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1.
Proc Natl Acad Sci U S A ; 120(28): e2210152120, 2023 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-37406102

RESUMO

Sepsis has emerged as a global health burden associated with multiple organ dysfunction and 20% mortality rate in patients. Numerous clinical studies over the past two decades have correlated the disease severity and mortality in septic patients with impaired heart rate variability (HRV), as a consequence of impaired chronotropic response of sinoatrial node (SAN) pacemaker activity to vagal/parasympathetic stimulation. However, the molecular mechanism(s) downstream to parasympathetic inputs have not been investigated yet in sepsis, particularly in the SAN. Based on electrocardiography, fluorescence Ca2+ imaging, electrophysiology, and protein assays from organ to subcellular level, we report that impaired muscarinic receptor subtype 2-G protein-activated inwardly-rectifying potassium channel (M2R-GIRK) signaling in a lipopolysaccharide-induced proxy septic mouse model plays a critical role in SAN pacemaking and HRV. The parasympathetic responses to a muscarinic agonist, namely IKACh activation in SAN cells, reduction in Ca2+ mobilization of SAN tissues, lowering of heart rate and increase in HRV, were profoundly attenuated upon lipopolysaccharide-induced sepsis. These functional alterations manifested as a direct consequence of reduced expression of key ion-channel components (GIRK1, GIRK4, and M2R) in the mouse SAN tissues and cells, which was further evident in the human right atrial appendages of septic patients and likely not mediated by the common proinflammatory cytokines elevated in sepsis.


Assuntos
Lipopolissacarídeos , Sepse , Humanos , Animais , Camundongos , Lipopolissacarídeos/toxicidade , Lipopolissacarídeos/metabolismo , Nó Sinoatrial/fisiologia , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Transdução de Sinais/fisiologia , Sepse/induzido quimicamente , Sepse/metabolismo
2.
PLoS Comput Biol ; 17(6): e1009091, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34157016

RESUMO

Lung cancer is still a leading cause of death worldwide. In recent years, knowledge has been obtained of the mechanisms modulating ion channel kinetics and thus of cell bioelectric properties, which is promising for oncological biomarkers and targets. The complex interplay of channel expression and its consequences on malignant processes, however, is still insufficiently understood. We here introduce the first approach of an in-silico whole-cell ion current model of a cancer cell, in particular of the A549 human lung adenocarcinoma, including the main functionally expressed ion channels in the plasma membrane as so far known. This hidden Markov-based model represents the electrophysiology behind proliferation of the A549 cell, describing its rhythmic oscillation of the membrane potential able to trigger the transition between cell cycle phases, and it predicts membrane potential changes over the cell cycle provoked by targeted ion channel modulation. This first A549 in-silico cell model opens up a deeper insight and understanding of possible ion channel interactions in tumor development and progression, and is a valuable tool for simulating altered ion channel function in lung cancer electrophysiology.


Assuntos
Adenocarcinoma de Pulmão/metabolismo , Adenocarcinoma de Pulmão/patologia , Canais Iônicos/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Modelos Biológicos , Células A549 , Ciclo Celular , Pontos de Checagem do Ciclo Celular , Proliferação de Células , Biologia Computacional , Simulação por Computador , Humanos , Transporte de Íons , Cadeias de Markov , Potenciais da Membrana , Técnicas de Patch-Clamp
3.
Biochem Biophys Res Commun ; 472(1): 40-5, 2016 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-26902113

RESUMO

We set out to determine the membrane potential (Vm) of the endothelial cell line EA.hy926 and its sensitivity to the antimycotic amphotericin B (AmB), a commonly used antifungal component in cell culture media. We measured the endothelial Vm under various experimental conditions by patch clamp technique and found that Vm of AmB-treated cells is (-12.1 ± 9.3) mV, while in AmB-untreated (control) cells it is (-57.1 ± 4.1) mV. In AmB-free extracellular solutions, Vm recovered toward control levels and this gain in Vm rapidly dissipated upon re-addition of AmB, demonstrating a rapid and reversible effect of AmB on endothelial Vm. The consequences of AmB dependent alterations in endothelial transmembrane potential were tested at the levels of Ca(2+) signaling, of nucleotide concentrations, and energy metabolism. In AmB-treated cells we found substantially reduced Ca(2+) entry (to about 60% of that in control cells) in response to histamine induced endoplasmic reticulum (ER) Ca(2+) depletion, and diminished the ATP-to-ADP ratio (by >30%). Our data demonstrate a marked and experimentally relevant dependence of basic functional parameters of cultured endothelial cells on the presence of the ionophoric antimycotic AmB. The profound and reversible effects of the widely used culture media component AmB need careful consideration when interpreting experimental data obtained under respective culture conditions.


