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BACKGROUND: Clonal hematopoiesis of indeterminate potential (CHIP) is an acquired genetic risk factor for both leukemia and cardiovascular disease. It results in proinflammatory myeloid cells in the bone marrow and blood; however, how these cells behave in the cardiovascular tissue remains unclear. Our study aimed at investigating whether CHIP-mutated macrophages accumulate preferentially in cardiovascular tissues and examining the transcriptome of tissue macrophages from DNMT3A (DNA methyltransferase 3 alpha) or TET2 (Tet methylcytosine dioxygenase 2) mutation carriers. METHODS: We recruited patients undergoing carotid endarterectomy or heart surgeries to screen for CHIP mutation carriers using targeted genomic sequencing. Myeloid and lymphoid cells were isolated from blood and cardiovascular tissue collected during surgeries using flow cytometry. DNA and RNA extracted from these sorted cells were subjected to variant allele frequency measurement using droplet digital polymerase chain reaction and transcriptomic profiling using bulk RNA sequencing, respectively. RESULTS: Using droplet digital polymerase chain reaction, we detected similar variant allele frequency of CHIP in monocytes from blood and macrophages from atheromas and heart tissues, even among heart macrophages with and without CCR2 (C-C motif chemokine receptor 2) expression. Bulk RNA sequencing revealed a proinflammatory gene profile of myeloid cells from DNMT3A or TET2 mutation carriers compared with those from noncarriers. CONCLUSIONS: Quantitatively, CHIP-mutated myeloid cells did not preferentially accumulate in cardiovascular tissues, but qualitatively, they expressed a more disease-prone phenotype.
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Doenças Cardiovasculares , Hematopoiese Clonal , Humanos , Hematopoiese Clonal/genética , Hematopoese/genética , Macrófagos/metabolismo , Doenças Cardiovasculares/genética , Doenças Cardiovasculares/metabolismo , MutaçãoRESUMO
Macrophages (MΦ) play pivotal roles in tissue homeostasis and repair. Their mechanical environment has been identified as a key modulator of various cell functions, and MΦ mechanosensitivity is likely to be critical - in particular in a rhythmically contracting organ such as the heart. Cultured MΦ, differentiated in vitro from bone marrow (MΦBM), form a popular research model. This study explores the activity of mechanosensitive ion channels (MSC) in murine MΦBM and compares it to MSC activity in MΦ enzymatically isolated from cardiac tissue (tissue-resident MΦ; MΦTR). We show that MΦBM and MΦTR have stretch-induced currents, indicating the presence of functional MSC in their plasma membrane. The current profiles in MΦBM and in MΦTR show characteristics of cation non-selective MSC such as Piezo1 or transient receptor potential channels. While Piezo1 ion channel activity is detectable in the plasma membrane of MΦBM using the patch-clamp technique, or by measuring cytosolic calcium concentration upon perfusion with the Piezo1 channel agonist Yoda1, no Piezo1 channel activity was observed in MΦTR. The selective transient receptor potential vanilloid 4 (TRPV4) channel agonist GSK1016790A induces calcium entry in MΦTR and in MΦBM. In MΦ isolated from left-ventricular scar tissue 28 days after cryoablation, stretch-induced current characteristics are not significantly different compared to non-injured control tissue, even though scarred ventricular tissue is expected to be mechanically remodelled and to contain an altered composition of pre-existing cardiac and circulation-recruited MΦ. Our data suggest that the in vitro differentiation protocols used to obtain MΦBM generate cells that differ from MΦ recruited from the circulation during tissue repair in vivo. Further investigations are needed to explore MSC identity in lineage-traced MΦ in scar tissue, and to compare mechanosensitivity of circulating monocytes with that of MΦBM. KEY POINTS: Bone marrow-derived (MΦBM) and tissue resident (MΦTR) macrophages have stretch-induced currents, indicating expression of functional mechanosensitive channels (MSC) in their plasma membrane. Stretch-activated current profiles show characteristics of cation non-selective MSC; and mRNA coding for MSC, including Piezo1 and TRPV4, is expressed in murine MΦBM and in MΦTR. Calcium entry upon pharmacological activation of TRPV4 confirms functionality of the channel in MΦTR and in MΦBM. Piezo1 ion channel activity is detected in the plasma membrane of MΦBM but not in MΦTR, suggesting that MΦBM may not be a good model to study the mechanotransduction of MΦTR. Stretch-induced currents, Piezo1 mRNA expression and response to pharmacological activation are not significantly changed in cardiac MΦ 28 days after cryoinjury compared to sham operated mice.
