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Abstract Introduction Cardiovascular diseases represent a major cause of death world-wide and one of their greatest complications is the development of cardiac arrhythmias, in which ventricular fibrillation (VF) stands out as the most severe one. The only therapy that reverses VF is defibrillation. However defibrillatory shock is capable of killing heart cells and it is known that the orientation of the cell major axis with respect to the electrical field (E) direction is a determining factor for cellular excitation and injury, which is leading to the development of new defibrillation protocols. The aim of this work is to fill the gap in information about cell lethality for intermediate cell orientation angles. Methods Ventricular myocytes were extracted from adult male Wistar rats and the cells were plated in a chamber for perfusion and stimulation with bipolar voltage pulses to determine the stimulation threshold (ET). Then, monopolar stimulus was applied and amplitude was increased until cell lethal injury. This protocol was performed on four experimental groups: cells oriented at 0°, 30°, 60° and 90°, with respect to E direction. Results 87 cells were analyzed and an increase in amplitude of E associated with 50% lethality (E50) was verified as the direction of E application and cell major axis orientation departed. Conclusion Taken the same probability of lethality, our data suggest a nonlinear increase of E amplitude from 0° to 90° similar to that of ET. These in-between data had not yet been shown and are important for service-based future defibrillation protocols.
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Multidirectional defibrillation protocols have shown better efficiency than monodirectional; still, no testing was performed to assess cell lethality. We investigated lethality of multidirectional defibrillator-like shocks on isolated cardiomyocytes. Cells were isolated from adult male Wistar rats and plated into a perfusion chamber. Electrical field stimulation threshold (ET) was obtained, and cells were paced with suprathreshold bipolar electrical field (E) pulses. Either one monodirectional high-intensity electrical field (HEF) pulse aligned at 0° (group Mono0) or 60° (group Mono60) to cell major axis or a multidirectional sequence of three HEF pulses aligned at 0°, 60°, and 120° each was applied. If cell recovered from shock, pacing was resumed, and a higher amplitude HEF, proportional to ET, was applied. The sequence was repeated until cell death. Lethality curves were built by means of survival analysis from sub-lethal and lethal E. Non-linear fit was performed, and E values corresponding to 50% probability of lethality (E50) were compared. Multidirectional groups presented lethality curves similar to Mono0. Mono60 displayed the highest E50. The novel data endorse the idea of multidirectional stimuli being safer because their effects on lethality of individual cells were equal to a single monodirectional stimulus, while their defibrillatory threshold is lower. Graphical abstract Monodirectional and multidirectional lethality protocol comparison on isolated rat cardiomyocytes. The heart image is a derivative of "3D Heart in zBrush" ( https://vimeo.com/65568770 ) by Laloxl, used under CC BY 3.0 ( https://creativecommons.org/licenses/by/3.0/legalcode )/image extracted from original video.
Assuntos
Estimulação Elétrica/métodos , Miócitos Cardíacos/fisiologia , Animais , Morte Celular , Cardioversão Elétrica , Estimulação Elétrica/instrumentação , Desenho de Equipamento , Masculino , Probabilidade , Ratos WistarRESUMO
Application of high intensity electric fields (HIEF) to the myocardium is commonly used for cardiac defibrillation/cardioversion. Although effective at reversing life-threatening arrhythmias, HIEF may cause myocyte damage due to membrane electropermeabilization. In this study, the influence of cell length and width on HIEF-induced lethal injury was analyzed in isolated rat cardiomyocytes in parallel alignment with the field. The field-induced maximum variation of membrane potential (ΔVmax) was estimated with the Klee-Plonsey model. The studied myocyte population was arranged in two group pairs for comparison: the longest vs. the shortest cells, and the widest vs. narrowest cells. Threshold field intensity was significantly lower in the longest vs. shortest myocytes, whereas cell width influence was not significant. The threshold ΔVmax was comparable in all groups. Likewise, a significant leftward shift of the lethality curve (i.e., relationship of the probability of lethality vs. field intensity) of the longest cells was observed, evidencing greater sensitivity to HIEF-induced damage. However, the lethality curve as a function of ΔVmax was similar in all groups, confirming a prediction of the Klee-Plonsey model. The similar results for excitation and injury at threshold and HIEF stimulation, respectively, indicate that: a) the effect of cell length on the sensitivity to the field would be attributable to differences in field-induced membrane polarization that lead to excitation or lethal electroporation; b) the Klee-Plonsey model seems to be reliable for analysis of cell interaction with HIEF; c) it is possible that increased cell length in hypertrophied hearts enhances myocyte fragility upon defibrillation/cardioversion.