Assuntos
Anfotericina B/toxicidade , Antifúngicos/toxicidade , Células Endoteliais/efeitos dos fármacos , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Técnicas de Cultura de Células/métodos , Linhagem Celular , Meios de Cultura/toxicidade , Células Endoteliais/metabolismo , Humanos , Potenciais da Membrana/efeitos dos fármacos , Técnicas de Patch-Clamp
4.
J Mol Cell Cardiol ; 72: 64-73, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24583250

RESUMO

Lower heart rate is associated with better survival in patients with multiple organ dysfunction syndrome (MODS), a disease mostly caused by sepsis. The benefits of heart rate reduction by ivabradine during MODS are currently being investigated in the MODIfY clinical trial. Ivabradine is a selective inhibitor of the pacemaker current If and since If is impaired by lipopolysaccharide (LPS, endotoxin), a trigger of sepsis, we aimed to explore If blocking potency of ivabradine under elevated endotoxin levels in human atrial cardiomyocytes. Treatment of myocytes with S-LPS (containing the lipid A moiety, a core oligosaccharide and an O-polysaccharide chain) but not R595 (an O-chain lacking LPS-form) caused If inhibition under acute and chronic septic conditions. The specific interaction of S-LPS but not R595 to pacemaker channels HCN2 and HCN4 proves the necessity of O-chain for S-LPS-HCN interaction. The efficacy of ivabradine to block If was reduced under septic conditions, an observation that correlated with lower intracellular ivabradine concentrations in S-LPS- but not R595-treated cardiomyocytes. Computational analysis using a sinoatrial pacemaker cell model revealed that despite a reduction of If under septic conditions, ivabradine further decelerated pacemaking activity. This novel finding, i.e. If inhibition by ivabradine under elevated endotoxin levels in vitro, may provide a molecular understanding for the efficacy of this drug on heart rate reduction under septic conditions in vivo, e.g. the MODIfY clinical trial.


Assuntos
Potenciais de Ação/efeitos dos fármacos , Benzazepinas/farmacologia , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/antagonistas & inibidores , Lipopolissacarídeos/farmacologia , Proteínas Musculares/antagonistas & inibidores , Miócitos Cardíacos/efeitos dos fármacos , Nó Sinoatrial/efeitos dos fármacos , Ensaios Clínicos como Assunto , Átrios do Coração/citologia , Átrios do Coração/efeitos dos fármacos , Átrios do Coração/metabolismo , Frequência Cardíaca/efeitos dos fármacos , Humanos , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Ivabradina , Modelos Biológicos , Proteínas Musculares/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Técnicas de Patch-Clamp , Canais de Potássio/metabolismo , Cultura Primária de Células , Nó Sinoatrial/citologia , Nó Sinoatrial/metabolismo
5.
Front Mol Biosci ; 11: 1394398, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38770217

RESUMO

Introduction: Advances in molecular targeting of ion channels may open up new avenues for therapeutic approaches in cancer based on the cells' bioelectric properties. In addition to in-vitro or in-vivo models, in silico models can provide deeper insight into the complex role of electrophysiology in cancer and reveal the impact of altered ion channel expression and the membrane potential on malignant processes. The A549 in silico model is the first computational cancer whole-cell ion current model that simulates the bioelectric mechanisms of the human non-small cell lung cancer cell line A549 during the different phases of the cell cycle. This work extends the existing model with a detailed mathematical description of the store-operated Ca2+ entry (SOCE) and the complex local intracellular calcium dynamics, which significantly affect the entire electrophysiological properties of the cell and regulate cell cycle progression. Methods: The initial model was extended by a multicompartmental approach, addressing the heterogenous calcium profile and dynamics in the ER-PM junction provoked by local calcium entry of store-operated calcium channels (SOCs) and uptake by SERCA pumps. Changes of cytosolic calcium levels due to diffusion from the ER-PM junction, release from the ER by RyR channels and IP3 receptors, as well as corresponding PM channels were simulated and the dynamics evaluated based on calcium imaging data. The model parameters were fitted to available data from two published experimental studies, showing the function of CRAC channels and indirectly of IP3R, RyR and PMCA via changes of the cytosolic calcium levels. Results: The proposed calcium description accurately reproduces the dynamics of calcium imaging data and simulates the SOCE mechanisms. In addition, simulations of the combined A549-SOCE model in distinct phases of the cell cycle demonstrate how Ca2+ - dynamics influence responding channels such as KCa, and consequently modulate the membrane potential accordingly. Discussion: Local calcium distribution and time evolution in microdomains of the cell significantly impact the overall electrophysiological properties and exert control over cell cycle progression. By providing a more profound description, the extended A549-SOCE model represents an important step on the route towards a valid model for oncological research and in silico supported development of novel therapeutic strategies.