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The transient receptor potential melastatin 4 (TRPM4) channel contributes extensively to cardiac electrical activity, especially cardiomyocyte action potential formation. Mechanical stretch can induce changes in heart rate and rhythm, and the mechanosensitive channel Piezo1 is expressed in many cell types within the myocardium. Our previous study showed that TRPM4 and Piezo1 are closely co-localized in the t-tubules of ventricular cardiomyocytes and contribute to the Ca2+ -dependent signalling cascade that underlies hypertrophy in response to mechanical pressure overload. However, there was no direct evidence showing that Piezo1 activation was related to TRPM4 activation in situ. In the present study, we employed the HL-1 mouse atrial myocyte-like cell line as an in vitro model to investigate whether Piezo1-TRPM4 coupling can affect action potential properties. We used the small molecule Piezo1 agonist, Yoda1, as a surrogate for mechanical stretch to activate Piezo1 and detected the action potential changes in HL-1 cells using FluoVolt, a fluorescent voltage sensitive dye. Our results demonstrate that Yoda1-induced activation of Piezo1 changes the action potential frequency in HL-1 cells. This change in action potential frequency is reduced by Piezo1 knockdown using small intefering RNA. Importantly knockdown or pharmacological inhibition of TRPM4 significantly affected the degree to which Yoda1-evoked Piezo1 activation influenced action potential frequency. Thus, the present study provides in vitro evidence of a functional coupling between Piezo1 and TRPM4 in a cardiomyocyte-like cell line. The coupling of a mechanosensitive Ca2+ permeable channel and a Ca2+ -activated TRP channel probably represents a ubiquitous model for the role of TRP channels in mechanosensory transduction. KEY POINTS: The transient receptor potential melastatin 4 (TRPM4) and Piezo1 channels have been confirmed to contribute to the Ca2+ -dependent signalling cascade that underlies cardiac hypertrophy in response to mechanical pressure overload. However, there was no direct evidence showing that Piezo1 activation was related to TRPM4 activation in situ. We employed the HL-1 mouse atrial myocyte-like cell line as an in vitro model to investigate the effect of Piezo1-TRPM4 coupling on cardiac electrical properties. The results show that both pharmacological and genetic inhibition of TRPM4 significantly affected the degree to which Piezo1 activation influenced action potential frequency in HL-1 cells. Our findings provide in vitro evidence of a functional coupling between Piezo1 and TRPM4 in a cardiomyocyte-like cell line. The coupling of a mechanosensitive Ca2+ permeable channel and a Ca2+ -activated TRP channel probably represents a ubiquitous model for the role of TRP channels in mechanosensory transduction in various (patho)physiological processes.
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RATIONALE: The sarcolemma of cardiomyocytes contains many proteins that are essential for electromechanical function in general, and excitation-contraction coupling in particular. The distribution of these proteins is nonuniform between the bulk sarcolemmal surface and membrane invaginations known as transverse tubules (TT). TT form an intricate network of fluid-filled conduits that support electromechanical synchronicity within cardiomyocytes. Although continuous with the extracellular space, the narrow lumen and the tortuous structure of TT can form domains of restricted diffusion. As a result of unequal ion fluxes across cell surface and TT membranes, limited diffusion may generate ion gradients within TT, especially deep within the TT network and at high pacing rates. OBJECTIVE: We postulate that there may be an advective component to TT content exchange, wherein cyclic deformation of TT during diastolic stretch and systolic shortening serves to mix TT luminal content and assists equilibration with bulk extracellular fluid. METHODS AND RESULTS: Using electron tomography, we explore the 3-dimensional nanostructure of TT in rabbit ventricular myocytes, preserved at different stages of the dynamic cycle of cell contraction and relaxation. We show that cellular deformation affects TT shape in a sarcomere length-dependent manner and on a beat-by-beat time-scale. Using fluorescence recovery after photobleaching microscopy, we show that apparent speed of diffusion is affected by the mechanical state of cardiomyocytes, and that cyclic contractile activity of cardiomyocytes accelerates TT diffusion dynamics. CONCLUSIONS: Our data confirm the existence of an advective component to TT content exchange. This points toward a novel mechanism of cardiac autoregulation, whereby the previously implied increased propensity for TT luminal concentration imbalances at high electrical stimulation rates would be countered by elevated advection-assisted diffusion at high mechanical beating rates. The relevance of this mechanism in health and during pathological remodeling (eg, cardiac hypertrophy or failure) forms an exciting target for further research.
