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1.
J Hepatol ; 2020 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-33276032

RESUMO

BACKGROUND: Intrahepatic cholestasis of pregnancy (ICP) is associated with increased stillbirth risk. This study aimed to assess the relationship between bile acid concentrations and fetal cardiac dysfunction in ICP with or without ursodeoxycholic acid (UDCA) treatment. METHODS: Bile acid profiles and NT-proBNP, a marker of ventricular dysfunction, were assayed in umbilical venous serum from 15 controls and 76 ICP cases (36 untreated, 40 UDCA-treated). Fetal ECG traces were obtained from 43 controls and 48 ICP cases (26 untreated, 22 UDCA-treated). PR interval length and heart rate variability parameters (RMSSD, SDNN) were measured in two behavioural states (quiet and active sleep). Partial correlation coefficients (r) and median [IQR] are reported. RESULTS: In untreated ICP, fetal total serum bile acids (TSBA, r=0.49, p=0.019), their hydrophobicity index (r=0.20, p=0.039), glycocholate (r=0.56, p=0.007) and taurocholate (r=0.44, p=0.039) positively correlated with fetal NT-proBNP. Maternal TSBA (r=0.40, p=0.026) and alanine aminotransferase (r=0.40, p=0.046) also positively correlated with fetal NT-proBNP. No significant correlations to NT-proBNP were observed in the UDCA-treated cohort. Fetal PR interval length positively correlated with maternal TSBA in untreated (r=0.46, p=0.027) and UDCA-treated ICP (r=0.54, p=0.026). Fetal RMSSD in active sleep (9.6 [8.8,11.3] vs. 8.7 [7.6,9.6] ms, p=0.028) and SDNN in quiet sleep (11.0 [9.5,14.9] vs. 7.9 [5.1,9.7] ms, p=0.013) and active sleep (25.4 [21.0,32.4] vs. 18.2 [14.7,25.7] ms, p=0.003) were significantly higher in untreated ICP cases than controls. Heart rate variability values in UDCA-treated cases did not differ to controls. CONCLUSIONS: Elevated fetal and maternal serum bile acid concentrations in untreated ICP are associated with an abnormal fetal cardiac phenotype characterised by increased NT-proBNP concentration, PR interval length and heart rate variability. UDCA treatment partially attenuates this phenotype.

2.
Cells ; 9(10)2020 10 12.
Artigo em Inglês | MEDLINE | ID: mdl-33053822

RESUMO

The ability to differentiate induced-pluripotent stem cells into cardiomyocytes (iPSC-CMs) has opened up novel avenues for potential cardiac therapies. However, iPSC-CMs exhibit a range of somewhat immature functional properties. This study explored the development of the beta-adrenergic receptor (ßAR) pathway, which is crucial in regulating contraction and signifying the health and maturity of myocytes. We explored the compartmentation of ß2AR-signalling and phosphodiesterases (PDEs) in caveolae, as functional nanodomains supporting the mature phenotype. Förster Resonance Energy Transfer (FRET) microscopy was used to study the cyclic adenosine monophosphate (cAMP) levels in iPSC-CMs at day 30, 60, and 90 following ßAR subtype-specific stimulation. Subsequently, the PDE2, PDE3, and PDE4 activity was investigated using specific inhibitors. Cells were treated with methyl-ß-cyclodextrin (MßCD) to remove cholesterol as a method of decompartmentalising ß2AR. As iPSC-CMs mature with a prolonged culture time, the caveolae density is increased, leading to a reduction in the overall cytoplasmic cAMP signal stimulated through ß2AR (but not ß1AR). Pan-phosphodiesterase inhibition or caveolae depletion leads to an increase in the ß2AR-stimulated cytoplasmic cAMP. Moreover, with time in culture, the increase in the ßAR-dependent cytoplasmic cAMP becomes more sensitive to cholesterol removal. The regulation of the ß2AR response by PDE2 and 4 is similarly increased with the time in culture. We conclude that both the ß2AR and PDEs are restricted to the caveolae nanodomains, and thereby exhibit a tighter spatial restriction over the cAMP signal in late-stage compared to early iPSC-CMs.

