RESUMEN

AIMS: Diseased atria are characterized by functional and structural heterogeneities, adding to abnormal impulse generation and propagation. These heterogeneities are thought to lie at the origin of fractionated electrograms recorded during sinus rhythm (SR) in atrial fibrillation (AF) patients and are assumed to be involved in the onset and perpetuation (e.g. by re-entry) of this disorder. The underlying mechanisms, however, remain incompletely understood. Here, we tested whether regions of dense fibrosis could create an electrically isolated conduction pathway (EICP) in which re-entry could be established via ectopy and local block to become 'trapped'. We also investigated whether this could generate local fractionated electrograms and whether the re-entrant wave could 'escape' and cause a global tachyarrhythmia due to dynamic changes at a connecting isthmus. METHODS AND RESULTS: To precisely control and explore the geometrical properties of EICPs, we used light-gated depolarizing ion channels and patterned illumination for creating specific non-conducting regions in silico and in vitro. Insight from these studies was used for complementary investigations in virtual human atria with localized fibrosis. We demonstrated that a re-entrant tachyarrhythmia can exist locally within an EICP with SR prevailing in the surrounding tissue and identified conditions under which re-entry could escape from the EICP, thereby converting a local latent arrhythmic source into an active driver with global impact on the heart. In a realistic three-dimensional model of human atria, unipolar epicardial pseudo-electrograms showed fractionation at the site of 'trapped re-entry' in coexistence with regular SR electrograms elsewhere in the atria. Upon escape of the re-entrant wave, acute arrhythmia onset was observed. CONCLUSIONS: Trapped re-entry as a latent source of arrhythmogenesis can explain the sudden onset of focal arrhythmias, which are able to transgress into AF. Our study might help to improve the effectiveness of ablation of aberrant cardiac electrical signals in clinical practice.

Asunto(s)

Fibrilación Atrial , Humanos , Atrios Cardíacos , Canales Iónicos , Taquicardia/patología , Fibrosis

RESUMEN

Aim: Channelrhodopsins (ChRs) are a large family of light-gated ion channels with distinct properties, which is of great importance in the selection of a ChR variant for a given application. However, data to guide such selection for cardiac optogenetic applications are lacking. Therefore, we investigated the functioning of different ChR variants in normal and pathological hypertrophic cardiomyocytes subjected to various illumination protocols. Methods and Results: Isolated neonatal rat ventricular cardiomyocytes (NRVMs) were transduced with lentiviral vectors to express one of the following ChR variants: H134R, CatCh, ReaChR, or GtACR1. NRVMs were treated with phenylephrine (PE) to induce pathological hypertrophy (PE group) or left untreated [control (CTL) group]. In these groups, ChR currents displayed unique and significantly different properties for each ChR variant on activation by a single 1-s light pulse (1 mW/mm2: 470, 565, or 617 nm). The concomitant membrane potential (V m) responses also showed a ChR variant-specific profile, with GtACR1 causing a slight increase in average V m during illumination (V plateau: -38 mV) as compared with a V plateau > -20 mV for the other ChR variants. On repetitive activation at increasing frequencies (10-ms pulses at 1-10 Hz for 30 s), peak currents, which are important for cardiac pacing, decreased with increasing activation frequencies by 17-78% (p < 0.05), while plateau currents, which are critical for arrhythmia termination, decreased by 10-75% (p < 0.05), both in a variant-specific manner. In contrast, the corresponding V plateau remained largely stable. Importantly, current properties and V m responses were not statistically different between the PE and CTL groups, irrespective of the variant used (p > 0.05). Conclusion: Our data show that ChR variants function equally well in cell culture models of healthy and pathologically hypertrophic myocardium but show strong, variant-specific use-dependence. This use-dependent nature of ChR function should be taken into account during the design of cardiac optogenetic studies and the interpretation of the experimental findings thereof.

RESUMEN

Homeostatic regulation protects organisms against hazardous physiological changes. However, such regulation is limited in certain organs and associated biological processes. For example, the heart fails to self-restore its normal electrical activity once disturbed, as with sustained arrhythmias. Here we present proof-of-concept of a biological self-restoring system that allows automatic detection and correction of such abnormal excitation rhythms. For the heart, its realization involves the integration of ion channels with newly designed gating properties into cardiomyocytes. This allows cardiac tissue to i) discriminate between normal rhythm and arrhythmia based on frequency-dependent gating and ii) generate an ionic current for termination of the detected arrhythmia. We show in silico, that for both human atrial and ventricular arrhythmias, activation of these channels leads to rapid and repeated restoration of normal excitation rhythm. Experimental validation is provided by injecting the designed channel current for arrhythmia termination in human atrial myocytes using dynamic clamp.

Asunto(s)

Arritmias Cardíacas/metabolismo , Fenómenos Electrofisiológicos/fisiología , Activación del Canal Iónico , Canales Iónicos/fisiología , Miocitos Cardíacos/fisiología , Línea Celular , Simulación por Computador , Humanos , Técnicas de Placa-Clamp , Reproducibilidad de los Resultados