Assuntos
Acoplamento Excitação-Contração , Frequência Cardíaca , Contração Miocárdica , Miócitos Cardíacos/metabolismo , Sarcolema/metabolismo , Potenciais de Ação , Animais , Difusão , Tomografia com Microscopia Eletrônica , Feminino , Recuperação de Fluorescência Após Fotodegradação , Miócitos Cardíacos/ultraestrutura , Coelhos , Sarcolema/ultraestruturaRESUMO
The cardiac cell mechanical environment changes on a beat-by-beat basis as well as in the course of various cardiac diseases. Cells sense and respond to mechanical cues via specialized mechano-sensors initiating adaptive signaling cascades. With the aim of revealing new candidates underlying mechano-transduction relevant to cardiac diseases, we investigated mechano-sensitive ion channels (MSC) in human hearts for their chamber- and disease-preferential mRNA expression. Based on a meta-analysis of RNA sequencing studies, we compared the mRNA expression levels of MSC in human atrial and ventricular tissue samples from transplant donor hearts (no cardiac disease), and from patients in sinus rhythm (underlying diseases: heart failure, coronary artery disease, heart valve disease) or with atrial fibrillation. Our results suggest that a number of MSC genes are expressed chamber preferentially, e.g., CHRNE in the atria (compared to the ventricles), TRPV4 in the right atrium (compared to the left atrium), CACNA1B and KCNMB1 in the left atrium (compared to the right atrium), as well as KCNK2 and KCNJ2 in ventricles (compared to the atria). Furthermore, 15 MSC genes are differentially expressed in cardiac disease, out of which SCN9A (lower expressed in heart failure compared to donor tissue) and KCNQ5 (lower expressed in atrial fibrillation compared to sinus rhythm) show a more than twofold difference, indicative of possible functional relevance. Thus, we provide an overview of cardiac MSC mRNA expression in the four cardiac chambers from patients with different cardiac diseases. We suggest that the observed differences in MSC mRNA expression may identify candidates involved in altered mechano-transduction in the respective diseases.
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Fibrilação Atrial , Cardiopatias , Insuficiência Cardíaca , Transplante de Coração , Humanos , Fibrilação Atrial/genética , Fibrilação Atrial/metabolismo , Doadores de Tecidos , Átrios do Coração/metabolismo , Ventrículos do Coração , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/metabolismo , Cardiopatias/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismoRESUMO
Efficient pumping of the healthy left ventricle (LV) requires heterogeneities in mechanical function of individual cardiomyocytes (CM). Deformation of sub-endocardial (Endo) tissue is greater than that of sub-epicardial (Epi) regions. Papillary muscles (PM), often considered to be part of Endo tissue, show lower beat-by-beat length variation than Epi (or Endo) regions, even though they contribute to the shift in atrio-ventricular valve plane, which is essential for LV pump function. Thus far, no comparative assessment of CM mechanics for PM and LV free wall has been published. Here, we investigate contractility and cytosolic calcium concentration ([Ca2+]c) transients in rabbit single CM, freshly isolated from PM, Endo and Epi regions of the LV (free wall tissue was further subdivided into near-basal [Base], equatorial [Centre], and near-apical [Apex] parts). Functional parameters were measured in the absence of external mechanical loads (non-loaded), or during afterloaded (auxotonic) CM contractions, initiated from different levels of preload (diastolic axial stretch), using the carbon fibre technique. We note significant differences in time-course and amplitudes of sarcomere shortening between PM, Endo and Epi CM. In non-loaded CM, sarcomere shortening between regions compares as follows: Endo > Epi and Endo > PM. During afterloaded contractions, the slope of auxotonic tension-length relation and the Frank-Starling gain index (preload-dependent increase in tension and shortening) follow the sequence of Endo > Epi > PM. In terms of apico-basal gradients, time-to-peak sarcomere shortening was greater in Apex compared to Centre and Base in non-loaded CM only. Thus, CM from PM show the least pronounced preload-dependent activation of force across the LV regions assessed, while CM from Endo regions show the strongest response. This is in keeping with prior in situ observations on the smaller extent of PM shortening and their thus lower functional requirement for sensitivity to preload, compared to LV free wall. The here identified regional differences in cellular Frank-Starling responses illustrate the extent to which CM mechanical responses appear to be in keeping with in situ differences in mechanical demand.