3.
Circ Res ; 2020 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-33092464

RESUMO

Rationale: Ca2+ induced Ca2+ release (CICR) in normal hearts requires close approximation of L-type calcium channels (LTCCs) within the transverse tubules (T-tubules), and Ryanodine receptors (RyR) within the junctional sarcoplasmic reticulum (jSR). CICR is disrupted in cardiac hypertrophy and heart failure, which is associated with loss of T-tubules and disruption of cardiac dyads. In these conditions, LTCCs are redistributed from the T-tubules to disrupt CICR. The molecular mechanism responsible for LTCCs recruitment to and from the T-tubules is not well known. Junctophilin-2 (JPH2) enables close association between T-tubules and the jSR to ensure efficient CICR. JPH2 has a so-called Joining region that is located near domains that interact with T-tubular plasma membrane, where LTCCs are housed. The idea that this Joining region directly interacts with LTCCs and contributes to LTCC recruitment to T-tubules is unknown. Objective:To determine if the Joining region in JPH2 recruits LTCCs to T-tubules through direct molecular interaction in cardiomyocytes to enable efficient CICR. Methods and Results: Modified abundance of JPH2 and redistribution of LTCC were studied in left ventricular hypertrophy in vivo and in cultured adult Feline and rat ventricular myocytes. Protein-protein interaction studies showed that the Joining region in JPH2 interacts with LTCC-α1C subunit and causes LTCCs distribution to the dyads, where they colocalize with RyRs. A JPH2 with induced mutations in the Joining region (mutPG1JPH2) caused T-tubule remodeling and dyad loss, showing that an interaction between LTCC and JPH2 is crucial for T-tubule stabilization. mutPG1JPH2 caused asynchronous Ca2+-release with impaired excitation-contraction (EC) coupling after ß-adrenergic stimulation. The disturbed Ca2+ regulation in mutPG1JPH2 overexpressing myocytes caused Calcium/calmodulin-dependent kinase-II activation and altered myocyte bioenergetics. Conclusions: The interaction between LTCC and the Joining region in JPH2 facilitates dyad assembly and maintains normal CIRC in cardiomyocytes.

4.
Sci Rep ; 10(1): 15284, 2020 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-32943714

RESUMO

Acute myocardial ischaemia and reperfusion (I-R) are major causes of ventricular arrhythmias in patients with a history of coronary artery disease. Ursodeoxycholic acid (UDCA) has previously been shown to be antiarrhythmic in fetal hearts. This study was performed to investigate if UDCA protects against ischaemia-induced and reperfusion-induced arrhythmias in the adult myocardium, and compares the effect of acute (perfusion only) versus prolonged (2 weeks pre-treatment plus perfusion) UDCA administration. Langendorff-perfused adult Sprague-Dawley rat hearts were subjected to acute regional ischaemia by ligation of the left anterior descending artery (10 min), followed by reperfusion (2 min), and arrhythmia incidence quantified. Prolonged UDCA administration reduced the incidence of acute ischaemia-induced arrhythmias (p = 0.028), with a reduction in number of ventricular ectopic beats during the ischaemic phase compared with acute treatment (10 ± 3 vs 58 ± 15, p = 0.036). No antiarrhythmic effect was observed in the acute UDCA administration group. Neither acute nor prolonged UDCA treatment altered the incidence of reperfusion arrhythmias. The antiarrhythmic effect of UDCA may be partially mediated by an increase in cardiac wavelength, due to the attenuation of conduction velocity slowing (p = 0.03), and the preservation of Connexin43 phosphorylation during acute ischaemia (p = 0.0027). The potential antiarrhythmic effects of prolonged UDCA administration merit further investigation.

