RESUMO
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
Recurrent episodes of decompensated heart failure (HF) represent an emerging cause of hospitalizations in developed countries with an urgent need for effective therapies. Recently, the pregnancy-related hormone relaxin (RLN) was found to mediate cardio-protective effects and act as a positive inotrope in the cardiovascular system. RLN binds to the RLN family peptide receptor 1 (RXFP1), which is predominantly expressed in atrial cardiomyocytes. We therefore hypothesized that ventricular RXFP1 expression might exert potential therapeutic effects in an in vivo model of cardiac dysfunction. Thus, mice were exposed to pressure overload by transverse aortic constriction and treated with AAV9 to ectopically express RXFP1. To activate RXFP1 signaling, RLN was supplemented subcutaneously. Ventricular RXFP1 expression was well tolerated. Additional RLN administration not only abrogated HF progression but restored left ventricular systolic function. In accordance, upregulation of fetal genes and pathological remodeling markers were significantly reduced. In vitro, RLN stimulation of RXFP1-expressing cardiomyocytes induced downstream signaling, resulting in protein kinase A (PKA)-specific phosphorylation of phospholamban (PLB), which was distinguishable from ß-adrenergic activation. PLB phosphorylation corresponded to increased calcium amplitude and contractility. In conclusion, our results demonstrate that ligand-activated cardiac RXFP1 gene therapy represents a therapeutic approach to attenuate HF with the potential to adjust therapy by exogenous RLN supplementation.
Assuntos
Terapia Genética/métodos , Insuficiência Cardíaca/terapia , Receptores Acoplados a Proteínas G/genética , Receptores de Peptídeos/genética , Relaxina/administração & dosagem , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Dependovirus/genética , Modelos Animais de Doenças , Vetores Genéticos/administração & dosagem , Insuficiência Cardíaca/etiologia , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/fisiopatologia , Humanos , Injeções Subcutâneas , Ligantes , Masculino , Camundongos , Fosforilação , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Peptídeos/metabolismo , Resultado do Tratamento , Função VentricularRESUMO
AIMS: Atrial fibrillation (AF) is a commonly occurring arrhythmia after cardiac surgery (postoperative AF, poAF) and is associated with poorer outcomes. Considering that reduced atrial contractile function is a predictor of poAF and that Ca2+ plays an important role in both excitation-contraction coupling and atrial arrhythmogenesis, this study aims to test whether alterations of intracellular Ca2+ handling contribute to impaired atrial contractility and to the arrhythmogenic substrate predisposing patients to poAF. METHODS AND RESULTS: Right atrial appendages were obtained from patients in sinus rhythm undergoing open-heart surgery. Cardiomyocytes were investigated by simultaneous measurement of [Ca2+]i and action potentials (APs, patch-clamp). Patients were followed-up for 6 days to identify those with and without poAF. Speckle-tracking analysis of preoperative echocardiography revealed reduced left atrial contraction strain in poAF patients. At the time of surgery, cellular Ca2+ transients (CaTs) and the sarcoplasmic reticulum (SR) Ca2+ content were smaller in the poAF group. CaT decay was slower in poAF, but the decay of caffeine-induced Ca2+ transients was unaltered, suggesting preserved sodium-calcium exchanger function. In agreement, western blots revealed reduced SERCA2a expression in poAF patients but unaltered phospholamban expression/phosphorylation. Computational modelling indicated that reduced SERCA activity promotes occurrence of CaT and AP alternans. Indeed, alternans of CaT and AP occurred more often and at lower stimulation frequencies in atrial myocytes from poAF patients. Resting membrane potential and AP duration were comparable between both groups at various pacing frequencies (0.25-8 Hz). CONCLUSIONS: Biochemical, functional, and modelling data implicate reduced SERCA-mediated Ca2+ reuptake into the SR as a major contributor to impaired preoperative atrial contractile function and to the pre-existing arrhythmogenic substrate in patients developing poAF.