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Ventrículos do Coração , Miócitos Cardíacos , Animais , Endocárdio/fisiologia , Contração Miocárdica/fisiologia , Miócitos Cardíacos/fisiologia , Músculos Papilares , CoelhosRESUMO
AIMS: Atrial Fibrillation (AF) is an arrhythmia of increasing prevalence in the aging populations of developed countries. One of the important indicators of AF is sustained atrial dilatation, highlighting the importance of mechanical overload in the pathophysiology of AF. The mechanisms by which atrial cells, including fibroblasts, sense and react to changing mechanical forces, are not fully elucidated. Here, we characterise stretch-activated ion channels (SAC) in human atrial fibroblasts and changes in SAC- presence and activity associated with AF. METHODS AND RESULTS: Using primary cultures of human atrial fibroblasts, isolated from patients in sinus rhythm or sustained AF, we combine electrophysiological, molecular and pharmacological tools to identify SAC. Two electrophysiological SAC- signatures were detected, indicative of cation-nonselective and potassium-selective channels. Using siRNA-mediated knockdown, we identified the cation-nonselective SAC as Piezo1. Biophysical properties of the potassium-selective channel, its sensitivity to calcium, paxilline or iberiotoxin (blockers), and NS11021 (activator), indicated presence of calcium-dependent 'big potassium channels' (BKCa). In cells from AF patients, Piezo1 activity and mRNA expression levels were higher than in cells from sinus rhythm patients, while BKCa activity (but not expression) was downregulated. Both Piezo1-knockdown and removal of extracellular calcium from the patch pipette resulted in a significant reduction of BKCa current during stretch. No co-immunoprecipitation of Piezo1 and BKCa was detected. CONCLUSIONS: Human atrial fibroblasts contain at least two types of ion channels that are activated during stretch: Piezo1 and BKCa. While Piezo1 is directly stretch-activated, the increase in BKCa activity during mechanical stimulation appears to be mainly secondary to calcium influx via SAC such as Piezo1. During sustained AF, Piezo1 is increased, while BKCa activity is reduced, highlighting differential regulation of both channels. Our data support the presence and interplay of Piezo1 and BKCa in human atrial fibroblasts in the absence of physical links between the two channel proteins.
Assuntos
Arritmia Sinusal/metabolismo , Fibrilação Atrial/metabolismo , Remodelamento Atrial/genética , Átrios do Coração/metabolismo , Canais Iônicos/metabolismo , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Miofibroblastos/metabolismo , Transdução de Sinais/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Arritmia Sinusal/patologia , Arritmia Sinusal/cirurgia , Fibrilação Atrial/patologia , Fibrilação Atrial/cirurgia , Remodelamento Atrial/efeitos dos fármacos , Cálcio/metabolismo , Células Cultivadas , Feminino , Técnicas de Silenciamento de Genes , Átrios do Coração/patologia , Humanos , Indóis/farmacologia , Canais Iônicos/genética , Transporte de Íons/efeitos dos fármacos , Transporte de Íons/genética , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/agonistas , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/antagonistas & inibidores , Masculino , Pessoa de Meia-Idade , Peptídeos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Tetrazóis/farmacologia , Tioureia/análogos & derivados , Tioureia/farmacologia , TransfecçãoRESUMO
AIMS: Short-QT syndrome 1 (SQT1) is an inherited channelopathy with accelerated repolarization due to gain-of-function in HERG/IKr. Patients develop atrial fibrillation, ventricular tachycardia (VT), and sudden cardiac death with pronounced inter-individual variability in phenotype. We generated and characterized transgenic SQT1 rabbits and investigated electrical remodelling. METHODS AND RESULTS: Transgenic rabbits were generated by oocyte-microinjection of ß-myosin-heavy-chain-promoter-KCNH2/HERG-N588K constructs. Short-QT syndrome 1 and wild type (WT) littermates were subjected to in vivo ECG, electrophysiological studies, magnetic resonance imaging, and ex vivo action potential (AP) measurements. Electrical remodelling was assessed using patch clamp, real-time PCR, and western blot. We generated three SQT1 founders. QT interval was shorter and QT/RR slope was shallower in SQT1 than in WT (QT, 147.8 ± 2 ms vs. 166.4 ± 3, P < 0.0001). Atrial and ventricular refractoriness and AP duration were shortened in SQT1 (vAPD90, 118.6 ± 5 ms vs. 154.4 ± 2, P < 0.0001). Ventricular tachycardia/fibrillation (VT/VF) inducibility was increased in SQT1. Systolic function was unaltered but diastolic relaxation was enhanced in SQT1. IKr-steady was increased with impaired inactivation in SQT1, while IKr-tail was reduced. Quinidine prolonged/normalized QT and action potential duration (APD) in SQT1 rabbits by reducing IKr. Diverse electrical remodelling was observed: in SQT1, IK1 was decreased-partially reversing the phenotype-while a small increase in IKs may partly contribute to an accentuation of the phenotype. CONCLUSION: Short-QT syndrome 1 rabbits mimic the human disease phenotype on all levels with shortened QT/APD and increased VT/VF-inducibility and show similar beneficial responses to quinidine, indicating their value for elucidation of arrhythmogenic mechanisms and identification of novel anti-arrhythmic strategies.