6.
IEEE Trans Biomed Eng ; 71(6): 1980-1992, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38498749

RESUMO

OBJECTIVE: This study aims to explore the potential of organic electrolytic photocapacitors (OEPCs), an innovative photovoltaic device, in mediating the activation of native voltage-gated Cav1.2 channels (ICa,L) in Guinea pig ventricular cardiomyocytes. METHODS: Whole-cell patch-clamp recordings were employed to examine light-triggered OEPC mediated ICa,L activation, integrating the channel's kinetic properties into a multicompartment cell model to take intracellular ion concentrations into account. A multidomain model was additionally incorporated to evaluate effects of OEPC-mediated stimulation. The final model combines external stimulation, multicompartmental cell simulation, and a patch-clamp amplifier equivalent circuit to assess the impact on achievable intracellular voltage changes. RESULTS: Light pulses activated ICa,L, with amplitudes similar to voltage-clamp activation and high sensitivity to the L-type Ca2+ channel blocker, nifedipine. Light-triggered ICa,L inactivation exhibited kinetic parameters comparable to voltage-induced inactivation. CONCLUSION: OEPC-mediated activation of ICa,L demonstrates their potential for nongenetic optical modulation of cellular physiology potentially paving the way for the development of innovative therapies in cardiovascular health. The integrated model proves the light-mediated activation of ICa,L and advances the understanding of the interplay between the patch-clamp amplifier and external stimulation devices. SIGNIFICANCE: Treating cardiac conduction disorders by minimal-invasive means without genetic modifications could advance therapeutic approaches increasing patients' quality of life compared with conventional methods employing electronic devices.


Assuntos
Canais de Cálcio Tipo L , Simulação por Computador , Miócitos Cardíacos , Animais , Cobaias , Miócitos Cardíacos/fisiologia , Canais de Cálcio Tipo L/metabolismo , Técnicas de Patch-Clamp , Modelos Cardiovasculares , Potenciais de Ação/fisiologia , Potenciais de Ação/efeitos da radiação , Luz
7.
Front Oncol ; 12: 998907, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36483038

RESUMO

A search in the GDC Data Portal revealed 304 documented somatic mutations of the KCNJ3 gene in primary tumors (out of 10.202 cases). Most affected tumor types were carcinomas from uterus, skin and lung, while breast cancer exerted the lowest number of somatic mutations. We focused our research on 15 missense mutations within the region between TM1 and TM2, comprising the pore helix and ion selectivity signature. Expression was measured by confocal laser scan microscopy of eGFP tagged GIRK1 subunits, expressed with and without GIRK4 in oocytes of Xenopus laevis. GIRK ion currents were activated via coexpressed m2Rs and measured by the Two Electrode Voltage Clamp technique. Magnitude of the total GIRK current, as well as the fraction of current inducible by the agonist, were measured. Ion selectivity was gauged by assessment of the PNa+/PK+ ratio, calculated by the GIRK current reversal potential in extracellular media at different Na+ and K+ concentrations. None of the tested mutations was able to form functional GIRK1 homooligomeric ion channels. One of the mutations, G145A, which locates directly to the ion selectivity signature, exerted an increased PNa+/PK+ ratio. Generally, the missense mutations studied can be categorized into three groups: (i) normal/reduced expression accompanied by reduced/absent function (S132Y, F136L, E139K, G145A, R149Q, R149P, G178D, S185Y, Q186R), (ii) normal/increased expression as well as increased function (E140M, A142T, M184I) and (iii) miniscule expression but increased function relative to expression levels (I151N, G158S). We conclude, that gain of function mutations, identical or similar to categories (ii) and (iii), may potentially be involved in genesis and progression of malignancies in tissues that exert a high rate of occurrence of somatic mutations of KCNJ3.