Campos elétricos de alta intensidade (HIEF) são aplicados ao miocárdio durante desfibrilação e cardioversão. Embora eficazes na reversão de arritmias potencialmente letais, HIEF podem lesar cardiomiócitos por eletropermeabilização da membrana. Neste estudo, a influência das dimensões celulares sobre o efeito letal de HIEF foi estudada em cardiomiócitos isolados de rato alinhados paralelamente ao campo. A máxima variação do potencial de membrana induzida pelo campo (ΔVmax) foi calculada com o modelo de Klee-Plonsey. As células estudadas foram distribuídas em dois pares de grupos de acordo com seu comprimento e largura. A intensidade limiar do campo não dependeu da largura celular, mas sim do comprimento (menor nas células mais longas, p < 0.001), enquanto ΔVmax no limiar foi comparável entre os grupos. Nas células mais longas, observou-se desvio à esquerda (p < 0.01) da curva que descreve a relação entre probabilidade de letalidade e a intensidade do campo, evidenciando maior sensibilidade à ação deletéria de HIEF. Porém, a curva de letalidade em função de ΔVmax foi semelhante em todos os grupos, o que confirma a predição pelo modelo de Klee-Plonsey. A similaridade de resultados com estimulação limiar e com HIEF indica que: a) o efeito do comprimento celular sobre a sensibilidade ao campo poderia ser atribuído a diferenças no grau de polarização da membrana durante a aplicação do estímulo; b) o modelo de Klee-Plonsey parece ser confiável para a análise da interação espacial da célula com HIEF; c) é possível que o maior comprimento celular em miócitos hipertrofiados os torne mais susceptíveis a lesão durante desfibrilação/cardioversão.
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Defibrillator-type shocks may cause electric and contractile dysfunction. In this study, we determined the relationship between probability of lethal injury and electric field intensity (E in isolated rat ventricular myocytes, with emphasis on field orientation and stimulus waveform. This relationship was sigmoidal with irreversible injury for E > 50 V/cm . During both threshold and lethal stimulation, cells were twofold more sensitive to the field when it was applied longitudinally (versus transversally) to the cell major axis. For a given E, the estimated maximum variation of transmembrane potential (Delta V(max)) was greater for longitudinal stimuli, which might account for the greater sensitivity to the field. Cell death, however, occurred at lower maximum Delta V(max) values for transversal shocks. This might be explained by a less steep spatial decay of transmembrane potential predicted for transversal stimulation, which would possibly result in occurrence of electroporation in a larger membrane area. For the same stimulus duration, cells were less sensitive to field-induced injury when shocks were biphasic (versus monophasic). Ours results indicate that, although significant myocyte death may occur in the E range expected during clinical defibrillation, biphasic shocks are less likely to produce irreversible cell injury.
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Estimulação Elétrica , Potenciais da Membrana , Miócitos Cardíacos/patologia , Miócitos Cardíacos/fisiologia , Algoritmos , Animais , Cálcio/metabolismo , Estimulação Elétrica/efeitos adversos , Estimulação Elétrica/métodos , Ventrículos do Coração/citologia , Masculino , Modelos Cardiovasculares , Ratos , Ratos WistarRESUMO
Cardiac inotropy depends, among other factors, on the interval between contractions. In this study, we developed instrumentation for cell shortening recording, which was used to investigate the influence of stimulatory rhythm on contraction amplitude of isolated rat ventricular myocytes. Peak cell shortening amplitude was recorded during electric stimulation at the average rate of 0.5 Hz with different stimulatory patterns: regular and pseudo-random rhythms, as well as double pulse stimulation. Cells were perfused at 23 ºC with modified Tyrodes solution with or without 10 nM isoproterenol (ISO). The main advantages of the developed microscopy system were its relatively low cost(~US$ 1,000.00), small size (150 × 170 × 300 mm), and absence of detectable optic distortions. We observed that average contraction amplitude was similar for all stimulatory patterns, in the absence and presence of ISO (p > 0.05), although the amplitude of individual contractions was highly dependent on the previous interval, and was significantly increased by ISO (p < 0.05). With the double pulse patterns, the amplitude ratio of contractions following the shorter and the longer intervals was ~0.55. ISO positive inotropic effect was more prominent for contractions after short intervals, which increased the ratio to ~0.80. This might be explained by acceleration of the recovery of sarcoplasmic reticulum Ca2+ release channels from the adapted state, possibly by proteinkinase A-dependent phosphorylation, which would resultin enhanced systolic Ca2+ release.
O inotropismo cardíaco depende de inúmeros fatores, entre eles o intervalo entre contrações. Neste trabalho, desenvolvemos instrumentação para registro de encurtamento celular e investigamos a influência do ritmo estimulatório sobre a atividade contrátil de miócitos ventriculares isolados de rato. A amplitude do encurtamento celular foi registrada durante estimulação elétrica à freqüência média de 0,5 Hz, com ritmo regular, ritmo pseudoaleatório e pulsos duplos. Os miócitos foram perfundidos a 23 ºC com solução de Tyrode modificada contendo ou não 10 nMde isoproterenol (ISO). O sistema de microscopia desenvolvido é de custo relativo baixo (~US$ 1.000,00), dimensões reduzidas(150 × 170 × 300 mm) e apresenta boa qualidade óptica (sem distorções ou paralaxe detectáveis). Observamos que a amplitude média das contrações foi semelhante em todos os ritmos estimulatórios na ausência e presença de ISO (p > 0,05), embora a amplitude de contrações individuais fosse dependente do intervalo precedente, e ISO tenha causado aumento da amplitude média das contrações (p < 0,05). Nos padrões com pulso duplo, a razão de amplitude das contrações que seguem o menor e o maior intervalo foi ~0,55. O efeito inotrópico positivo de ISO foi mais pronunciado para contrações após intervalos curtos, o que levou a razão para ~0,80. Isto poderia ser explicado por aceleração da recuperação dos canais de liberação de Ca2+ do retículo sarcoplasmático do estado adaptado, causada possivelmente por fosforilação pela proteína quinase A, o que aumentaria a quantidade de Ca2+ liberada durante a sístole.