RESUMEN

Aims: The generation of homogeneous cardiomyocyte populations from fresh tissue or stem cells is laborious and costly. A potential solution to this problem would be to establish lines of immortalized cardiomyocytes. However, as proliferation and (terminal) differentiation of cardiomyocytes are mutually exclusive processes, their permanent immortalization causes loss of electrical and mechanical functions. We therefore aimed at developing conditionally immortalized atrial myocyte (iAM) lines allowing toggling between proliferative and contractile phenotypes by a single-component change in culture medium composition. Methods and results: Freshly isolated neonatal rat atrial cardiomyocytes (AMs) were transduced with a lentiviral vector conferring doxycycline (dox)-controlled expression of simian virus 40 large T antigen. Under proliferative conditions (i.e. in the presence of dox), the resulting cells lost most cardiomyocyte traits and doubled every 38 h. Under differentiation conditions (i.e. in the absence of dox), the cells stopped dividing and spontaneously reacquired a phenotype very similar to that of primary AMs (pAMs) in gene expression profile, sarcomeric organization, contractile behaviour, electrical properties, and response to ion channel-modulating compounds (as assessed by patch-clamp and optical voltage mapping). Moreover, differentiated iAMs had much narrower action potentials and propagated them at >10-fold higher speeds than the widely used murine atrial HL-1 cells. High-frequency electrical stimulation of confluent monolayers of differentiated iAMs resulted in re-entrant conduction resembling atrial fibrillation, which could be prevented by tertiapin treatment, just like in monolayers of pAMs. Conclusion: Through controlled expansion and differentiation of AMs, large numbers of functional cardiomyocytes were generated with properties superior to the differentiated progeny of existing cardiomyocyte lines. iAMs provide an attractive new model system for studying cardiomyocyte proliferation, differentiation, metabolism, and (electro)physiology as well as to investigate cardiac diseases and drug responses, without using animals.

Asunto(s)

Diferenciación Celular , Proliferación Celular , Atrios Cardíacos/metabolismo , Desarrollo de Músculos , Miocitos Cardíacos/metabolismo , Animales , Animales Recién Nacidos , Fibrilación Atrial/metabolismo , Fibrilación Atrial/fisiopatología , Línea Celular Transformada , Regulación de la Expresión Génica , Atrios Cardíacos/citología , Frecuencia Cardíaca , Potenciales de la Membrana , Fenotipo , Ratas , Transducción de Señal , Factores de Tiempo

RESUMEN

Aims: Anatomical re-entry is an important mechanism of ventricular tachycardia, characterized by circular electrical propagation in a fixed pathway. It's current investigative and therapeutic approaches are non-biological, rather unspecific (drugs), traumatizing (electrical shocks), or irreversible (ablation). Optogenetics is a new biological technique that allows reversible modulation of electrical function with unmatched spatiotemporal precision using light-gated ion channels. We therefore investigated optogenetic manipulation of anatomical re-entry in ventricular cardiac tissue. Methods and results: Transverse, 150-µm-thick ventricular slices, obtained from neonatal rat hearts, were genetically modified with lentiviral vectors encoding Ca2+-translocating channelrhodopsin (CatCh), a light-gated depolarizing ion channel, or enhanced yellow fluorescent protein (eYFP) as control. Stable anatomical re-entry was induced in both experimental groups. Activation of CatCh was precisely controlled by 470-nm patterned illumination, while the effects on anatomical re-entry were studied by optical voltage mapping. Regional illumination in the pathway of anatomical re-entry resulted in termination of arrhythmic activity only in CatCh-expressing slices by establishing a local and reversible, depolarization-induced conduction block in the illuminated area. Systematic adjustment of the size of the light-exposed area in the re-entrant pathway revealed that re-entry could be terminated by either wave collision or extinction, depending on the depth (transmurality) of illumination. In silico studies implicated source-sink mismatches at the site of subtransmural conduction block as an important factor in re-entry termination. Conclusions: Anatomical re-entry in ventricular tissue can be manipulated by optogenetic induction of a local and reversible conduction block in the re-entrant pathway, allowing effective re-entry termination. These results provide distinctively new mechanistic insight into re-entry termination and a novel perspective for cardiac arrhythmia management.

Asunto(s)

Arritmias Cardíacas/prevención & control , Canales de Calcio/efectos de la radiación , Luz , Miocitos Cardíacos/efectos de la radiación , Optogenética , Rodopsina/efectos de la radiación , Potenciales de Acción , Animales , Animales Recién Nacidos , Arritmias Cardíacas/genética , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/fisiopatología , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/genética , Canales de Calcio/biosíntesis , Canales de Calcio/genética , Simulación por Computador , Vectores Genéticos , Lentivirus/genética , Proteínas Luminiscentes/biosíntesis , Proteínas Luminiscentes/genética , Modelos Cardiovasculares , Miocitos Cardíacos/metabolismo , Ratas Wistar , Rodopsina/biosíntesis , Rodopsina/genética , Factores de Tiempo , Técnicas de Cultivo de Tejidos , Transfección , Imagen de Colorante Sensible al Voltaje

RESUMEN

Atrial fibrillation (AF) is the most frequent form of arrhythmia occurring in the industrialized world. Because of its complex nature, each identified form of AF requires specialized treatment. Thus, an in-depth understanding of the bases of these arrhythmias is essential for therapeutic development. A variety of experimental studies aimed at understanding the mechanisms of AF are performed using primary cultures of neonatal rat atrial cardiomyocytes (NRAMs). Previously, we have shown that the distinct advantage of NRAM cultures is that they allow standardized, systematic, robust re-entry induction in the presence of a constitutively-active acetylcholine-mediated K+ current (IKACh-c). Experimental studies dedicated to mechanistic explorations of AF, using these cultures, often use computer models for detailed electrophysiological investigations. However, currently, no mathematical model for NRAMs is available. Therefore, in the present study we propose the first model for the action potential (AP) of a NRAM with constitutively-active acetylcholine-mediated K+ current (IKACh-c). The descriptions of the ionic currents were based on patch-clamp data obtained from neonatal rats. Our monolayer model closely mimics the action potential duration (APD) restitution and conduction velocity (CV) restitution curves presented in our previous in vitro studies. In addition, the model reproduces the experimentally observed dynamics of spiral wave rotation, in the absence and in the presence of drug interventions, and in the presence of localized myofibroblast heterogeneities.

