subcutaneously (Aldo, n = 39; controls, n = 38). After 8 wk, left ventricular pressure-volume analysis of isolated working hearts was performed, and left atrial systolic and diastolic function was also assessed by atrial pressure-diameter loops. Moreover, left atrial myocytes were isolated to investigate their global and local Ca2+ handling and contractility. At similar heart rates, pressure-volume analysis of isolated hearts and in vivo hemodynamic measurements revealed neither systolic nor diastolic left ventricular dysfunction in Aldo. In particular, atrial filling pressures and atrial size were not increased in Aldo. Aldo rats showed a significant reduction of atrial late diastolic A wave, atrial active work index, and increased V waves. Consistently, in Aldo rats, sarcomere shortening and the amplitude of electrically evoked global Ca2+ transients were substantially reduced. Sarcoplasmic reticulum-Ca2+ content and fractional Ca2+ release were decreased, substantiated by a reduced sarcoplasmic reticulum calcium ATPase activity, resulting from a reduced CAMKII-evoked phosphorylation of phospholamban. Hyperaldosteronism induced atrial systolic and diastolic dysfunction, while atrial size and left ventricular hemodynamics, including filling pressures, were unaffected in rats. The described model suggests a direct causal link between hyperaldosteronism and decreased atrial contractility and diastolic compliance.
Assuntos
Aldosterona/farmacologia , Função do Átrio Esquerdo/efeitos dos fármacos , Átrios do Coração/efeitos dos fármacos , Hemodinâmica/efeitos dos fármacos , Contração Miocárdica/efeitos dos fármacos , Pressão , Animais , Cálcio/metabolismo , Diástole , Hiperaldosteronismo/fisiopatologia , Ratos , Ratos Sprague-Dawley , Retículo Sarcoplasmático/efeitos dos fármacos , Retículo Sarcoplasmático/metabolismo , Sístole , Função Ventricular Esquerda/efeitos dos fármacos , Pressão Ventricular/efeitos dos fármacosRESUMO
This study focuses on characterization of the cardiac N-methyl D-aspartate receptors (NMDARs) as a target for endogenous and synthetic agonists and antagonists. Using isolated perfused rat hearts, we have shown that intracoronary administration of the NMDAR agonists and antagonists has a pronounced effect on autonomous heart function. Perfusion of rat hearts with autologous blood supplemented with NMDAR agonists was associated with induction of tachycardia, sinus arrhythmia, and ischemia occurring within physiological plasma concentration range for glutamate and glycine. Intracoronary administration of the NMDAR antagonists exerted an antiarrhythmic effect and resulted in bradycardia and improvement of capillary perfusion. Action of antagonists eliprodil, Ro25-6981, memantine, ketamine, and MK-801 on autonomous heart function diverged strikingly from that of L-type Ca channel blockers. Cardiac NMDAR subunit composition differed from that of neuronal receptors and was age specific and chamber specific. Transcripts of the GluN3A and GluN2D were found in all heart chambers, whereas expression of GluN1 and GluN2A and 2C was restricted to the atria. Expression of the GluN2B protein in ventricles increased markedly with age of the animals. The obtained data reveal that NMDARs are expressed in rat heart contributing to the autonomic heart rate regulation and the function of the cardiac conduction system.
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Sistemas de Liberação de Medicamentos/métodos , Agonistas de Aminoácidos Excitatórios/administração & dosagem , Antagonistas de Aminoácidos Excitatórios/administração & dosagem , Miócitos Cardíacos/efeitos dos fármacos , Receptores de N-Metil-D-Aspartato/agonistas , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Animais , Feminino , Coração/efeitos dos fármacos , Humanos , Masculino , Miócitos Cardíacos/metabolismo , Técnicas de Cultura de Órgãos , Ratos , Ratos Wistar , Receptores de N-Metil-D-Aspartato/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.
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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
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.