9.
Antioxidants (Basel) ; 11(1)2021 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-35052529

RESUMO

Neutrophil-derived myeloperoxidase (MPO) and its potent oxidant, hypochlorous acid (HOCl), gained attention as important oxidative mediators in cardiac damage and dysfunction. As cardiomyocytes generate low-density lipoprotein (LDL)-like particles, we aimed to identify the footprints of proatherogenic HOCl-LDL, which adversely affects cellular signalling cascades in various cell types, in the human infarcted myocardium. We performed immunohistochemistry for MPO and HOCl-LDL in human myocardial tissue, investigated the impact of HOCl-LDL on electrophysiology and contractility in primary cardiomyocytes, and explored underlying mechanisms in HL-1 cardiomyocytes and human atrial appendages using immunoblot analysis, qPCR, and silencing experiments. HOCl-LDL reduced ICa,L and IK1, and increased INaL, leading to altered action potential characteristics and arrhythmic events including early- and delayed-afterdepolarizations. HOCl-LDL altered the expression and function of CaV1.2, RyR2, NCX1, and SERCA2a, resulting in impaired contractility and Ca2+ homeostasis. Elevated superoxide anion levels and oxidation of CaMKII were mediated via LOX-1 signaling in HL-1 cardiomyocytes. Furthermore, HOCl-LDL-mediated alterations of cardiac contractility and electrophysiology, including arrhythmic events, were ameliorated by the CaMKII inhibitor KN93 and the INaL blocker, ranolazine. This study provides an explanatory framework for the detrimental effects of HOCl-LDL compared to native LDL and cardiac remodeling in patients with high MPO levels during the progression of cardiovascular disease.

10.
Proteomics ; 10(1): 141-58, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19899077

RESUMO

Microglia, the immunocompetent cells of the CNS, are rapidly activated in response to injury and microglia migration towards and homing at damaged tissue plays a key role in CNS regeneration. Lysophosphatidic acid (LPA) is involved in signaling events evoking microglia responses through cognate G protein-coupled receptors. Here we show that human immortalized C13NJ microglia express LPA receptor subtypes LPA(1), LPA(2), and LPA(3) on mRNA and protein level. LPA activation of C13NJ cells induced Rho and extracellular signal-regulated kinase activation and enhanced cellular ATP production. In addition, LPA induced process retraction, cell spreading, led to pronounced changes of the actin cytoskeleton and reduced cell motility, which could be reversed by inhibition of Rho activity. To get an indication about LPA-induced global alterations in protein expression patterns a 2-D DIGE/LC-ESI-MS proteomic approach was applied. On the proteome level the most prominent changes in response to LPA were observed for glycolytic enzymes and proteins regulating cell motility and/or cytoskeletal dynamics. The present findings suggest that naturally occurring LPA is a potent regulator of microglia biology. This might be of particular relevance in the pathophysiological context of neurodegenerative disorders where LPA concentrations can be significantly elevated in the CNS.


Assuntos
Movimento Celular , Citoesqueleto/metabolismo , Microglia/citologia , Microglia/metabolismo , Proteoma/metabolismo , Receptores de Ácidos Lisofosfatídicos/metabolismo , Linhagem Celular , Regulação da Expressão Gênica , Glicólise , Humanos , Lisofosfolipídeos/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Receptores de Ácidos Lisofosfatídicos/genética , Transdução de Sinais , Proteínas rho de Ligação ao GTP/metabolismo
11.
Prog Biophys Mol Biol ; 157: 3-10, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32217074