Asunto(s)

Acetilcolina/metabolismo , Potenciales de Acción/fisiología , Función Atrial/fisiología , Modelos Cardiovasculares , Miocitos Cardíacos/fisiología , Potasio/metabolismo , Animales , Animales Recién Nacidos , Simulación por Computador , Activación del Canal Iónico/fisiología , Ratas , Canales de Sodio/fisiología

RESUMEN

AIMS: Fibrosis increases arrhythmogenicity in myocardial tissue by causing structural and functional disruptions in the cardiac syncytium. Forced fusion of fibroblastic cells with adjacent cardiomyocytes may theoretically resolve these disruptions. Therefore, the electrophysiological effects of such electrical and structural integration of fibroblastic cells into a cardiac syncytium were studied. METHODS AND RESULTS: Human ventricular scar cells (hVSCs) were transduced with lentiviral vectors encoding enhanced green fluorescent protein alone (eGFP↑-hVSCs) or together with the fusogenic vesicular stomatitis virus G protein (VSV-G/eGFP↑-hVSCs) and subsequently co-cultured (1:4 ratio) with neonatal rat ventricular cardiomyocytes (NRVMs) in confluent monolayers yielding eGFP↑- and VSV-G/eGFP↑-co-cultures, respectively. Cellular fusion was induced by brief exposure to pH = 6.0 medium. Optical mapping experiments showed eGFP↑-co-cultures to be highly arrhythmogenic [43.3% early afterdepolarization (EAD) incidence vs. 7.7% in control NRVM cultures, P < 0.0001], with heterogeneous prolongation of action potential (AP) duration (APD). Fused VSV-G/eGFP↑-co-cultures displayed markedly lower EAD incidence (4.6%, P < 0.001) than unfused co-cultures, associated with decreases in APD, APD dispersion, and decay time of cytosolic Ca(2+) waves. Heterokaryons strongly expressed connexin43 (Cx43). Also, maximum diastolic potential in co-cultures was more negative after fusion, while heterokaryons exhibited diverse mixed NRVM/hVSC whole-cell current profiles, but consistently showed increased outward Kv currents compared with NRVMs or hVSCs. Inhibition of Kv channels by tetraethylammonium chloride abrogated the anti-arrhythmic effects of fusion in VSV-G/eGFP↑-co-cultures raising EAD incidence from 7.9 to 34.2% (P < 0.001). CONCLUSION: Forced fusion of cultured hVSCs with NRVMs yields electrically functional heterokaryons and reduces arrhythmogenicity by preventing EADs, which is, at least partly, attributable to increased repolarization force.

Asunto(s)

Antiarrítmicos/farmacología , Arritmias Cardíacas/metabolismo , Técnicas de Cocultivo , Ventrículos Cardíacos/citología , Miocardio/metabolismo , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Potenciales de Acción/fisiología , Animales , Células Cultivadas , Ventrículos Cardíacos/efectos de los fármacos , Humanos , Ratas

RESUMEN

AIMS: Atrial fibrillation (AF) is the most common cardiac arrhythmia and often involves reentrant electrical activation (e.g. spiral waves). Drug therapy for AF can have serious side effects including proarrhythmia, while electrical shock therapy is associated with discomfort and tissue damage. Hypothetically, forced expression and subsequent activation of light-gated cation channels in cardiomyocytes might deliver a depolarizing force sufficient for defibrillation, thereby circumventing the aforementioned drawbacks. We therefore investigated the feasibility of light-induced spiral wave termination through cardiac optogenetics. METHODS AND RESULTS: Neonatal rat atrial cardiomyocyte monolayers were transduced with lentiviral vectors encoding light-activated Ca(2+)-translocating channelrhodopsin (CatCh; LV.CatCh∼eYFP↑) or eYFP (LV.eYFP↑) as control, and burst-paced to induce spiral waves rotating around functional cores. Effects of CatCh activation on reentry were investigated by optical and multi-electrode array (MEA) mapping. Western blot analyses and immunocytology confirmed transgene expression. Brief blue light pulses (10 ms/470 nm) triggered action potentials only in LV.CatCh∼eYFP↑-transduced cultures, confirming functional CatCh-mediated current. Prolonged light pulses (500 ms) resulted in reentry termination in 100% of LV.CatCh∼eYFP↑-transduced cultures (n = 31) vs. 0% of LV.eYFP↑-transduced cultures (n = 11). Here, CatCh activation caused uniform depolarization, thereby decreasing overall excitability (MEA peak-to-peak amplitude decreased 251.3 ± 217.1 vs. 9.2 ± 9.5 µV in controls). Consequently, functional coresize increased and phase singularities (PSs) drifted, leading to reentry termination by PS-PS or PS-boundary collisions. CONCLUSION: This study shows that spiral waves in atrial cardiomyocyte monolayers can be terminated effectively by a light-induced depolarizing current, produced by the arrhythmogenic substrate itself, upon optogenetic engineering. These results provide proof-of-concept for shockless defibrillation.

Asunto(s)

Fibrilación Atrial/terapia , Luz , Miocitos Cardíacos/efectos de la radiación , Optogenética , Potenciales de Acción , Animales , Animales Recién Nacidos , Fibrilación Atrial/genética , Fibrilación Atrial/metabolismo , Fibrilación Atrial/fisiopatología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Western Blotting , Estimulación Cardíaca Artificial , Células Cultivadas , Channelrhodopsins , Estudios de Factibilidad , Técnica del Anticuerpo Fluorescente , Vectores Genéticos , Atrios Cardíacos/metabolismo , Atrios Cardíacos/fisiopatología , Atrios Cardíacos/efectos de la radiación , Lentivirus/genética , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Miocitos Cardíacos/metabolismo , Técnicas de Placa-Clamp , Ratas Wistar , Factores de Tiempo , Transducción Genética , Transfección , Imagen de Colorante Sensible al Voltaje