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Potenciais de Ação , Arritmias Cardíacas , Átrios do Coração/fisiopatologia , Sistema de Condução Cardíaco/anormalidades , Cardiopatias Congênitas , Ventrículos do Coração/fisiopatologia , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/genética , Potenciais de Ação/fisiologia , Animais , Animais Geneticamente Modificados , Antiarrítmicos/farmacologia , Arritmias Cardíacas/genética , Arritmias Cardíacas/fisiopatologia , Modelos Animais de Doenças , Eletrocardiografia , Feminino , Sistema de Condução Cardíaco/fisiopatologia , Cardiopatias Congênitas/genética , Cardiopatias Congênitas/fisiopatologia , Humanos , Masculino , Fenótipo , Quinidina/farmacologia , CoelhosRESUMO
Atria-selective antiarrhythmic drugs in need of alliance partners. Guideline-based treatment of atrial fibrillation (AF) comprises prevention of thromboembolism and stroke, as well as antiarrhythmic therapy by drugs, electrical rhythm conversion, ablation and surgical procedures. Conventional antiarrhythmic drugs are burdened with unwanted side effects including a propensity of triggering life-threatening ventricular fibrillation. In order to solve this therapeutic dilemma, 'atria-selective' antiarrhythmic drugs have been developed for the treatment of supraventricular arrhythmias. These drugs are designed to aim at atrial targets, taking advantage of differences in atrial and ventricular ion channel expression and function. However it is not clear, whether such drugs are sufficiently antiarrhythmic or whether they are in need of an alliance partner for clinical efficacy. Atria-selective Na+ channel blockers display fast dissociation kinetics and high binding affinity to inactivated channels. Compounds targeting atria-selective K+ channels include blockers of ultra rapid delayed rectifier (Kv1.5) or acetylcholine-activated inward rectifier K+ channels (Kir3.x), inward rectifying K+ channels (Kir2.x), Ca2+-activated K+ channels of small conductance (SK), weakly rectifying two-pore domain K+ channels (K2P), and transient receptor potential channels (TRP). Despite good antiarrhythmic data from in-vitro and animal model experiments, clinical efficacy of atria-selective antiarrhythmic drugs remains to be demonstrated. In the present review we will briefly summarize the novel compounds and their proposed antiarrhythmic action. In addition, we will discuss the evidence for putative improvement of antiarrhythmic efficacy and potency by addressing multiple pathophysiologically relevant targets as possible alliance partners.
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Antiarrítmicos/uso terapêutico , Fibrilação Atrial/tratamento farmacológico , Átrios do Coração/efeitos dos fármacos , Animais , Fibrilação Atrial/fisiopatologia , Humanos , Canais Iônicos/fisiologiaRESUMO
Mechanical forces will have been omnipresent since the origin of life, and living organisms have evolved mechanisms to sense, interpret, and respond to mechanical stimuli. The cardiovascular system in general, and the heart in particular, is exposed to constantly changing mechanical signals, including stretch, compression, bending, and shear. The heart adjusts its performance to the mechanical environment, modifying electrical, mechanical, metabolic, and structural properties over a range of time scales. Many of the underlying regulatory processes are encoded intracardially and are, thus, maintained even in heart transplant recipients. Although mechanosensitivity of heart rhythm has been described in the medical literature for over a century, its molecular mechanisms are incompletely understood. Thanks to modern biophysical and molecular technologies, the roles of mechanical forces in cardiac biology are being explored in more detail, and detailed mechanisms of mechanotransduction have started to emerge. Mechano-gated ion channels are cardiac mechanoreceptors. They give rise to mechano-electric feedback, thought to contribute to normal function, disease development, and, potentially, therapeutic interventions. In this review, we focus on acute mechanical effects on cardiac electrophysiology, explore molecular candidates underlying observed responses, and discuss their pharmaceutical regulation. From this, we identify open research questions and highlight emerging technologies that may help in addressing them.