RESUMO

Electrophysiologists routinely use simple voltage steps to evaluate cell membrane capacitance derived from corresponding current responses. Frequently, the resting membrane voltage Vrest is employed as holding potential for the subsequent command voltage step and more or less accurate methods are utilised to analyse the transient current. Another choice as holding potential is the peak of the "quasi steady-state" current to voltage relationship, Vpeak. The aim of this study is the systematic evaluation of capacitance estimation accuracy from voltage step experiments depending on the choice of holding potential and analysis method. In this paper, a simulation approach is employed to analyse the current response of a model patch-clamp circuit. Four commonly accepted methods are implemented, utilizing different aspects of the transient current (charge, membrane time constant, and influence of the series resistance) in various combinations and with various degrees of refinement. This simulation study indicates an acceptable accuracy of the elaborated methods for capacitance estimation at holding potentials Vrest and Vpeak over a broad range of capacitance as well as series resistance values. Simple integration of the current transient provides sufficient accuracy at holding potentials, which effectively minimizes changes in resistive membrane current flow during command voltage steps (particularly around Vpeak). However, biphasic command protocols performed at Vpeak activate voltage dependent sodium channels, thereby possibly leading to the threshold voltage for an action potential. Compared to Vrest, all methods utilizing monophasic step protocols, gain additional accuracy, when applied at Vpeak as holding potential.


Assuntos
Potenciais de Ação , Simulação por Computador , Miócitos Cardíacos/citologia , Técnicas de Patch-Clamp , Membrana Celular , Condutividade Elétrica , Humanos , Potenciais da Membrana/fisiologia , Neurônios , Reprodutibilidade dos Testes
12.
Sci Rep ; 9(1): 19277, 2019 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-31848385

RESUMO

Excessive expression of subunit 1 of GIRK1 in ER+ breast tumors is associated with reduced survival times and increased lymph node metastasis in patients. To investigate possible tumor-initiating properties, benign MCF10A and malign MCF7 mammary epithelial cells were engineered to overexpress GIRK1 neoplasia associated vital parameters and resting potentials were measured and compared to controls. The presence of GIRK1 resulted in resting potentials negative to the controls. Upon GIRK1 overexpression, several cellular pathways were regulated towards pro-tumorigenic action as revealed by comparison of transcriptomes of MCF10AGIRK1 with the control (MCF10AeGFP). According to transcriptome analysis, cellular migration was promoted while wound healing and extracellular matrix interactions were impaired. Vital parameters in MCF7 cells were affected akin the benign MCF10A lines, but to a lesser extent. Thus, GIRK1 regulated cellular pathways in mammary epithelial cells are likely to contribute to the development and progression of breast cancer.


Assuntos
Neoplasias da Mama/genética , Carcinogênese/genética , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética , Neoplasias/genética , Neoplasias da Mama/patologia , Movimento Celular/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Metástase Linfática , Células MCF-7 , Glândulas Mamárias Humanas/metabolismo , Glândulas Mamárias Humanas/patologia , Neoplasias/patologia , Transcriptoma/genética
13.
Front Physiol ; 9: 546, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29867582

RESUMO

Beat to beat variability of cardiac tissue or isolated cells is frequently investigated by determining time intervals from electrode measurements in order to compute scale dependent or scale independent parameters. In this study, we utilize high-speed video camera recordings to investigate the variability of intervals as well as mechanical contraction strengths and relative contraction strengths with nonlinear analyses. Additionally, the video setup allowed us simultaneous electrode registrations of extracellular potentials. Sinoatrial node tissue under control and acetylcholine treated conditions was used to perform variability analyses by computing sample entropies and Higuchi dimensions. Beat to beat interval variabilities measured by the two recording techniques correlated very well, and therefore, validated the video analyses for this purpose. Acetylcholine treatment induced a reduction of beating rate and contraction strength, but the impact on interval variability was negligible. Nevertheless, the variability analyses of contraction strengths revealed significant differences in sample entropies and Higuchi dimensions between control and acetylcholine treated tissue. Therefore, the proposed high-speed video camera technique might represent a non-invasive tool that allows long-lasting recordings for detecting variations in beating behavior over a large range of scales.

14.
Shock ; 28(6): 655-661, 2007 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-18092381

RESUMO

LPSs trigger the development of sepsis by gram-negative bacteria and cause a variety of biological effects on host cells, including alterations on ionic channels. Because heart rate variability is reduced in human sepsis and endotoxemia, we hypothesized that LPS affects the pacemaker current I(f) in human heart, which might--at least in part--explain this phenomenon. Isolated human myocytes from right atrial appendages were incubated for 6 to 10 h with LPS (1 and 10 microg/mL) and afterwards used to investigate the pacemaker current I(f). I(f) was measured with the whole-cell patch-clamp technique (at 37 degrees C). Incubation of atrial myocytes with 10 microg/mL LPS was found to significantly impair I(f) by suppressing the current at membrane potentials positive to -80 mV and slowing down current activation, but without effecting maximal current conductance. Furthermore, in incubated cells (10 microg/mL), the response of I(f) to [beta]-adrenergic stimulation (1 microM isoproterenol) was significantly larger compared with control cells (shift of half-maximal activation voltage to more positive potentials amounted to -10 and -14 mV in untreated and treated cells, respectively). Simulations using a spontaneously active sinoatrial cell model demonstrated that LPS-induced I(f) impairment reduced the responsiveness of the model cell to fluctuations of autonomic input. This study showed a direct impact of LPS on the cardiac pacemaker current I(f). The LPS-induced I(f) impairment may contribute to the clinically observed reduction in heart rate variability under septic conditions and in cardiac diseases such as heart failure, where endotoxin can be of pathophysiological relevance.