RESUMEN

BACKGROUND: Atrial fibrillation is the most common cardiac arrhythmia. Ventricular proarrhythmia hinders pharmacological atrial fibrillation treatment. Modulation of atrium-specific Kir3.x channels, which generate a constitutively active current (I(K,ACh-c)) after atrial remodeling, might circumvent this problem. However, it is unknown whether and how I(K,ACh-c) contributes to atrial fibrillation induction, dynamics, and termination. Therefore, we investigated the effects of I(K,ACh-c) blockade and Kir3.x downregulation on atrial fibrillation. METHODS AND RESULTS: Neonatal rat atrial cardiomyocyte cultures and intact atria were burst paced to induce reentry. To study the effects of Kir3.x on action potential characteristics and propagation patterns, cultures were treated with tertiapin or transduced with lentiviral vectors encoding Kcnj3- or Kcnj5-specific shRNAs. Kir3.1 and Kir3.4 were expressed in atrial but not in ventricular cardiomyocyte cultures. Tertiapin prolonged action potential duration (APD; 54.7±24.0 to 128.8±16.9 milliseconds; P<0.0001) in atrial cultures during reentry, indicating the presence of I(K,ACh-c). Furthermore, tertiapin decreased rotor frequency (14.4±7.4 to 6.6±2.0 Hz; P<0.05) and complexity (6.6±7.7 to 0.6±0.8 phase singularities; P<0.0001). Knockdown of Kcnj3 or Kcnj5 gave similar results. Blockade of I(K,ACh-c) prevented/terminated reentry by prolonging APD and changing APD and conduction velocity restitution slopes, thereby altering the probability of APD alternans and rotor destabilization. Whole-heart mapping experiments confirmed key findings (e.g., >50% reduction in atrial fibrillation inducibility after I(K,ACh-c) blockade). CONCLUSIONS: Atrium-specific Kir3.x controls the induction, dynamics, and termination of fibrillation by modulating APD and APD/conduction velocity restitution slopes in atrial tissue with I(K,ACh-c). This study provides new molecular and mechanistic insights into atrial tachyarrhythmias and identifies Kir3.x as a promising atrium-specific target for antiarrhythmic strategies.

Asunto(s)

Fibrilación Atrial/fisiopatología , Regulación hacia Abajo/fisiología , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/fisiología , Atrios Cardíacos/fisiopatología , Miocitos Cardíacos/fisiología , Acetilcolina/farmacología , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Animales , Venenos de Abeja/farmacología , Células Cultivadas , Modelos Animales de Enfermedad , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/efectos de los fármacos , Atrios Cardíacos/efectos de los fármacos , Miocitos Cardíacos/citología , Miocitos Cardíacos/efectos de los fármacos , Ratas , Ratas Wistar , Factores de Tiempo , Imagen de Colorante Sensible al Voltaje

RESUMEN

AIMS: Cardiac hypertrophy and fibrosis are associated with potentially lethal arrhythmias. As these substrates often occur simultaneously in one patient, distinguishing between pro-arrhythmic mechanisms is difficult. This hampers understanding of underlying pro-arrhythmic mechanisms and optimal treatment. This study investigates and compares arrhythmogeneity and underlying pro-arrhythmic mechanisms of either cardiac hypertrophy or fibrosis in in vitro models. METHODS AND RESULTS: Fibrosis was mimicked by free myofibroblast (MFB) proliferation in neonatal rat ventricular monolayers. Cultures with inhibited MFB proliferation were used as control or exposed to phenylephrine to induce hypertrophy. At Day 9, cultures were studied with patch-clamp and optical-mapping techniques and assessed for protein expression. In hypertrophic (n = 111) and fibrotic cultures (n = 107), conduction and repolarization were slowed. Triggered activity was commonly found in these substrates and led to high incidences of spontaneous re-entrant arrhythmias [67.5% hypertrophic, 78.5% fibrotic vs. 2.9% in controls (n = 102)] or focal arrhythmias (39.1, 51.7 vs. 8.8%, respectively). Kv4.3 and Cx43 protein expression levels were decreased in hypertrophy but unaffected in fibrosis. Depolarization of cardiomyocytes (CMCs) was only found in fibrotic cultures (-48 ± 7 vs. -66 ± 7 mV in control, P < 0.001). L-type calcium-channel blockade prevented arrhythmias in hypertrophy, but caused conduction block in fibrosis. Targeting heterocellular coupling by low doses of gap-junction uncouplers prevented arrhythmias by accelerating repolarization only in fibrotic cultures. CONCLUSION: Cultured hypertrophic or fibrotic myocardial tissues generated similar focal and re-entrant arrhythmias. These models revealed electrical remodelling of CMCs as a pro-arrhythmic mechanism of hypertrophy and MFB-induced depolarization of CMCs as a pro-arrhythmic mechanism of fibrosis. These findings provide novel mechanistic insight into substrate-specific arrhythmicity.

Asunto(s)

Arritmias Cardíacas/etiología , Cardiomegalia/complicaciones , Miocitos Cardíacos/metabolismo , Potenciales de Acción , Animales , Animales Recién Nacidos , Antiarrítmicos/farmacología , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/patología , Arritmias Cardíacas/fisiopatología , Arritmias Cardíacas/prevención & control , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio Tipo L/efectos de los fármacos , Canales de Calcio Tipo L/metabolismo , Cardiomegalia/metabolismo , Cardiomegalia/patología , Cardiomegalia/fisiopatología , Células Cultivadas , Técnicas de Cocultivo , Conexina 43/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patología , Fibrosis , Uniones Comunicantes/efectos de los fármacos , Uniones Comunicantes/metabolismo , Cinética , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/patología , Técnicas de Placa-Clamp , Fenilefrina/farmacología , Ratas , Canales de Potasio Shal/metabolismo , Imagen de Colorante Sensible al Voltaje