5.
Front Cell Dev Biol ; 8: 695, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32850816

RESUMO

Mediastinal lymphadenopathy and auto-antibodies are clinical phenomena during ischemic heart failure pointing to an autoimmune response against the heart. T and B cells have been convincingly demonstrated to be activated after myocardial infarction, a prerequisite for the generation of mature auto-antibodies. Yet, little is known about the immunoglobulin isotype repertoire thus pathological potential of anti-heart auto-antibodies during heart failure. We obtained human myocardial tissue from ischemic heart failure patients and induced experimental MI in rats. We found that anti-heart autoimmunity persists during heart failure. Rat mediastinal lymph nodes are enlarged and contain active secondary follicles with mature isotype-switched IgG2a B cells. Mature IgG2a auto-antibodies specific for cardiac antigens are present in rat heart failure serum, and IgG and complement C3 deposits are evident in heart failure tissue of both rats and human patients. Previously established myocardial inflammation, and the herein provided proof of B cell maturation in lymph nodes and myocardial deposition of mature auto-antibodies, provide all the hallmark signs of an established autoimmune response in chronic heart failure.

6.
Nanoscale ; 12(30): 16315-16329, 2020 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-32720664

RESUMO

Mechanical properties of single myocytes contribute to the whole heart performance, but the measurement of mechanics in living cells at high resolution with minimal force interaction remains challenging. Angiotensin II (AngII) is a peptide hormone that regulates a number of physiological functions, including heart performance. It has also been shown to contribute to cell mechanics by inducing cell stiffening. Using non-contact high-resolution Scanning Ion Conductance Microscopy (SICM), we determine simultaneously cell topography and membrane transverse Young's modulus (YM) by a constant pressure application through a nanopipette. While applying pressure, the vertical position is recorded and a deformation map is generated from which YM can be calculated and corrected for the uneven geometry. High resolution of this method also allows studying specific membrane subdomains, such as Z-grooves and crests. We found that short-term AngII treatment reduces the transversal YM in isolated adult rat cardiomyocytes acting via an AT1 receptor. Blocking either a TGF-ß1 receptor or Rho kinase abolishes this effect. Analysis of the cytoskeleton showed that AngII depletes microtubules by decreasing long-lived detyrosinated and acetylated microtubule populations. Interestingly, in the failing cardiomyocytes, which are stiffer than controls, the short-term AngII treatment also reduces the YM, thus normalizing the mechanical state of cells. This suggests that the short-term softening effect of AngII on cardiac cells is opposite to the well-characterized long-term hypertrophic effect. In conclusion, we generate a precise nanoscale indication map of location-specific transverse cortical YM within the cell and this can substantially advance our understanding of cellular mechanics in a physiological environment, for example in isolated cardiac myocytes.

7.
EBioMedicine ; 57: 102845, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32580140

RESUMO

BACKGROUND: Subcellular localization and function of L-type calcium channels (LTCCs) play an important role in regulating contraction of cardiomyocytes. Understanding how this is affected by the disruption of transverse tubules during heart failure could lead to new insights into the disease. METHODS: Cardiomyocytes were isolated from healthy donor hearts, as well as from patients with cardiomyopathies and with left ventricular assist devices. Scanning ion conductance and confocal microscopy was used to study membrane structures in the cells. Super-resolution scanning patch-clamp was used to examine LTCC function in different microdomains. Computational modeling predicted the impact of these changes to arrhythmogenesis at the whole-heart level. FINDINGS: We showed that loss of structural organization in failing myocytes leads to re-distribution of functional LTCCs from the T-tubules to the sarcolemma. In ischemic cardiomyopathy, the increased LTCC open probability in the T-tubules depends on the phosphorylation by protein kinase A, whereas in dilated cardiomyopathy, the increased LTCC opening probability in the sarcolemma results from enhanced phosphorylation by calcium-calmodulin kinase II. LVAD implantation corrected LTCCs pathophysiological activity, although it did not improve their distribution. Using computational modeling in a 3D anatomically-realistic human ventricular model, we showed how LTCC location and activity can trigger heart rhythm disorders of different severity. INTERPRETATION: Our findings demonstrate that LTCC redistribution and function differentiate between disease aetiologies. The subcellular changes observed in specific microdomains could be the consequence of the action of distinct protein kinases. FUNDING: This work was supported by NIH grant (ROI-HL 126802 to NT-JG) and British Heart Foundation (grant RG/17/13/33173 to JG, project grant PG/16/17/32069 to RAC). Funders had no role in study design, data collection, data analysis, interpretation, writing of the report.