Assuntos
Potenciais de Ação , Apêndice Atrial/metabolismo , Fibrilação Atrial/etiologia , Sinalização do Cálcio , Cálcio/metabolismo , Procedimentos Cirúrgicos Cardíacos/efeitos adversos , Frequência Cardíaca , Miócitos Cardíacos/metabolismo , Idoso , Apêndice Atrial/fisiopatologia , Fibrilação Atrial/metabolismo , Fibrilação Atrial/fisiopatologia , Proteínas de Ligação ao Cálcio/metabolismo , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fosforilação , Retículo Sarcoplasmático/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Fatores de TempoRESUMO
Epidemiological studies of the COVID-19 pandemic have revealed evidence of cardiac involvement and documented that myocardial injury and myocarditis are predictors of poor outcomes. Nonetheless, little is understood regarding SARS-CoV-2 tropism within the heart and whether cardiac complications result directly from myocardial infection. Here, we develop a human engineered heart tissue model and demonstrate that SARS-CoV-2 selectively infects cardiomyocytes. Viral infection is dependent on expression of angiotensin-I converting enzyme 2 (ACE2) and endosomal cysteine proteases, suggesting an endosomal mechanism of cell entry. After infection with SARS-CoV-2, engineered tissues display typical features of myocarditis, including cardiomyocyte cell death, impaired cardiac contractility, and innate immune cell activation. Consistent with these findings, autopsy tissue obtained from individuals with COVID-19 myocarditis demonstrated cardiomyocyte infection, cell death, and macrophage-predominate immune cell infiltrate. These findings establish human cardiomyocyte tropism for SARS-CoV-2 and provide an experimental platform for interrogating and mitigating cardiac complications of COVID-19.
RESUMO
BACKGROUND: Despite an increasing understanding of atrial fibrillation (AF) pathophysiology, translation into mechanism-based treatment options is lacking. In atrial cardiomyocytes of patients with chronic AF, expression, and function of tandem of P domains in a weak inward rectifying TASK-1 (K+ channel-related acid-sensitive K+ channel-1) (K2P3.1) atrial-specific 2-pore domain potassium channels is enhanced, resulting in action potential duration shortening. TASK-1 channel inhibition prevents action potential duration shortening to maintain values observed among sinus rhythm subjects. The present preclinical study used a porcine AF model to evaluate the antiarrhythmic efficacy of TASK-1 inhibition by adeno-associated viral anti-TASK-1-siRNA (small interfering RNA) gene transfer. METHODS: AF was induced in domestic pigs by atrial burst stimulation via implanted pacemakers. Adeno-associated viral vectors carrying anti-TASK-1-siRNA were injected into both atria to suppress TASK-1 channel expression. After the 14-day follow-up period, porcine cardiomyocytes were isolated from right and left atrium, followed by electrophysiological and molecular characterization. RESULTS: AF was associated with increased TASK-1 transcript, protein and ion current levels leading to shortened action potential duration in atrial cardiomyocytes compared to sinus rhythm controls, similar to previous findings in humans. Anti-TASK-1 adeno-associated viral application significantly reduced AF burden in comparison to untreated AF pigs. Antiarrhythmic effects of anti-TASK-1-siRNA were associated with reduction of TASK-1 currents and prolongation of action potential durations in atrial cardiomyocytes to sinus rhythm values. Conclusions Adeno-associated viral-based anti-TASK-1 gene therapy suppressed AF and corrected cellular electrophysiological remodeling in a porcine model of AF. Suppression of AF through selective reduction of TASK-1 currents represents a new option for antiarrhythmic therapy.
Assuntos
Antiarrítmicos/uso terapêutico , Fibrilação Atrial/genética , Remodelamento Atrial/fisiologia , Regulação da Expressão Gênica , Terapia Genética/métodos , Átrios do Coração/fisiopatologia , Proteínas do Tecido Nervoso/genética , Canais de Potássio de Domínios Poros em Tandem/genética , Potenciais de Ação/fisiologia , Animais , Fibrilação Atrial/metabolismo , Fibrilação Atrial/terapia , Modelos Animais de Doenças , Eletrocardiografia , Miócitos Cardíacos/metabolismo , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/biossíntese , Canais de Potássio de Domínios Poros em Tandem/antagonistas & inibidores , Canais de Potássio de Domínios Poros em Tandem/biossíntese , RNA/genética , Ratos , SuínosRESUMO
BACKGROUND/AIMS: TASK channels belong to the two-pore-domain potassium (K2P) channel family. TASK-1 is discussed to contribute to chronic atrial fibrillation (AFib) and has been together with uncoupling protein 1 found as a marker protein of brown adipose tissue (BAT) fat. In addition, TASK-1 was linked in a genome-wide association study to an increased body mass index. A recent study showed that TASK-1 inhibition is causing obesity in mice by a BAT whitening and that these effects are linked to the mineralocorticoid receptor pathway, albeit the mechanism remained elusive. Therefore, we aimed to probe whether K2P channels are regulated by serum- and glucocorticoid-inducible kinases (SGKs) which are known to modify many cellular functions by modulating ion channels. METHODS: To this end we used functional co-expression studies and chemiluminescence-assays in Xenopus oocytes, together with fluorescence imaging and quantitative PCR experiments. RESULTS: SGKs and proteinkinase B (PKB) induced a strong, dose- and time-dependent current reduction of TASK-1 and TASK-3. SGK co-expression reduced the surface expression of TASK-1/3, leading to a predominant localization of the channels into late endosomes. The down regulation of TASK-3 channels was abrogated by the dynamin inhibitor dynasore, confirming a role of SGKs in TASK-1/3 channel endocytosis. CONCLUSION: Stress-mediated changes in SGK expression pattern or activation is likely to alter TASK-1/3 expression at the surface membrane. The observed TASK-1 regulation might contribute to the pathogenesis of chronic AFib and provide a mechanistic link between increased mineralocorticoid levels and TASK-1 reduction, both linked to BAT whitening.