RESUMEN

AIMS: Sustained ventricular fibrillation (VF) is maintained by multiple stable rotors. Destabilization of sustained VF could be beneficial by affecting VF complexity (defined by the number of rotors). However, underlying mechanisms affecting VF stability are poorly understood. Therefore, the aim of this study was to correlate changes in arrhythmia complexity with changes in specific electrophysiological parameters, allowing a search for novel factors and underlying mechanisms affecting stability of sustained VF. METHODS AND RESULTS: Neonatal rat ventricular cardiomyocyte monolayers and Langendorff-perfused adult rat hearts were exposed to increasing dosages of the gap junctional uncoupler 2-aminoethoxydiphenyl borate (2-APB) to induce arrhythmias. Ion channel blockers/openers were added to study effects on VF stability. Electrophysiological parameters were assessed by optical mapping and patch-clamp techniques. Arrhythmia complexity in cardiomyocyte cultures increased with increasing dosages of 2-APB (n > 38), leading to sustained VF: 0.0 ± 0.1 phase singularities/cm(2) in controls vs. 0.0 ± 0.1, 1.0 ± 0.9, 3.3 ± 3.2, 11.0 ± 10.1, and 54.3 ± 21.7 in 5, 10, 15, 20, and 25 µmol/L 2-APB, respectively. Arrhythmia complexity inversely correlated with wavelength. Lengthening of wavelength during fibrillation could only be induced by agents (BaCl(2)/BayK8644) increasing the action potential duration (APD) at maximal activation frequencies (minimal APD); 123 ± 32%/117 ± 24% of control. Minimal APD prolongation led to transient VF destabilization, shown by critical wavefront collision leading to rotor termination, followed by significant decreases in VF complexity and activation frequency (52%/37%). These key findings were reproduced ex vivo in rat hearts (n = 6 per group). CONCLUSION: These results show that stability of sustained fibrillation is regulated by minimal APD. Minimal APD prolongation leads to transient destabilization of fibrillation, ultimately decreasing VF complexity, thereby providing novel insights into anti-fibrillatory mechanisms.

Asunto(s)

Potenciales de Acción , Uniones Comunicantes/metabolismo , Miocitos Cardíacos/metabolismo , Fibrilación Ventricular/metabolismo , Potenciales de Acción/efectos de los fármacos , Animales , Animales Recién Nacidos , Compuestos de Boro/toxicidad , Células Cultivadas , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Uniones Comunicantes/efectos de los fármacos , Canales Iónicos/efectos de los fármacos , Canales Iónicos/metabolismo , Cinética , Moduladores del Transporte de Membrana/toxicidad , Miocitos Cardíacos/efectos de los fármacos , Técnicas de Placa-Clamp , Perfusión , Ratas , Fibrilación Ventricular/inducido químicamente , Fibrilación Ventricular/fisiopatología , Imagen de Colorante Sensible al Voltaje

RESUMEN

Depolarization-induced automaticity (DIA) of cardiomyocytes is the property of those cells to generate pacemaker cell-like spontaneous electrical activity when subjected to a depolarizing current. This property provides a candidate mechanism for generation of pathogenic ectopy in cardiac tissue. The purpose of this study was to determine the biophysical mechanism of DIA in terms of the ion conductance properties of the cardiomyocyte membrane. First, we determined, by use of the conventional whole-cell patch-clamp technique, the membrane conductance and DIA properties of ventricular cardiomyocytes isolated from adult rat heart. Second, we reproduced and analysed DIA properties by using an adapted version of the experimentally based mathematical cardiomyocyte model of Pandit et al. (Biophys J 81:3029-3051 2001, Biophys J 84:832-841 2003) and Padmala and Demir (J Cardiovasc Electrophysiol 14:990-995 2003). DIA in 23 rat cardiomyocytes was a damped membrane potential oscillation with a variable number of action potentials and/or waves, depending on the strength of the depolarizing current and the particular cell. The adapted model was used to reconstruct the DIA properties of a particular cardiomyocyte from its whole-cell voltage-clamp currents. The main currents involved in DIA were an L-type calcium current (I CaL) and a slowly activating and inactivating Kv current (I ss), with linear (I B) and inward rectifier (I K1) currents acting as background currents and I Na and I t as modulators. Essential for DIA is a sufficiently large window current of a slowly inactivating I CaL combined with a critically sized repolarizing current I ss. Slow inactivation of I ss makes DIA transient. In conclusion, we established a membrane mechanism of DIA primarily based on I CaL, I ss and inward rectifier properties; this may be helpful in understanding cardiac ectopy and its treatment.

Asunto(s)

Canales de Calcio Tipo L/metabolismo , Ventrículos Cardíacos/citología , Activación del Canal Iónico , Potenciales de la Membrana , Miocitos Cardíacos/citología , Canales de Potasio con Entrada de Voltaje/metabolismo , Potenciales de Acción , Animales , Conductividad Eléctrica , Femenino , Modelos Biológicos , Miocitos Cardíacos/metabolismo , Ratas , Ratas Wistar

RESUMEN

BACKGROUND: Right ventricular failure (RVF) in pulmonary hypertension (PH) is associated with increased incidence of sudden death by a poorly explored mechanism. We test the hypothesis that PH promotes spontaneous ventricular fibrillation (VF) during a critical post-PH onset period characterized by a sudden increase in mortality. METHODS AND RESULTS: Rats received either a single subcutaneous dose of monocrotaline (MCT, 60 mg/kg) to induce PH-associated RVF (PH, n=24) or saline (control, n=17). Activation pattern of the RV-epicardial surface was mapped using voltage-sensitive dye in isolated Langendorff-perfused hearts along with single glass-microelectrode and ECG-recordings. MCT-injected rats developed severe PH by day 21 and progressed to RVF by approximately day 30. Rats manifested increased mortality, and ≈30% rats died suddenly and precipitously during 23-32 days after MCT. This fatal period was associated with the initiation of spontaneous VF by a focal mechanism in the RV, which was subsequently maintained by both focal and incomplete reentrant wave fronts. Microelectrode recordings from the RV-epicardium at the onset of focal activity showed early afterdepolarization-mediated triggered activity that led to VF. The onset of the RV cellular triggered beats preceded left ventricular depolarizations by 23±8 ms. The RV but not the left ventricular cardiomyocytes isolated during this fatal period manifested significant action potential duration prolongation, dispersion, and an increased susceptibility to depolarization-induced repetitive activity. No spontaneous VF was observed in any of the control hearts. RVF was associated with significantly reduced RV ejection fraction (P<0.001), RV hypertrophy (P<0.001), and RV fibrosis (P<0.01). The hemodynamic function of the LV and its structure were preserved. CONCLUSIONS: PH-induced RVF is associated with a distinct phase of increased mortality characterized by spontaneous VF arising from the RV by an early afterdepolarization-mediated triggered activity.