10.
Elife ; 92020 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-32228862

RESUMO

Cardiomyocyte ß3-adrenoceptors (ß3-ARs) coupled to soluble guanylyl cyclase (sGC)-dependent production of the second messenger 3',5'-cyclic guanosine monophosphate (cGMP) have been shown to protect from heart failure. However, the exact localization of these receptors to fine membrane structures and subcellular compartmentation of ß3-AR/cGMP signals underpinning this protection in health and disease remain elusive. Here, we used a Förster Resonance Energy Transfer (FRET)-based cGMP biosensor combined with scanning ion conductance microscopy (SICM) to show that functional ß3-ARs are mostly confined to the T-tubules of healthy rat cardiomyocytes. Heart failure, induced via myocardial infarction, causes a decrease of the cGMP levels generated by these receptors and a change of subcellular cGMP compartmentation. Furthermore, attenuated cGMP signals led to impaired phosphodiesterase two dependent negative cGMP-to-cAMP cross-talk. In conclusion, topographic and functional reorganization of the ß3-AR/cGMP signalosome happens in heart failure and should be considered when designing new therapies acting via this receptor.

11.
Cells ; 9(3)2020 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-32131460

RESUMO

Fibrosis is a significant global health problem associated with many inflammatory and degenerative diseases affecting multiple organs, individually or simultaneously. Fibrosis develops when extracellular matrix (ECM) remodeling becomes excessive or uncontrolled and is associated with nearly all forms of heart disease. Cardiac fibroblasts and myofibroblasts are the main effectors of ECM deposition and scar formation. The heart is a complex multicellular organ, where the various resident cell types communicate between themselves and with cells of the blood and immune systems. Exosomes, which are small extracellular vesicles, (EVs), contribute to cell-to-cell communication and their pathophysiological relevance and therapeutic potential is emerging. Here, we will critically review the role of endogenous exosomes as possible fibrosis mediators and discuss the possibility of using stem cell-derived and/or engineered exosomes as anti-fibrotic agents.

12.
Biochem Soc Trans ; 48(1): 61-70, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-32104883

RESUMO

Multiple intra-cellular signalling pathways rely on calcium and 3'-5' cyclic adenosine monophosphate (cAMP) to act as secondary messengers. This is especially true in cardiomyocytes which act as the force-producing units of the cardiac muscle and are required to react rapidly to environmental stimuli. The specificity of functional responses within cardiomyocytes and other cell types is produced by the organellar compartmentation of both calcium and cAMP. In this review, we assess the role of molecular localisation and relative contribution of active and passive processes in producing compartmentation. Active processes comprise the creation and destruction of signals, whereas passive processes comprise the release or sequestration of signals. Cardiomyocytes display a highly articulated membrane structure which displays significant cell-to-cell variability. Special attention is paid to the way in which cell membrane caveolae and the transverse-axial tubule system allow molecular localisation. We explore the effects of cell maturation, pathology and regional differences in the organisation of these processes. The subject of signal compartmentation has had a significant amount of attention within the cardiovascular field and has undergone a revolution over the past two decades. Advances in the area have been driven by molecular imaging using fluorescent dyes and genetically encoded constructs based upon fluorescent proteins. We also explore the use of scanning probe microscopy in the area. These techniques allow the analysis of molecular compartmentation within specific organellar compartments which gives researchers an entirely new perspective.