Assuntos
Lipopolissacarídeos/farmacologia , Miócitos Cardíacos/efeitos dos fármacos , Adulto , Idoso , Células Cultivadas , Eletrofisiologia , Feminino , Frequência Cardíaca/efeitos dos fármacos , Humanos , Canais Iônicos/fisiologia , Isoproterenol/farmacologia , Masculino , Potenciais da Membrana/efeitos dos fármacos , Pessoa de Meia-Idade , Miócitos Cardíacos/citologia , Miócitos Cardíacos/fisiologia , Técnicas de Patch-Clamp , Sepse/patologia , Sepse/fisiopatologia
15.
Biochem Pharmacol ; 145: 64-80, 2017 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-28859968

RESUMO

Saxagliptin treatment has been associated with increased rate of hospitalization for heart failure in type 2 diabetic patients, though the underlying mechanism(s) remain elusive. To address this, we assessed the effects of saxagliptin on human atrial trabeculae, guinea pig hearts and cardiomyocytes. We found that the primary target of saxagliptin, dipeptidyl peptidase-4, is absent in cardiomyocytes, yet saxagliptin internalized into cardiomyocytes and impaired cardiac contractility via inhibition of the Ca2+/calmodulin-dependent protein kinase II-phospholamban-sarcoplasmic reticulum Ca2+-ATPase 2a axis and Na+-Ca2+ exchanger function in Ca2+ extrusion. This resulted in reduced sarcoplasmic reticulum Ca2+ content, diastolic Ca2+ overload, systolic dysfunction and impaired contractile force. Furthermore, saxagliptin reduced protein kinase C-mediated delayed rectifier K+ current that prolonged action potential duration and consequently QTc interval. Importantly, saxagliptin aggravated pre-existing cardiac dysfunction induced by ischemia/reperfusion injury. In conclusion, our novel results provide mechanisms for the off-target deleterious effects of saxagliptin on cardiac function and support the outcome of SAVOR-TIMI 53 trial that linked saxagliptin with the risk of heart failure.


Assuntos
Adamantano/análogos & derivados , Dipeptídeos/toxicidade , Dipeptidil Peptidase 4/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Átrios do Coração/citologia , Miócitos Cardíacos/efeitos dos fármacos , Adamantano/toxicidade , Idoso , Animais , Linhagem Celular , Dipeptidil Peptidase 4/genética , Inibidores da Dipeptidil Peptidase IV/farmacologia , Feminino , Regulação Enzimológica da Expressão Gênica/fisiologia , Cardiopatias/metabolismo , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Contração Miocárdica/efeitos dos fármacos , Miócitos Cardíacos/enzimologia
16.
Cardiovasc Res ; 66(1): 74-83, 2005 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-15769450

RESUMO

OBJECTIVE: It was our aim to investigate effects of human LDL, copper-, or AAPH-oxidized over different periods of time to different degrees (ox-LDL), on viability and electrophysiological parameters of isolated ventricular myocytes of guinea pigs. METHODS: Guinea pig ventricular myocytes were incubated with ox-LDL or native LDL (at 0.5 mg/ml) for 12 h, and afterwards myocyte damage, action potentials, and transmembrane ion currents were studied (at 37 degrees C). RESULTS: Ox-LDL was found to induce severe myocyte damage, whereas native LDL had no effect. Myocyte damage was dependent on the content of total lipid hydroperoxides in both copper-oxidized and AAPH-oxidized LDL. Incubation with ox-LDL led to intense contractile and electrophysiological effects including prolongation of action potential duration, depolarization of resting membrane potential, spontaneous activity, generation of afterdepolarizations, and modification of transmembrane ion currents (e.g. inward rectifier, calcium, and background currents). CONCLUSIONS: Ox-LDL induced cell damage and irregular electrical activity in ventricular myocytes. These effects were dependent on the lipid hydroperoxide content of ox-LDL and were similar to oxidative stress (OS) induced by various OS-generating systems. The observed effects may play a role for functional cardiac abnormalities in patients with increased ox-LDL levels.