Assuntos
Proteínas do Tecido Nervoso/metabolismo , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Proteínas Quinases Dependentes de 3-Fosfoinositídeo/metabolismo , Animais , Células COS , Chlorocebus aethiops , Clatrina/metabolismo , Endocitose , Endossomos/metabolismo , Células HeLa , Humanos , Hidrazonas/farmacologia , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/metabolismo , Medições Luminescentes , Microscopia de Fluorescência , Proteínas do Tecido Nervoso/genética , Oócitos/química , Oócitos/fisiologia , Técnicas de Patch-Clamp , Plasmídeos/genética , Plasmídeos/metabolismo , Canais de Potássio de Domínios Poros em Tandem/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Imagem com Lapso de Tempo , Xenopus laevis/crescimento & desenvolvimentoRESUMO
Two-pore-domain potassium (K2P) channels modulate cellular excitability. The significance of stretch-activated cardiac K2P channels (K2P2.1, TREK-1, KCNK2; K2P4.1, TRAAK, KCNK4; K2P10.1, TREK-2, KCNK10) in heart disease has not been elucidated in detail. The aim of this work was to assess expression and remodeling of mechanosensitive K2P channels in atrial fibrillation (AF) and heart failure (HF) patients in comparison to murine models. Cardiac K2P channel levels were quantified in atrial (A) and ventricular (V) tissue obtained from patients undergoing open heart surgery. In addition, control mice and mouse models of AF (cAMP-response element modulator (CREM)-IbΔC-X transgenic animals) or HF (cardiac dysfunction induced by transverse aortic constriction, TAC) were employed. Human and murine KCNK2 displayed highest mRNA abundance among mechanosensitive members of the K2P channel family (V > A). Disease-associated K2P2.1 remodeling was studied in detail. In patients with impaired left ventricular function, atrial KCNK2 (K2P2.1) mRNA and protein expression was significantly reduced. In AF subjects, downregulation of atrial and ventricular KCNK2 (K2P2.1) mRNA and protein levels was observed. AF-associated suppression of atrial Kcnk2 (K2P2.1) mRNA and protein was recapitulated in CREM-transgenic mice. Ventricular Kcnk2 expression was not significantly altered in mouse models of disease. In conclusion, mechanosensitive K2P2.1 and K2P10.1 K+ channels are expressed throughout the heart. HF- and AF-associated downregulation of KCNK2 (K2P2.1) mRNA and protein levels suggest a mechanistic contribution to cardiac arrhythmogenesis.