Asunto(s)

Insuficiencia Cardíaca/complicaciones , Hipertensión Pulmonar/complicaciones , Disfunción Ventricular Derecha/complicaciones , Fibrilación Ventricular/etiología , Animales , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Electrocardiografía , Estudios de Seguimiento , Insuficiencia Cardíaca/fisiopatología , Frecuencia Cardíaca/fisiología , Hipertensión Pulmonar/fisiopatología , Masculino , Pronóstico , Ratas , Ratas Sprague-Dawley , Disfunción Ventricular Derecha/fisiopatología , Fibrilación Ventricular/fisiopatología

RESUMEN

AIMS: Arrhythmogenesis in cardiac fibrosis remains incompletely understood. Therefore, this study aims to investigate how heterocellular coupling between cardiomyocytes (CMCs) and myofibroblasts (MFBs) affects arrhythmogeneity of fibrotic myocardial cultures. Potentially, this may lead to the identification of novel anti-arrhythmic strategies. METHODS AND RESULTS: Co-cultures of neonatal rat CMCs and MFBs in a 1:1 ratio were used as a model of cardiac fibrosis, with purified CMC cultures as control. Arrhythmogeneity was studied at day 9 of culture by voltage-sensitive dye mapping. Heterocellular coupling was reduced by transducing MFBs with lentiviral vectors encoding shRNA targeting connexin43 (Cx43) or luciferase (pLuc) as control. In fibrotic cultures, conduction velocity (CV) was lowered (11.2 ± 1.6 cm/s vs. 23.9 ± 2.1 cm/s; P < 0.0001), while action potential duration and ectopic activity were increased. Maximal diastolic membrane potential (MDP) of CMCs was less negative in fibrotic cultures. In fibrotic cultures, (n = 30) 30.0% showed spontaneous re-entrant tachyarrhythmias compared with 5% in controls (n = 60). Cx43 silencing in MFBs made the MDP in CMCs more negative, increased excitability and CV by 51% (P < 0.001), and reduced action potential duration and ectopic activity (P < 0.01), thereby reducing re-entry incidence by 40% compared with pLuc-silenced controls. Anti-arrhythmic effects of Cx43 down-regulation in MFBs was reversed by depolarization of CMCs through I(k1) inhibition or increasing extracellular [K(+)]. CONCLUSION: Arrhythmogeneity of fibrotic myocardial cultures is mediated by Cx43 expression in MFBs. Reduced expression of Cx43 causes a more negative MDP of CMCs. This preserves CMC excitability, limits prolongation of repolarization and thereby strongly reduces the incidence of spontaneous re-entrant tachyarrhythmias.

Asunto(s)

Conexina 43/genética , Diástole/fisiología , Terapia Genética/métodos , Miocitos Cardíacos/fisiología , Taquicardia/prevención & control , Potenciales de Acción/fisiología , Animales , Animales Recién Nacidos , Conexina 43/metabolismo , Fibroblastos/patología , Fibroblastos/fisiología , Fibrosis/patología , Fibrosis/fisiopatología , Sistema de Conducción Cardíaco/fisiología , Lentivirus/genética , Miocardio/patología , Miocitos Cardíacos/patología , Técnicas de Placa-Clamp , Cultivo Primario de Células , ARN Interferente Pequeño/genética , Ratas , Ratas Wistar , Taquicardia/genética , Taquicardia/fisiopatología

RESUMEN

Mesenchymal progenitor cells can be differentiated in vitro into myotubes that exhibit many characteristic features of primary mammalian skeletal muscle fibers. However, in general, they do not show the functional excitation-contraction coupling or the striated sarcomere arrangement typical of mature myofibers. Epigenetic modifications have been shown to play a key role in regulating the progressional changes in transcription necessary for muscle differentiation. In this study, we demonstrate that treatment of murine C2C12 mesenchymal progenitor cells with 10 µM of the DNA methylation inhibitor 5-azacytidine (5AC) promotes myogenesis, resulting in myotubes with enhanced maturity as compared to untreated myotubes. Specifically, 5AC treatment resulted in the up-regulation of muscle genes at the myoblast stage, while at later stages nearly 50% of the 5AC-treated myotubes displayed a mature, well-defined sarcomere organization, as well as spontaneous contractions that coincided with action potentials and intracellular calcium transients. Both the percentage of striated myotubes and their contractile activity could be inhibited by 20 nM TTX, 10 µM ryanodine, and 100 µM nifedipine, suggesting that action potential-induced calcium transients are responsible for these characteristics. Our data suggest that genomic demethylation induced by 5AC overcomes an epigenetic barrier that prevents untreated C2C12 myotubes from reaching full maturity.