Assuntos
Potenciais de Ação/efeitos dos fármacos , Canais Iônicos/efeitos dos fármacos , Lipoproteínas LDL/farmacologia , Miócitos Cardíacos/metabolismo , Adulto , Amidinas/metabolismo , Animais , Canais de Cálcio/efeitos dos fármacos , Técnicas de Cultura de Células , Sobrevivência Celular , Cobre/metabolismo , Cobaias , Ventrículos do Coração , Humanos , Peróxidos Lipídicos/metabolismo , Masculino , Potenciais da Membrana/efeitos dos fármacos , Miócitos Cardíacos/patologia , Oxirredução , Canais de Potássio Corretores do Fluxo de Internalização/efeitos dos fármacos
17.
Cardiovasc Res ; 64(2): 250-9, 2004 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-15485684

RESUMO

OBJECTIVE: The left human atrium plays an important role in initiation of atrial fibrillation (AF) and the hyperpolarization activated cation current (I(f)) is a candidate for contributing to abnormal automaticity. However, electrophysiological data concerning I(f) are not available in this cardiac region and we therefore investigated I(f) in human left atrial tissue. METHODS: Human atrial myocytes were isolated from the left atrial appendage (LAA) and the left atrial wall (LAW) obtained from patients undergoing open heart surgery. I(f) was measured with the whole-cell patch-clamp technique. RESULTS: I(f) densities between -70 and -110 mV were found to be significantly higher in LAA than in LAW cells. Furthermore, in the group of LAA cells the half maximal activation potential (V(1/2)) was found to be less negative (V(1/2) of -84.3+/-1.9 mV, n=14/9) compared to LAW cells (V(1/2) of -97.8+/-2.1 mV, n=28/9). Beta-adrenergic receptor stimulation with isoproterenol (1 microM) caused an acceleration of current activation and a V(1/2) shift to more positive potentials in cells of both regions (LAA: 8.8+/-2.3 mV, n=6/4 and LAW: 8.9+/-2.6 mV, n=6/4). Simulations using a mathematical model of the human atrial myocyte demonstrated that I(f) was able to induce spontaneous activity in the model at a regular rhythm due to the interplay of I(f), Na(+)/Ca(2+) exchange current and Ca(2+) release of the sarcoplasmic reticulum (SR). CONCLUSIONS: Our study revealed the presence of I(f) in left atrial myocytes and showed that I(f) parameters depend on atrial region. I(f) current densities were sufficient to convert the mathematical model of a quiescent human atrial cell into a "pacemaker cell". These data support the hypothesis of I(f) as a contributor to abnormal automaticity in human atrial tissue.


Assuntos
Complexos Atriais Prematuros/fisiopatologia , Simulação por Computador , Canais Iônicos/fisiologia , Modelos Cardiovasculares , Miócitos Cardíacos/metabolismo , Agonistas Adrenérgicos beta/farmacologia , Cátions , Estimulação Elétrica , Átrios do Coração , Humanos , Isoproterenol/farmacologia , Técnicas de Patch-Clamp
18.
Artigo em Inglês | MEDLINE | ID: mdl-24110998

RESUMO

Extracardiac factors of heart rate variability have commonly been investigated using linear and nonlinear methods for a long time. Recently, intracardiac mechanisms on an electrophysiological basis have been found to be also important. This work is focused on the evaluation of complex measures of temporal signals gained with microelectrode measurements of embryonic chick heart aggregates. Septic conditions were mimicked in vitro by lipopolysaccharide (LPS) administration in order to investigate the influence on beat to beat variability. Surrogate data analysis revealed high statistical significances for normalized complexity measures.