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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
AIMS: Pathological cardiac hypertrophy is a major predictor for the development of cardiac diseases. It is associated with chronic neurohumoral stimulation and with altered cardiac Ca(2+) signalling in cardiomyocytes. TRPC proteins form agonist-induced cation channels, but their functional role for Ca(2+) homeostasis in cardiomyocytes during fast cytosolic Ca(2+) cycling and neurohumoral stimulation leading to hypertrophy is unknown. METHODS AND RESULTS: In a systematic analysis of multiple knockout mice using fluorescence imaging of electrically paced adult ventricular cardiomyocytes and Mn(2+)-quench microfluorimetry, we identified a background Ca(2+) entry (BGCE) pathway that critically depends on TRPC1/C4 proteins but not others such as TRPC3/C6. Reduction of BGCE in TRPC1/C4-deficient cardiomyocytes lowers diastolic and systolic Ca(2+) concentrations both, under basal conditions and under neurohumoral stimulation without affecting cardiac contractility measured in isolated hearts and in vivo. Neurohumoral-induced cardiac hypertrophy as well as the expression of foetal genes (ANP, BNP) and genes regulated by Ca(2+)-dependent signalling (RCAN1-4, myomaxin) was reduced in TRPC1/C4 knockout (DKO), but not in TRPC1- or TRPC4-single knockout mice. Pressure overload-induced hypertrophy and interstitial fibrosis were both ameliorated in TRPC1/C4-DKO mice, whereas they did not show alterations in other cardiovascular parameters contributing to systemic neurohumoral-induced hypertrophy such as renin secretion and blood pressure. CONCLUSIONS: The constitutively active TRPC1/C4-dependent BGCE fine-tunes Ca(2+) cycling in beating adult cardiomyocytes. TRPC1/C4-gene inactivation protects against development of maladaptive cardiac remodelling without altering cardiac or extracardiac functions contributing to this pathogenesis.
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Canais de Cálcio/fisiologia , Sinalização do Cálcio/fisiologia , Cardiomegalia/metabolismo , Miócitos Cardíacos/metabolismo , Canais de Cátion TRPC/fisiologia , Angiotensina II/metabolismo , Angiotensinogênio/metabolismo , Animais , Cálcio/metabolismo , Cardiomegalia/fisiopatologia , Hemodinâmica/fisiologia , Homeostase/fisiologia , Camundongos Knockout , Remodelação VentricularRESUMO
Atrial fibrillation (AF) is characterized by irregular contractions of atrial cardiomyocytes and increased energy demand. The aim of this study was to characterize the influence of arrhythmia on glucose and fatty acid (FA) metabolism in cardiomyocytes, mice and human left atrial myocardium. Compared to regular pacing, irregular (pseudo-random variation at the same number of contractions/min) pacing of neonatal rat cardiomyocytes induced shorter action potential durations and effective refractory periods and increased diastolic [Ca(2+)]c. This was associated with the activation of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and AMP-activated protein kinase (AMPK). Membrane expression of fatty acid translocase (FAT/CD36) and (14)C-palmitic acid uptake were augmented while membrane expression of glucose transporter subtype 4 (GLUT-4) as well as (3)H-glucose uptake were reduced. Inhibition of AMPK and CaMKII prevented these arrhythmia-induced metabolic changes. Similar alterations of FA metabolism were observed in a transgenic mouse model (RacET) for spontaneous AF. Consistent with these findings samples of left atrial myocardium of patients with AF compared to matched samples of patients with sinus rhythm showed up-regulation of CaMKII and AMPK and increased membrane expression of FAT/CD36, resulting in lipid accumulation. These changes of FA metabolism were accompanied by decreased membrane expression of GLUT-4, increased glycogen content and increased expression of the pro-apoptotic protein bax. Irregular pacing of cardiomyocytes increases diastolic [Ca(2+)]c and activation of CaMKII and AMPK resulting in lipid accumulation, reduced glucose uptake and increased glycogen synthesis. These metabolic changes are accompanied by an activation of pro-apoptotic signalling pathways.