Asunto(s)

Azacitidina/farmacología , Metilación de ADN/fisiología , Desarrollo de Músculos/efectos de los fármacos , Músculo Esquelético/citología , Animales , Señalización del Calcio/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Línea Celular , Quelantes/farmacología , Metilación de ADN/efectos de los fármacos , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Epigénesis Genética , Perfilación de la Expresión Génica , Células Madre Mesenquimatosas , Ratones , Contracción Muscular/efectos de los fármacos , Fibras Musculares Esqueléticas , Nifedipino/farmacología , Rianodina/farmacología , Sarcómeros/efectos de los fármacos , Sarcómeros/ultraestructura , Tetrodotoxina/farmacología

RESUMEN

AIMS: Cardiac fibrosis is associated with increased incidence of cardiac arrhythmias, but the underlying proarrhythmic mechanisms remain incompletely understood and antiarrhythmic therapies are still suboptimal. This study tests the hypothesis that myofibroblast (MFB) proliferation leads to tachyarrhythmias by altering the excitability of cardiomyocytes (CMCs) and that inhibition of MFB proliferation would thus lower the incidence of such arrhythmias. METHODS AND RESULTS: Endogenous MFBs in neonatal rat CMC cultures proliferated freely or under control of different dosages of antiproliferative agents (mitomycin-C and paclitaxel). At Days 4 and 9, arrhythmogeneity of these cultures was studied by optical and multi-electrode mapping. Cultures were also studied for protein expression and electrophysiological properties. MFB proliferation slowed conduction from 15.3 ± 3.5 cm/s (Day 4) to 8.8 ± 0.3 cm/s (Day 9) (n = 75, P < 0.01), whereas MFB numbers increased to 37.4 ± 1.7 and 62.0 ± 2%. At Day 9, 81.3% of these cultures showed sustained spontaneous reentrant arrhythmias. However, only 2.6% of mitomycin-C-treated cultures (n = 76, P < 0.0001) showed tachyarrhythmias, and ectopic activity was decreased. Arrhythmia incidence was drug-dose dependent and strongly related to MFB proliferation. Paclitaxel treatment yielded similar results. CMCs were functionally coupled to MFBs and more depolarized in cultures with ongoing MFB proliferation in which only L-type Ca(2+)-channel blockade terminated 100% of reentrant arrhythmias, in contrast to Na(+) blockade (36%, n = 12). CONCLUSION: Proliferation of MFBs in myocardial cultures gives rise to spontaneous, sustained reentrant tachyarrhythmias. Antiproliferative treatment of such cultures prevents the occurrence of arrhythmias by limiting MFB-induced depolarization, conduction slowing, and ectopic activity. This study could provide a rationale for a new treatment option for cardiac arrhythmias.

Asunto(s)

Fibroblastos , Mitomicina/farmacología , Miocardio/citología , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Taquicardia , Animales , Antineoplásicos Fitogénicos/farmacología , División Celular/efectos de los fármacos , División Celular/fisiología , Células Cultivadas , Técnicas de Cocultivo , Relación Dosis-Respuesta a Droga , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/fisiología , Ventrículos Cardíacos/citología , Miocitos Cardíacos/citología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/fisiología , Paclitaxel/farmacología , Técnicas de Placa-Clamp , Ratas , Taquicardia/patología , Taquicardia/fisiopatología , Taquicardia/prevención & control

RESUMEN

Alignment of cardiomyocytes (CMCs) contributes to the anisotropic (direction-related) tissue structure of the heart, thereby facilitating efficient electrical and mechanical activation of the ventricles. This study aimed to investigate the effects of forced alignment of stem cells during cardiomyogenic differentiation on their functional integration with CMC cultures. Labeled neonatal rat (nr) mesenchymal stem cells (nrMSCs) were allowed to differentiate into functional heart muscle cells in different cell-alignment patterns during 10 days of coculture with nrCMCs. Development of functional cellular properties was assessed by measuring impulse transmission across these stem cells between 2 adjacent nrCMC fields, cultured onto microelectrode arrays and previously separated by a laser-dissected channel (230+/-10 microm) for nrMSC transplantation. Coatings in these channels were microabraded in a direction (1) parallel or (2) perpendicular to the channel or were (3) left unabraded to establish different cell patterns. Application of cells onto microabraded coatings resulted in anisotropic cell alignment within the channel. Application on unabraded coatings resulted in isotropic (random) alignment. After coculture, conduction across seeded nrMSCs occurred from day 1 (perpendicular and isotropic) or day 6 (parallel) onward. Conduction velocity across nrMSCs at day 10 was highest in the perpendicular (11+/-0.9 cm/sec; n=12), intermediate in the isotropic (7.1+/-1 cm/sec; n=11) and lowest in the parallel configuration (4.9+/-1 cm/sec; n=11) (P<0.01). nrCMCs and fibroblasts served as positive and negative control, respectively. Also, immunocytochemical analysis showed alignment-dependent increases in connexin 43 expression. In conclusion, forced alignment of nrMSCs undergoing cardiomyogenic differentiation affects the time course and degree of functional integration with surrounding cardiac tissue.

Asunto(s)

Comunicación Celular/fisiología , Diferenciación Celular/fisiología , Sistema de Conducción Cardíaco/fisiología , Células Madre Mesenquimatosas/citología , Miocitos Cardíacos/citología , Animales , Células Cultivadas , Técnicas de Cocultivo , Conexina 43/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Masculino , Células Madre Mesenquimatosas/metabolismo , Microelectrodos , Modelos Animales , Miocardio/metabolismo , Miocitos Cardíacos/metabolismo , Técnicas de Placa-Clamp , Ratas , Ratas Wistar

RESUMEN

BACKGROUND: Butamben or n-butyl-p-aminobenzoate is a long-acting experimental local anesthetic for the treatment of chronic pain when given as an epidural suspension. We have investigated whether Cav1.2/L-type calcium channels may be a target of this butamben action. METHODS: The effect of butamben on these channels was studied in undifferentiated rat PC12-cells with the whole-cell patch-clamp technique in voltage-clamp. Ba(2+) ions were used as the charge carriers in the calcium channel currents, whereas K(+) currents were removed using K(+) free solutions. RESULTS: Butamben 500 microM reversibly suppressed the total whole-cell barium current by 90% +/- 3% (n = 15), whereas 10 microM nifedipine suppressed this barium current by 75% +/- 7% (n = 6). Preexposure to butamben followed by washout decreased the inhibition by nifidepine to 47% +/- 5% (n = 10). These suppressive effects were not due to the measurement procedure and the drug vehicles in the solutions (<0.1% ethanol; n = 6). CONCLUSIONS: Butamben inhibits the total barium current through expressed calcium channel types in PC12 cells, including Cav1.2/L-type channels. Because Cav1.2 channels may also occur in human nociceptive C fibers, this result allows the possibility that these L-type channels are involved in the analgesic action of butamben.