Assuntos
Fibrilação Atrial/metabolismo , Insuficiência Cardíaca/metabolismo , Fenômenos Mecânicos , Miocárdio/metabolismo , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Idoso , Fibrilação Atrial/genética , Fenômenos Biomecânicos , Regulação para Baixo , Feminino , Insuficiência Cardíaca/genética , Humanos , Masculino , Pessoa de Meia-Idade , Modelos Moleculares , Canais de Potássio de Domínios Poros em Tandem/química , Canais de Potássio de Domínios Poros em Tandem/genética , Conformação Proteica , Transporte Proteico , Regulação para CimaRESUMO
AIMS: Atrial fibrillation (AF) prevalence increases with advanced stages of left ventricular (LV) dysfunction. Remote proarrhythmic effects of ventricular dysfunction on atrial electrophysiology remain incompletely understood. We hypothesized that repolarizing K2P3.1 K+ channels, previously implicated in AF pathophysiology, may contribute to shaping the atrial action potential (AP), forming a specific electrical substrate with LV dysfunction that might represent a target for personalized antiarrhythmic therapy. METHODS AND RESULTS: A total of 175 patients exhibiting different stages of LV dysfunction were included. Ion channel expression was quantified by real-time polymerase chain reaction and Western blot. Membrane currents and APs were recorded from atrial cardiomyocytes using the patch-clamp technique. Severely reduced LV function was associated with decreased atrial K2P3.1 expression in sinus rhythm patients. In contrast, chronic (c)AF resulted in increased K2P3.1 levels, but paroxysmal (p)AF was not linked to significant K2P3.1 remodelling. LV dysfunction-related suppression of K2P3.1 currents prolonged atrial AP duration (APD) compared with patients with preserved LV function. In individuals with concomitant LV dysfunction and cAF, APD was determined by LV dysfunction-associated prolongation and by cAF-dependent shortening, respectively, consistent with changes in K2P3.1 abundance. K2P3.1 inhibition attenuated APD shortening in cAF patients irrespective of LV function, whereas in pAF subjects with severely reduced LV function, K2P3.1 blockade resulted in disproportionately high APD prolongation. CONCLUSION: LV dysfunction is associated with reduction of atrial K2P3.1 channel expression, while cAF leads to increased K2P3.1 abundance. Differential remodelling of K2P3.1 and APD provides a basis for patient-tailored antiarrhythmic strategies.
Assuntos
Potenciais de Ação/fisiologia , Antiarrítmicos/uso terapêutico , Fibrilação Atrial/fisiopatologia , Proteínas do Tecido Nervoso/metabolismo , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Disfunção Ventricular Esquerda/fisiopatologia , Idoso , Fibrilação Atrial/tratamento farmacológico , Índice de Massa Corporal , Doença do Sistema de Condução Cardíaco/etiologia , Doença do Sistema de Condução Cardíaco/fisiopatologia , Cardiomiopatia Dilatada/fisiopatologia , Regulação para Baixo/fisiologia , Feminino , Humanos , Masculino , Proteínas do Tecido Nervoso/antagonistas & inibidores , Canais de Potássio de Domínios Poros em Tandem/antagonistas & inibidores , Distribuição por Sexo , Fumar/efeitos adversos , Fumar/fisiopatologia , Regulação para Cima/fisiologia , Remodelação Ventricular/fisiologiaRESUMO
The human KCNK18 gene is predominantly expressed in brain, spinal cord, and dorsal root ganglion neurons. Encoded K2P18.1K(+) channels are functionally implicated in migraine, pain and anesthesia. Data delineating the in vivo significance of K2P18.1 are still limited owing to a lack of model systems allowing for rapid, whole organism phenotypic analyses. We hypothesized that zebrafish (Danio rerio) might close this scientific gap. This work was designed to characterize the zebrafish ortholog of K2P18.1 in comparison to human K2P18.1 channels. The complete coding sequence of zKCNK18 was amplified from zebrafish cDNA. Zebrafish KCNK18 expression was assessed by in situ hybridization. Human and zebrafish K2P18.1 currents were functionally analyzed using two-electrode voltage clamp electrophysiology and the Xenopus oocyte expression system. KCNK18 mRNA is expressed in zebrafish brain and eyes. Human and zebrafish K2P18.1 proteins share 32 % identity. Zebrafish K2P18.1 channels mediate K(+)-selective background currents that stabilize the negative resting membrane potential. Functional similarities between human and zK2P18.1 currents include open rectification properties, inhibition by barium, and regulation by signaling molecules protein kinase (PK)C, PKA, and phospholipase C. In contrast to the human ortholog, zK2P18.1 exhibited reduced sensitivity to elevation of intracellular calcium levels by ionomycin and was virtually insensitive to inhibition by quinidine. Zebrafish and human K2P18.1 channels share functional and regulatory properties, indicating that the zebrafish may serve as model to assess K2P18.1 function in vivo. However, distinct differences in K2P18.1 current regulation require careful consideration when zebrafish data are extrapolated to human physiology.