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Arritmias Cardíacas/metabolismo , Frequência Cardíaca , Ativação do Canal Iônico , Canais Iônicos/metabolismo , Mecanorreceptores/metabolismo , Mecanotransdução Celular , Miócitos Cardíacos/metabolismo , Potenciais de Ação , Animais , Arritmias Cardíacas/fisiopatologia , Forma Celular , Tamanho Celular , HumanosRESUMO
Caveolae are signal transduction centers, yet their subcellular distribution and preservation in cardiac myocytes after cell isolation are not well documented. Here, we quantify caveolae located within 100 nm of the outer cell surface membrane in rabbit single-ventricular cardiomyocytes over 8 h post-isolation and relate this to the presence of caveolae in intact tissue. Hearts from New Zealand white rabbits were either chemically fixed by coronary perfusion or enzymatically digested to isolate ventricular myocytes, which were subsequently fixed at 0, 3, and 8 h post-isolation. In live cells, the patch-clamp technique was used to measure whole-cell plasma membrane capacitance, and in fixed cells, caveolae were quantified by transmission electron microscopy. Changes in cell-surface topology were assessed using scanning electron microscopy. In fixed ventricular myocardium, dual-axis electron tomography was used for three-dimensional reconstruction and analysis of caveolae in situ. The presence and distribution of surface-sarcolemmal caveolae in freshly isolated cells matches that of intact myocardium. With time, the number of surface-sarcolemmal caveolae decreases in isolated cardiomyocytes. This is associated with a gradual increase in whole-cell membrane capacitance. Concurrently, there is a significant increase in area, diameter, and circularity of sub-sarcolemmal mitochondria, indicative of swelling. In addition, electron tomography data from intact heart illustrate the regular presence of caveolae not only at the surface sarcolemma, but also on transverse-tubular membranes in ventricular myocardium. Thus, caveolae are dynamic structures, present both at surface-sarcolemmal and transverse-tubular membranes. After cell isolation, the number of surface-sarcolemmal caveolae decreases significantly within a time frame relevant for single-cell research. The concurrent increase in cell capacitance suggests that membrane incorporation of surface-sarcolemmal caveolae underlies this, but internalization and/or micro-vesicle loss to the extracellular space may also contribute. Given that much of the research into cardiac caveolae-dependent signaling utilizes isolated cells, and since caveolae-dependent pathways matter for a wide range of other study targets, analysis of isolated cell data should take the time post-isolation into account.
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Cavéolas , Ventrículos do Coração/citologia , Miócitos Cardíacos/citologia , Animais , Cavéolas/fisiologia , Separação Celular , Células Cultivadas , Capacitância Elétrica , Tomografia com Microscopia Eletrônica , Imageamento Tridimensional , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Mitocôndrias/fisiologia , Modelos Biológicos , Miócitos Cardíacos/fisiologia , Técnicas de Patch-Clamp , Coelhos , Sarcolema/fisiologia , Propriedades de Superfície , Fixação de TecidosRESUMO
The collecting duct (CD) is the final segment of the kidney involved in the fine regulation of osmotic and ionic balance. During dehydration, arginine vasopressin (AVP) stimulates the expression and trafficking of aquaporin 2 (AQP2) to the apical membrane of CD principal cells, thereby allowing water reabsorption from the primary urine. Conversely, when the secretion of AVP is lowered, as for instance upon water ingestion or as a consequence of diabetes insipidus, the CD remains water impermeable leading to enhanced diuresis and urine dilution. In addition, an AVP-independent mechanism of urine dilution is also at play when fasting. Piezo1/2 are recently discovered essential components of the non-selective mechanically activated cationic channels. Using quantitative PCR analysis and taking advantage of a ß-galactosidase reporter mouse, we demonstrate that Piezo1 is preferentially expressed in CD principal cells of the inner medulla at the adult stage, unlike Piezo2. Remarkably, siRNAs knock-down or conditional genetic deletion of Piezo1 specifically in renal cells fully suppresses activity of the stretch-activated non-selective cationic channels (SACs). Piezo1 in CD cells is dispensable for urine concentration upon dehydration. However, urinary dilution and decrease in urea concentration following rehydration are both significantly delayed in the absence of Piezo1. Moreover, decreases in urine osmolarity and urea concentration associated with fasting are fully impaired upon Piezo1 deletion in CD cells. Altogether, these findings indicate that Piezo1 is critically required for SAC activity in CD principal cells and is implicated in urinary osmoregulation.