Assuntos
Compartimento Celular/fisiologia , Miócitos Cardíacos/metabolismo , Transdução de Sinais/fisiologia , Animais , Sinalização do Cálcio , Cavéolas/metabolismo , AMP Cíclico/metabolismo , Transferência Ressonante de Energia de Fluorescência/métodos , Insuficiência Cardíaca/metabolismo
13.
Cardiovasc Res ; 2020 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-32053184

RESUMO

AIM: In cardiomyocytes, transverse tubules (T-tubules) associate with the sarcoplasmic reticulum (SR), forming junctional membrane complexes (JMCs) where L-type calcium channels (LTCCs) are juxtaposed to Ryanodine receptors (RyR). Junctophilin-2 (JPH2) supports the assembly of JMCs by tethering T-tubules to the SR membrane. T-tubule remodeling in cardiac diseases is associated with down-regulation of JPH2 expression suggesting that JPH2 plays a crucial role in T-tubule stability. Furthermore, increasing evidence indicate that JPH2 might additionally act as a modulator of calcium signaling by directly regulating RyR and LTCCs. This study aimed at determining whether JPH2 overexpression restores normal T-tubule structure and LTCC function in cultured cardiomyocytes. METHODS AND RESULTS: Rat ventricular myocytes kept in culture for 4 days showed extensive T-tubule remodeling with impaired JPH2 localization and relocation of the scaffolding protein Caveolin3 (Cav3) from the T-tubules to the outer membrane. Overexpression of JPH2 restored T-tubule structure and Cav3 relocation. Depletion of membrane cholesterol by chronic treatment with Methyl-ß-cyclodextrin (MßCD) countered the stabilizing effect of JPH2 overexpression on T-tubules and Cav3. Super-resolution scanning patch-clamp showed that JPH2 overexpression greatly increased the number of functional LTCCs at the plasma membrane. Treatment with MßCD reduced LTCC open probability and activity. Proximity ligation assays showed that MßCD did not affect JPH2 interaction with RyR and the pore-forming LTCC subunit Cav1.2, but strongly impaired JPH2 association with Cav3 and the accessory LTCC subunit Cavß2. CONCLUSIONS: JPH2 promotes T-tubule structural stability and recruits functional LTCCs to the membrane, most likely by directly binding to the channel. Cholesterol is involved in the binding of JPH2 to T-tubules as well as in the modulation of LTCC activity. We propose a model where cholesterol and Cav3 support the assembly of lipid rafts which provide an anchor for JPH2 to form JMCs and a platform for signaling complexes to regulate LTCC activity. TRANSLATIONAL PERSPECTIVE: By tethering T-tubules to the sarcoplasmic reticulum, junctophilin-2 (JPH2) supports the assembly of junctional membrane complexes (JMCs), major sites of excitation-contraction coupling in cardiomyocytes. JPH2 downregulation underlies the disruption of JMCs and T-tubules in cardiomyopathies and enhancing JPH2 expression is a promising therapeutic approach for the treatment of heart failure. Our study demonstrates a new role for JPH2 in the recruitment of calcium channels to the membrane and provides innovative insights into the formation and organization of JMCs as well as into the regulation of excitation-contraction coupling. Our results are of significant importance when considering JPH2 as a therapeutic target.

14.
Elife ; 92020 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-31934859

RESUMO

We investigated targeting mechanisms of Na+ and KATP channels to the intercalated disk (ICD) of cardiomyocytes. Patch clamp and surface biotinylation data show reciprocal downregulation of each other's surface density. Mutagenesis of the Kir6.2 ankyrin binding site disrupts this functional coupling. Duplex patch clamping and Angle SICM recordings show that INa and IKATP functionally co-localize at the rat ICD, but not at the lateral membrane. Quantitative STORM imaging show that Na+ and KATP channels are localized close to each other and to AnkG, but not to AnkB, at the ICD. Peptides corresponding to Nav1.5 and Kir6.2 ankyrin binding sites dysregulate targeting of both Na+ and KATP channels to the ICD, but not to lateral membranes. Finally, a clinically relevant gene variant that disrupts KATP channel trafficking also regulates Na+ channel surface expression. The functional coupling between these two channels need to be considered when assessing clinical variants and therapeutics.