Assuntos
Entropia , Fractais , Frequência Cardíaca/fisiologia , Coração/embriologia , Coração/fisiopatologia , Sepse/embriologia , Sepse/fisiopatologia , Animais , Embrião de Galinha , Lipopolissacarídeos/farmacologia , Miócitos Cardíacos/efeitos dos fármacos , Processamento de Sinais Assistido por Computador
19.
Naunyn Schmiedebergs Arch Pharmacol ; 382(5-6): 433-40, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-20838990

RESUMO

Thienopyridines (ticlopidine, clopidogrel) are frequently used drugs in antiplatelet therapy and have been shown to exert a more pronounced negative inotropic effect than thienopyrimidinones. We hypothesized that these differences are due to a differential impact of thienopyridines and thienopyrimidinones on L-type calcium current at the single-cell level. The effects of thienopyridines and thienopyrimidinones were studied on L-type calcium current and action potential parameters with the whole-cell patch-clamp technique in isolated myocytes from guinea pig ventricle and human atrial appendage. Ticlopidine showed the greatest impact on the L-type calcium current in guinea pig myocytes. It significantly reduced L-type calcium current density as well as shifted half maximal inactivation potential to more negative potentials compared to clopidogrel (at 30 µmol/L) and to all thienopyrimidinones (30 and 100 µmol/L). Clopidogrel significantly reduced the L-type calcium current density as well as shifted the half maximal inactivation potential to more negative potentials compared to all thienopyrimidinones at 100 µmol/L only. In contrast, thienopyrimidinones did not affect L-type calcium current properties. The significant different effects of thienopyridines and thienopyrimidinones could also be demonstrated in human atrial myocytes. The more pronounced negative inotropic effect of thienopyridines is well explained by our results demonstrating a differential impairment of L-type calcium current by thienopyridines and thienopyrimidinones. L-type calcium current impairment by thienopyridines may be of special relevance for patients with cardiac diseases characterized by ionic remodelling.


Assuntos
Potenciais de Ação/efeitos dos fármacos , Canais de Cálcio Tipo L/fisiologia , Miócitos Cardíacos/efeitos dos fármacos , Inibidores da Agregação Plaquetária/farmacologia , Pirimidinas/farmacologia , Tienopiridinas/farmacologia , Tiofenos/farmacologia , Animais , Apêndice Atrial/citologia , Células Cultivadas , Clopidogrel , Cobaias , Ventrículos do Coração/citologia , Humanos , Técnicas In Vitro , Miócitos Cardíacos/fisiologia , Técnicas de Patch-Clamp , Ticlopidina/análogos & derivados , Ticlopidina/farmacologia
20.
Can J Physiol Pharmacol ; 87(4): 266-74, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19370080

RESUMO

In sepsis, systemic inflammatory response syndrome (SIRS), and multiorgan dysfunction syndrome (MODS), a severe prognostically relevant cardiac autonomic dysfunction exists, as manifested by a strong attenuation of sympathetically and vagally mediated heart rate variability (HRV). The mechanisms underlying this attenuation are not limited to the nervous system. They also include alterations of the cardiac pacemaker cells on a cellular level. As shown in human atrial cardiomyocytes, endotoxin interacts with cardiac hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels, which mediate the pacemaker current If and play an important role in transmitting sympathetic and vagal signals on heart rate and HRV. Moreover, endotoxin sensitizes cardiac HCN channels to sympathetic signals. These findings identify endotoxin as a pertinent modulator of the autonomic nervous regulation of heart function. In MODS, the vagal pathway of the autonomic nervous system is particularly compromised, leading to an attenuation of the cholinergic antiinflammatory reflex. An amelioration of the blunted vagal activity appears to be a promising novel therapeutic target to achieve a suppression of the inflammatory state and thereby an improvement of prognosis in MODS patients. Preliminary data revealed therapeutic benefits (increased survival rates and improvements of the depressed vagal activity) of the administration of statins, beta-blockers, and angiotensin-converting enzyme inhibitors in patients with MODS.


Assuntos
Coração/fisiopatologia , Insuficiência de Múltiplos Órgãos/fisiopatologia , Sepse/fisiopatologia , Síndrome de Resposta Inflamatória Sistêmica/fisiopatologia , Antagonistas Adrenérgicos beta/uso terapêutico , Inibidores da Enzima Conversora de Angiotensina/uso terapêutico , Animais , Frequência Cardíaca/efeitos dos fármacos , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Insuficiência de Múltiplos Órgãos/tratamento farmacológico , Prognóstico , Sepse/tratamento farmacológico , Síndrome de Resposta Inflamatória Sistêmica/tratamento farmacológico , Nervo Vago/fisiologia
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