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Canais Iônicos/metabolismo , Túbulos Renais Coletores/metabolismo , Túbulos Renais Coletores/fisiologia , Equilíbrio Hidroeletrolítico/fisiologia , Animais , Aquaporina 2/metabolismo , Arginina Vasopressina/farmacologia , Linhagem Celular , Desidratação/metabolismo , Desidratação/fisiopatologia , Diurese/fisiologia , Túbulos Renais Coletores/efeitos dos fármacos , Camundongos , Concentração Osmolar , Equilíbrio Hidroeletrolítico/efeitos dos fármacosRESUMO
Members of the K(2P) potassium channel family regulate neuronal excitability and are implicated in pain, anaesthetic responses, thermosensation, neuroprotection, and mood. Unlike other potassium channels, K(2P)s are gated by remarkably diverse stimuli that include chemical, thermal, and mechanical modalities. It has remained unclear whether the various gating inputs act through separate or common channel elements. Here, we show that protons, heat, and pressure affect activity of the prototypical, polymodal K(2P), K(2P)2.1 (KCNK2/TREK-1), at a common molecular gate that comprises elements of the pore-forming segments and the N-terminal end of the M4 transmembrane segment. We further demonstrate that the M4 gating element is conserved among K(2P)s and is employed regardless of whether the gating stimuli are inhibitory or activating. Our results define a unique gating mechanism shared by K(2P) family members and suggest that their diverse sensory properties are achieved by coupling different molecular sensors to a conserved core gating apparatus.
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Ativação do Canal Iônico , Canais de Potássio de Domínios Poros em Tandem/fisiologia , Sequência de Aminoácidos , Animais , Temperatura Alta , Camundongos , Dados de Sequência Molecular , Canais de Potássio de Domínios Poros em Tandem/genética , Pressão , PrótonsRESUMO
Mechanical forces associated with fluid flow and/or circumferential stretch are sensed by renal epithelial cells and contribute to both adaptive or disease states. Non-selective stretch-activated ion channels (SACs), characterized by a lack of inactivation and a remarkably slow deactivation, are active at the basolateral side of renal proximal convoluted tubules. Knockdown of Piezo1 strongly reduces SAC activity in proximal convoluted tubule epithelial cells. Similarly, overexpression of Polycystin-2 (PC2) or, to a greater extent its pathogenic mutant PC2-740X, impairs native SACs. Moreover, PC2 inhibits exogenous Piezo1 SAC activity. PC2 coimmunoprecipitates with Piezo1 and deletion of its N-terminal domain prevents both this interaction and inhibition of SAC activity. These findings indicate that renal SACs depend on Piezo1, but are critically conditioned by PC2.
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Células Epiteliais/metabolismo , Canais Iônicos/metabolismo , Mecanotransdução Celular , Canais de Cátion TRPP/metabolismo , Potenciais de Ação , Animais , Sítios de Ligação , Células COS , Células Cultivadas , Chlorocebus aethiops , Células Epiteliais/fisiologia , Túbulos Renais/citologia , Mutação , Ligação Proteica , Canais de Cátion TRPC/metabolismo , Canais de Cátion TRPP/química , Canais de Cátion TRPP/genéticaRESUMO
BACKGROUND: Arrhythmias may originate from surgically unaffected right ventricular (RV) regions in patients with tetralogy of Fallot (TOF). We aimed to investigate action potential (AP) remodelling and arrhythmia susceptibility in RV myocardium of patients with repaired and with unrepaired TOF, identify possible correlations with clinical phenotype and myocardial fibrosis, and compare findings with data from patients with atrial septal defect (ASD), a less severe congenital heart disease. METHODS: Intracellular AP were recorded ex vivo in RV outflow tract samples from 22 TOF and three ASD patients. Arrhythmias were provoked by superfusion with solutions containing reduced potassium and barium chloride, or isoprenaline. Myocardial fibrosis was quantified histologically and associations between clinical phenotype, AP shape, tissue arrhythmia propensity, and fibrosis were examined. RESULTS: Electrophysiological abnormalities (arrhythmias, AP duration [APD] alternans, impaired APD shortening at increased stimulation frequencies) were generally present in TOF tissue, even from infants, but rare or absent in ASD samples. More severely diseased and acyanotic patients, pronounced tissue susceptibility to arrhythmogenesis, and greater fibrosis extent were associated with longer APD. In contrast, APD was shorter in tissue from patients with pre-operative cyanosis. Increased fibrosis and repaired-TOF status were linked to tissue arrhythmia inducibility. CONCLUSIONS: Functional and structural tissue remodelling may explain arrhythmic activity in TOF patients, even at a very young age. Surprisingly, clinical acyanosis appears to be associated with more severe arrhythmogenic remodelling. Further research into the clinical drivers of structural and electrical myocardial alterations, and the relation between them, is needed to identify predictive factors for patients at risk.