15.
Proc Natl Acad Sci U S A ; 117(6): 2764-2766, 2020 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-31988123

RESUMO

The field of cardiomyocyte mechanobiology is gaining significant attention, due to accumulating evidence concerning the significant role of cellular mechanical effects on the integrated function of the heart. To date, the protein titin has been demonstrated as a major contributor to the cardiomyocytes Young's modulus (YM). The microtubular network represents another potential regulator of cardiac mechanics. However, the contribution of microtubules (MTs) to the membrane YM is still understudied and has not been interrogated in the context of myocardial infarction (MI) or mechanical loading and unloading. Using nanoscale mechanoscanning ion conductance microscopy, we demonstrate that MTs contribute to cardiomyocyte transverse YM in healthy and pathological states with different mechanical loading. Specifically, we show that posttranslational modifications of MTs have differing effects on cardiomyocyte YM: Acetylation provides flexibility, whereas detyrosination imparts rigidity. Further studies demonstrate that there is no correlation between the total protein amount of acetylated and detyrosinated MT. Yet, in the polymerized-only populations, an increased level of acetylation results in a decline of detyrosinated MTs in an MI model.


Assuntos
Microtúbulos/metabolismo , Miócitos Cardíacos/química , Miócitos Cardíacos/metabolismo , Acetilação , Animais , Fenômenos Biomecânicos , Membrana Celular/química , Membrana Celular/metabolismo , Células Cultivadas , Módulo de Elasticidade , Masculino , Microtúbulos/química , Processamento de Proteína Pós-Traducional , Ratos , Ratos Endogâmicos Lew , Ratos Sprague-Dawley
16.
Cells ; 8(12)2019 11 29.
Artigo em Inglês | MEDLINE | ID: mdl-31795419

RESUMO

Förster resonance energy transfer (FRET) is increasingly used for non-invasive measurement of fluorescently tagged molecules in live cells. In this study, we have developed a freely available software tool MultiFRET, which, together with the use of a motorised microscope stage, allows multiple single cells to be studied in one experiment. MultiFRET is a Java plugin for Micro-Manager software, which provides real-time calculations of ratio-metric signals during acquisition and can simultaneously record from multiple cells in the same experiment. It can also make other custom-determined live calculations that can be easily exported to Excel at the end of the experiment. It is flexible and can work with multiple spectral acquisition channels. We validated this software by comparing the output of MultiFRET to that of a previously established and well-documented method for live ratio-metric FRET experiments and found no significant difference between the data produced with the use of the new MultiFRET and other methods. In this validation, we used several cAMP FRET sensors and cell models: i) isolated adult cardiomyocytes from transgenic mice expressing the cytosolic epac1-camps and targeted pmEpac1 and Epac1-PLN sensors, ii) isolated neonatal mouse cardiomyocytes transfected with the AKAP79-CUTie sensor, and iii) human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) transfected with the Epac-SH74 sensor. The MultiFRET plugin is an open source freely available package that can be used in a wide area of live cell imaging when live ratio-metric calculations are required.


Assuntos
AMP Cíclico/metabolismo , Transferência Ressonante de Energia de Fluorescência , Miócitos Cardíacos/metabolismo , Software , Algoritmos , Animais , Biomarcadores , Transferência Ressonante de Energia de Fluorescência/métodos , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Reprodutibilidade dos Testes
17.
Elife ; 82019 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-31793877

RESUMO

Stable cell-cell contacts underpin tissue architecture and organization. Quantification of junctions of mammalian epithelia requires laborious manual measurements that are a major roadblock for mechanistic studies. We designed Junction Mapper as an open access, semi-automated software that defines the status of adhesiveness via the simultaneous measurement of pre-defined parameters at cell-cell contacts. It identifies contacting interfaces and corners with minimal user input and quantifies length, area and intensity of junction markers. Its ability to measure fragmented junctions is unique. Importantly, junctions that considerably deviate from the contiguous staining and straight contact phenotype seen in epithelia are also successfully quantified (i.e. cardiomyocytes or endothelia). Distinct phenotypes of junction disruption can be clearly differentiated among various oncogenes, depletion of actin regulators or stimulation with other agents. Junction Mapper is thus a powerful, unbiased and highly applicable software for profiling cell-cell adhesion phenotypes and facilitate studies on junction dynamics in health and disease.