Asunto(s)

Analgésicos/farmacología , Anestésicos Locales/farmacología , Bario/metabolismo , Benzocaína/análogos & derivados , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio Tipo L/efectos de los fármacos , Neuronas/efectos de los fármacos , Animales , Benzocaína/farmacología , Canales de Calcio Tipo L/metabolismo , Neuronas/metabolismo , Nifedipino/farmacología , Células PC12 , Técnicas de Placa-Clamp , Ratas

RESUMEN

BACKGROUND: Nonresponse to cardiac resynchronization therapy is associated with the presence of slow or nonconducting scar tissue. Genetic modification of scar tissue, aimed at improving conduction, may be a novel approach to achieve effective resynchronization. Therefore, the feasibility of resynchronization with genetically modified human ventricular scar fibroblasts was studied in a coculture model. METHODS AND RESULTS: An in vitro model was used to study the effects of forced expression of the myocardin (MyoC) gene in human ventricular scar fibroblasts (hVSFs) on resynchronization of 2 rat cardiomyocyte fields separated by a strip of hVSFs. Furthermore, the effects of MyoC expression on the capacity of hVSFs to serve as pacing sites were studied. MyoC-dependent gene activation in hVSFs was examined by gene and immunocytochemical analysis. Forced MyoC expression in hVSFs decreased dyssynchrony, expressed as the activation delay between 2 cardiomyocyte fields (control hVSFs 27.6+/-0.2 ms [n=11] versus MyoC-hVSFs 3.6+/-0.3 ms [n=11] at day 8, P<0.01). Also, MyoC-hVSFs could be stimulated electrically, which resulted in simultaneous activation of the 2 adjacent cardiomyocyte fields. Forced MyoC expression in hVSFs led to the expression of various connexin and cardiac ion channel genes. Intracellular measurements of MyoC-hVSFs coupled to surrounding cardiomyocytes showed strongly improved action potential conduction. CONCLUSIONS: Forced MyoC gene expression in hVSFs allowed electrical stimulation of these cells and conferred the ability to conduct an electrical impulse at high velocity, which resulted in resynchronization of 2 separated cardiomyocyte fields. Both phenomena appear mediated mainly by MyoC-dependent activation of genes that encode connexins, strongly enforcing intercellular electrical coupling.

Asunto(s)

Potenciales de Acción/fisiología , Cicatriz/fisiopatología , Fibroblastos/fisiología , Miocitos Cardíacos/fisiología , Animales , Células Cultivadas , Cicatriz/metabolismo , Cicatriz/patología , Técnicas de Cocultivo/métodos , Conexinas/biosíntesis , Conexinas/genética , Estimulación Eléctrica/métodos , Fibroblastos/metabolismo , Fibroblastos/patología , Regulación de la Expresión Génica/fisiología , Ventrículos Cardíacos/citología , Ventrículos Cardíacos/metabolismo , Humanos , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Proteínas Nucleares/fisiología , Ratas , Ratas Wistar , Transactivadores/fisiología , Activación Transcripcional , Función Ventricular

RESUMEN

Zervamicin IIB (ZER) is a 16-mer peptaibol that produces voltage-dependent conductances in artificial membranes, a property considered responsible for its antimicrobial activity to mainly Gram-positive microorganisms. In addition, ZER appears to inhibit the locomotor activity of the mouse (see elsewhere in this Issue), probably by affecting the brain. To examine whether the electrophysiological properties of the neuronal cells of the central neural system might be possibly influenced by the pore forming ZER, the present study was undertaken as a first attempt to unravel the molecular mechanism of this biological activity. To this end, membrane permeabilization of the neuron-like rat pheochromocytoma cell (PC12) by the channel-forming ZER was studied with the whole-cell patch-clamp technique, and compared with the permeabilizations of the well-known voltage-gated peptaibol alamethicin F50/5 (ALA) and the cation channel-forming peptide-antibiotic gramicidin D (GRAM). While 1 muM GRAM addition to PC12 cells kept at a membrane potential V(m)=0 mV causes an undelayed gradual increase of a leak conductance with a negative reversal potential of ca. -24 mV, ZER and ALA are ineffective at that concentration and potential. However, if ZER and ALA are added in 5-10 microM concentrations while V(m) is kept at -60 mV, they cause a sudden and strong permeabilization of the PC12 cell membrane after a delay of 1-2 min, usually leading to disintegrating morphology changes of the patched cell but not of the surrounding cells of the culture at that time scale. The zero reversal potential of the established conductance is consistent with the known aselectivity of the channels formed. This sudden permeabilization does not occur within 10-20 min at V(m)=0 mV, in accordance with the known voltage dependency of ZER and ALA channel formation in artificial lipid membranes. The permeabilizing action of these peptaibols on the culture as a whole is further supported by K(+)-release measurements from a PC12 suspension with a K(+)-selective electrode. Further analysis suggested that the permeabilizing action is associated with extra- or intracellular calcium effects, because barium inhibited the permeabilizing effects of ZER and ALA. We conclude, for the membrane of the mammalian neuron-like PC12 cell, that the permeabilizing effects of the peptides ZER and ALA are different from those of GRAM, consistent with earlier studies of these peptides in other (artificial) membrane systems. They are increased by cis-positive membrane potentials in the physiological range and may include calcium entry into the PC12 cell.

Asunto(s)

Alameticina/farmacología , Antibacterianos/farmacología , Permeabilidad de la Membrana Celular/efectos de los fármacos , Gramicidina/farmacología , Sistemas Neurosecretores/efectos de los fármacos , Péptidos/farmacología , Secuencia de Aminoácidos , Animales , Pruebas de Sensibilidad Microbiana , Sistemas Neurosecretores/citología , Células PC12 , Peptaiboles , Ratas