Assuntos
Comunicação Interatrial , Tetralogia de Fallot , Humanos , Tetralogia de Fallot/complicações , Tetralogia de Fallot/cirurgia , Potenciais de Ação , Arritmias Cardíacas , Fibrose , Comunicação Interatrial/complicações , Gravidade do PacienteRESUMO
Vascular smooth muscle cells (VSMCs) play a key role in aortic aneurysm formation. Bone morphogenetic proteins (BMPs) have been implicated as important regulators of VSMC phenotype, and dysregulation of the BMP pathway has been shown to be associated with vascular diseases. The aim of this study was to investigate for the first time the effects of BMP-4 on the VSMC phenotype and to understand its role in the development of thoracic aortic aneurysms (TAAs). Using the angiotensin II (AngII) osmotic pump model in mice, aortas from mice with VSMC-specific BMP-4 deficiency showed changes similar to AngII-infused aortas, characterised by a loss of contractile markers, increased fibrosis, and activation of matrix metalloproteinase 9. When BMP-4 deficiency was combined with AngII infusion, there was a significantly higher rate of apoptosis and aortic dilatation. In vitro, VSMCs with mRNA silencing of BMP-4 displayed a dedifferentiated phenotype with activated canonical BMP signalling. In contrast, BMP-2-deficient VSMCs exhibited the opposite phenotype. The compensatory regulation between BMP-2 and BMP-4, with BMP-4 promoting the contractile phenotype, appeared to be independent of the canonical signalling pathway. Taken together, these results demonstrate the impact of VSMC-specific BMP-4 deficiency on TAA development.
Assuntos
Aneurisma da Aorta Torácica , Proteína Morfogenética Óssea 2 , Proteína Morfogenética Óssea 4 , Músculo Liso Vascular , Miócitos de Músculo Liso , Animais , Masculino , Camundongos , Angiotensina II/farmacologia , Aneurisma da Aorta Torácica/metabolismo , Aneurisma da Aorta Torácica/patologia , Aneurisma da Aorta Torácica/genética , Apoptose/efeitos dos fármacos , Proteína Morfogenética Óssea 2/metabolismo , Proteína Morfogenética Óssea 4/metabolismo , Modelos Animais de Doenças , Camundongos Endogâmicos C57BL , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Fenótipo , Transdução de SinaisRESUMO
In the original publication [...].
RESUMO
The human heart responds to various diseases with structural, mechanical, and electrical remodelling processes [...].
Assuntos
Remodelamento Atrial , Cardiopatias , Insuficiência Cardíaca , Humanos , Remodelação Ventricular , CoraçãoRESUMO
Cardiomyocytes sense and shape their mechanical environment, contributing to its dynamics by their passive and active mechanical properties. While axial forces generated by contracting cardiomyocytes have been amply investigated, the corresponding radial mechanics remain poorly characterized. Our aim is to simultaneously monitor passive and active forces, both axially and radially, in cardiomyocytes freshly isolated from adult mouse ventricles. To do so, we combine a carbon fibre (CF) set-up with a custom-made atomic force microscope (AFM). CF allows us to apply stretch and to record passive and active forces in the axial direction. The AFM, modified for frontal access to fit in CF, is used to characterize radial cell mechanics. We show that stretch increases the radial elastic modulus of cardiomyocytes. We further find that during contraction, cardiomyocytes generate radial forces that are reduced, but not abolished, when cells are forced to contract near isometrically. Radial forces may contribute to ventricular wall thickening during contraction, together with the dynamic re-orientation of cells and sheetlets in the myocardium. This new approach for characterizing cell mechanics allows one to obtain a more detailed picture of the balance of axial and radial mechanics in cardiomyocytes at rest, during stretch, and during contraction. This article is part of the theme issue 'The cardiomyocyte: new revelations on the interplay between architecture and function in growth, health, and disease'.
Assuntos
Miócitos Cardíacos , Animais , Fibra de Carbono , Camundongos , Microscopia de Força Atômica/métodosRESUMO
In the rapidly expanding field of molecular optogenetics, the performance of the engineered systems relies on the switching properties of the underlying genetically encoded photoreceptors. In this study, the bacterial phytochromes Cph1 and DrBphP are engineered, recombinantly produced in Escherichia coli, and characterized regarding their switching properties in order to synthesize biohybrid hydrogels with increased light-responsive stiffness modulations. The R472A mutant of the cyanobacterial phytochrome 1 (Cph1) is identified to confer the phytochrome-based hydrogels with an increased dynamic range for the storage modulus but a different light-response for the loss modulus compared to the original Cph1-based hydrogel. Stiffness measurements of human atrial fibroblasts grown on these hydrogels suggest that differences in the loss modulus at comparable changes in the storage modulus affect cell stiffness and thus underline the importance of matrix viscoelasticity on cellular mechanotransduction. The hydrogels presented here are of interest for analyzing how mammalian cells respond to dynamic viscoelastic cues. Moreover, the Cph1-R472A mutant, as well as the benchmarking of the other phytochrome variants, are expected to foster the development and performance of future optogenetic systems.