Assuntos
Comunicação Celular/fisiologia , Biologia Computacional/métodos , Células Endoteliais/fisiologia , Junções Intercelulares/fisiologia , Queratinócitos/fisiologia , Miócitos Cardíacos/fisiologia , Animais , Caderinas/metabolismo , Adesão Celular/fisiologia , Células Cultivadas , Células Endoteliais/metabolismo , Humanos , Junções Intercelulares/metabolismo , Queratinócitos/metabolismo , Microscopia Confocal , Miócitos Cardíacos/metabolismo , Fenótipo , Ratos Sprague-Dawley , Software
18.
FASEB J ; 33(9): 10453-10468, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31253057

RESUMO

Healthy cardiomyocytes are electrically coupled at the intercalated discs by gap junctions. In infarcted hearts, adverse gap-junctional remodeling occurs in the border zone, where cardiomyocytes are chemically and electrically influenced by myofibroblasts. The physical movement of these contacts remains unquantified. Using scanning ion conductance microscopy, we show that intercellular contacts between cardiomyocytes and myofibroblasts are highly dynamic, mainly owing to the edge dynamics (lamellipodia) of the myofibroblasts. Decreasing the amount of functional connexin-43 (Cx43) at the membrane through Cx43 silencing, suppression of Cx43 trafficking, or hypoxia-induced Cx43 internalization attenuates heterocellular contact dynamism. However, we found decreased dynamism and stabilized membrane contacts when cellular coupling was strengthened using 4-phenylbutyrate (4PB). Fluorescent-dye transfer between cells showed that the extent of functional coupling between the 2 cell types correlated with contact dynamism. Intercellular calcein transfer from myofibroblasts to cardiomyocytes is reduced after myofibroblast-specific Cx43 down-regulation. Conversely, 4PB-treated myofibroblasts increased their functional coupling to cardiomyocytes. Consistent with lamellipodia-mediated contacts, latrunculin-B decreases dynamism, lowers physical communication between heterocellular pairs, and reduces Cx43 intensity in contact regions. Our data show that heterocellular cardiomyocyte-myofibroblast contacts exhibit high dynamism. Therefore, Cx43 is a potential target for prevention of aberrant cardiomyocyte coupling and myofibroblast proliferation in the infarct border zone.-Schultz, F., Swiatlowska, P., Alvarez-Laviada, A., Sanchez-Alonso, J. L., Song, Q., de Vries, A. A. F., Pijnappels, D. A., Ongstad, E., Braga, V. M. M., Entcheva, E., Gourdie, R. G., Miragoli, M., Gorelik, J. Cardiomyocyte-myofibroblast contact dynamism is modulated by connexin-43.


Assuntos
Adesão Celular , Comunicação Celular , Movimento Celular , Conexina 43/metabolismo , Miócitos Cardíacos/fisiologia , Miofibroblastos/fisiologia , Animais , Antineoplásicos/farmacologia , Células Cultivadas , Junções Comunicantes , Masculino , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Miofibroblastos/citologia , Miofibroblastos/efeitos dos fármacos , Fenilbutiratos/farmacologia , Ratos , Ratos Sprague-Dawley
19.
FASEB J ; 33(7): 8504-8518, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31017801