Assuntos
Fibrilação Atrial/metabolismo , Glucose/metabolismo , Metabolismo dos Lipídeos , Proteínas Quinases Ativadas por AMP/metabolismo , Potenciais de Ação , Idoso , Animais , Cálcio/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Feminino , Transportador de Glucose Tipo 4/fisiologia , Humanos , Masculino , Pessoa de Meia-Idade , Miócitos Cardíacos/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de SinaisRESUMO
During cardiogenesis, most myocytes arise from cardiac progenitors expressing the transcription factors Isl1 and Nkx2-5. Here, we show that a direct repression of Isl1 by Nkx2-5 is necessary for proper development of the ventricular myocardial lineage. Overexpression of Nkx2-5 in mouse embryonic stem cells (ESCs) delayed specification of cardiac progenitors and inhibited expression of Isl1 and its downstream targets in Isl1(+) precursors. Embryos deficient for Nkx2-5 in the Isl1(+) lineage failed to downregulate Isl1 protein in cardiomyocytes of the heart tube. We demonstrated that Nkx2-5 directly binds to an Isl1 enhancer and represses Isl1 transcriptional activity. Furthermore, we showed that overexpression of Isl1 does not prevent cardiac differentiation of ESCs and in Xenopus laevis embryos. Instead, it leads to enhanced specification of cardiac progenitors, earlier cardiac differentiation, and increased cardiomyocyte number. Functional and molecular characterization of Isl1-overexpressing cardiomyocytes revealed higher beating frequencies in both ESC-derived contracting areas and Xenopus Isl1-gain-of-function hearts, which associated with upregulation of nodal-specific genes and downregulation of transcripts of working myocardium. Immunocytochemistry of cardiomyocyte lineage-specific markers demonstrated a reduction of ventricular cells and an increase of cells expressing the pacemaker channel Hcn4. Finally, optical action potential imaging of single cardiomyocytes combined with pharmacological approaches proved that Isl1 overexpression in ESCs resulted in normally electrophysiologically functional cells, highly enriched in the nodal subtype at the expense of the ventricular lineage. Our findings provide an Isl1/Nkx2-5-mediated mechanism that coordinately regulates the specification of cardiac progenitors toward the different myocardial lineages and ensures proper acquisition of myocyte subtype identity.
Assuntos
Proteínas de Homeodomínio/biossíntese , Proteínas com Homeodomínio LIM/antagonistas & inibidores , Proteínas com Homeodomínio LIM/biossíntese , Miócitos Cardíacos/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/biossíntese , Animais , Linhagem da Célula/fisiologia , Células-Tronco Embrionárias/metabolismo , Células HEK293 , Proteína Homeobox Nkx-2.5 , Humanos , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Ligação Proteica/fisiologia , XenopusRESUMO
Asynchronous release of calcium (Ca(2+))-for example, the generation of Ca(2+) alternans in cardiac myocytes-is a phenomenon important in the development of cardiac arrhythmogenesis. The development of a failure to release Ca(2+) at individual release sites can be regarded as a major contributor to cardiac pathologies such as hypertrophy. Although confocal linescans provide sufficient temporal resolution to investigate the physiological and pathological cardiac excitation-contraction (EC) coupling, linescans can only image â¼1.5% of the cross section of myocytes, which raises doubts about how representative such recordings are, especially in light of nonhomogeneous uncoupling of Ca(2+) channels and ryanodine receptors. Nowadays, the speed of confocal microscopes has been greatly improved, enabling two-dimensional (2D) imaging at sufficient image rates (>100 frames/sec). To understand better the physiological and pathophysiological EC coupling of cardiomyocytes, we describe here a protocol to monitor fast intracellular Ca(2+) signals using fast 2D confocal scanning.