RESUMO

Dynamin 2 (DNM2) is a GTP-binding protein that controls endocytic vesicle scission and defines a whole class of dynamin-dependent endocytosis, including clathrin-mediated endocytosis by caveoli. It has been suggested that mutations in the DNM2 gene, associated with 3 inherited diseases, disrupt endocytosis. However, how exactly mutations affect the nanoscale morphology of endocytic machinery has never been studied. In this paper, we used live correlative scanning ion conductance microscopy (SICM) and fluorescence confocal microscopy (FCM) to study how disease-associated mutations affect the morphology and kinetics of clathrin-coated pits (CCPs) by directly following their dynamics of formation, maturation, and internalization in skin fibroblasts from patients with centronuclear myopathy (CNM) and in Cos-7 cells expressing corresponding dynamin mutants. Using SICM-FCM, which we have developed, we show how p.R465W mutation disrupts pit structure, preventing its maturation and internalization, and significantly increases the lifetime of CCPs. Differently, p.R522H slows down the formation of CCPs without affecting their internalization. We also found that CNM mutations in DNM2 affect the distribution of caveoli and reduce dorsal ruffling in human skin fibroblasts. Collectively, our SICM-FCM findings at single CCP level, backed up by electron microscopy data, argue for the impairment of several forms of endocytosis in DNM2-linked CNM.-Ali, T., Bednarska, J., Vassilopoulos, S., Tran, M., Diakonov, I. A., Ziyadeh-Isleem, A., Guicheney, P., Gorelik, J., Korchev, Y. E., Reilly, M. M., Bitoun, M., Shevchuk, A. Correlative SICM-FCM reveals changes in morphology and kinetics of endocytic pits induced by disease-associated mutations in dynamin.


Assuntos
Dinamina II/genética , Endocitose/genética , Mutação/genética , Miopatias Congênitas Estruturais/genética , Adulto , Animais , Células COS , Linhagem Celular , Chlorocebus aethiops , Clatrina/genética , Feminino , Fibroblastos/patologia , Humanos , Cinética , Masculino , Microscopia Confocal/métodos , Microscopia Eletrônica de Varredura/métodos , Microscopia de Fluorescência/métodos
20.
Cardiovasc Res ; 115(3): 546-555, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30165515

RESUMO

AIMS: Cyclic adenosine monophosphate (cAMP) regulates cardiac excitation-contraction coupling by acting in microdomains associated with sarcolemmal ion channels. However, local real time cAMP dynamics in such microdomains has not been visualized before. We sought to directly monitor cAMP in a microdomain formed around sodium-potassium ATPase (NKA) in healthy and failing cardiomyocytes and to better understand alterations of cAMP compartmentation in heart failure. METHODS AND RESULTS: A novel Förster resonance energy transfer (FRET)-based biosensor termed phospholemman (PLM)-Epac1 was developed by fusing a highly sensitive cAMP sensor Epac1-camps to the C-terminus of PLM. Live cell imaging in PLM-Epac1 and Epac1-camps expressing adult rat ventricular myocytes revealed extensive regulation of NKA/PLM microdomain-associated cAMP levels by ß2-adrenoceptors (ß2-ARs). Local cAMP pools stimulated by these receptors were tightly controlled by phosphodiesterase (PDE) type 3. In chronic heart failure following myocardial infarction, dramatic reduction of the microdomain-specific ß2-AR/cAMP signals and ß2-AR dependent PLM phosphorylation was accompanied by a pronounced loss of local PDE3 and an increase in PDE2 effects. CONCLUSIONS: NKA/PLM complex forms a distinct cAMP microdomain which is directly regulated by ß2-ARs and is under predominant control by PDE3. In heart failure, local changes in PDE repertoire result in blunted ß2-AR signalling to cAMP in the vicinity of PLM.


Assuntos
AMP Cíclico/metabolismo , Proteínas de Membrana/metabolismo , Miócitos Cardíacos/enzimologia , Fosfoproteínas/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Sarcolema/enzimologia , Sistemas do Segundo Mensageiro , ATPase Trocadora de Sódio-Potássio/metabolismo , Agonistas Adrenérgicos beta/farmacologia , Animais , Técnicas Biossensoriais , Células Cultivadas , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 2/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/metabolismo , Modelos Animais de Doenças , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Insuficiência Cardíaca/enzimologia , Insuficiência Cardíaca/patologia , Insuficiência Cardíaca/fisiopatologia , Masculino , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , Domínios e Motivos de Interação entre Proteínas , Ratos Sprague-Dawley , Receptores Adrenérgicos beta 2/efeitos dos fármacos , Sarcolema/efeitos dos fármacos , Sarcolema/patologia , Sistemas do Segundo Mensageiro/efeitos dos fármacos , Fatores de Tempo
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