Cellular Electrophysiological Changes Research Articles

Determinants of electrical propagation and propagation block in Arrhythmogenic Cardiomyopathy

Gap junction and ion channel remodeling occur early in Arrhythmogenic Cardiomyopathy (ACM), but their pathogenic consequences have not been elucidated. Here, we identified the arrhythmogenic substrate, consisting of propagation slowing and conduction block, in ACM models expressing two different desmosomal gene variants. Neonatal rat ventricular myocytes were transduced to express variants in genes encoding desmosomal proteins plakoglobin or plakophilin-2. Studies were performed in engineered cells and anisotropic tissues to quantify changes in conduction velocity, formation of unidirectional propagation, cell–cell electrical coupling, and ion currents. Conduction velocity decreased by 71% and 63% in the two ACM models. SB216763, an inhibitor of glycogen synthase kinase-3 beta, restored conduction velocity to near normal levels. Compared to control, both ACM models showed greater propensity for unidirectional conduction block, which increased further at greater stimulation frequencies. Cell–cell electrical conductance measured in cell pairs was reduced by 86% and 87% in the two ACM models. Computer modeling showed close correspondence between simulated and experimentally determined changes in conduction velocity. The simulation identified that reduced cell–cell electrical coupling was the dominant factor leading to slow conduction, while the combination of reduced cell–cell electrical coupling, reduced sodium current and inward rectifier potassium current explained the development of unidirectional block. Expression of two different ACM variants markedly reduced cell–cell electrical coupling and conduction velocity, and greatly increased the likelihood of developing unidirectional block – both key features of arrhythmogenesis. This study provides the first quantitative analysis of cellular electrophysiological changes leading to the substrate of reentrant arrhythmias in early stage ACM.

Characterization of atrial cardiomyopathy subtypes - A cellular electrophysiological comparison and evaluation of appropriate treatment options

Abstract Background Atrial fibrillation occurs in the context of atrial remodeling processes which are the result of an underlying atrial cardiomyopathy (AC). Insufficient understanding of the pathophysiological mechanisms behind AC contributes significantly to the fact that current clinical strategies for the treatment of atrial fibrillation are often ineffective. A systematic investigation of these cellular remodeling processes in the context of the various cardiac diseases underlying AC is still missing. Purpose The aim of this study was to characterize different subtypes of AC associated with common cardiac disease entities by systematic cellular electrophysiological characterization of the atria in combination with molecular and structural investigations. Methods For a comprehensive analysis of the molecular changes associated with AC, we characterized several mouse models of common heart diseases, including models for dilated cardiomyopathy (DCM), HFpEF, mitral regurgitation and immune checkpoint inhibitor (ICI-)mediated myocarditis. Patch clamp experiments were performed on isolated atrial cardiomyocytes to study atrial action potentials (aAP) and different ion currents contributing to the aAP. The cellular electrophysiological parameters of the different models were correlated to clinical parameters (ecg, echocardiography of the atria) of the mouse models and with data from RNA sequencing of the mice’ atria. Results Patch clamp measurements performed on isolated left and right atrial cardiomyocytes revealed distinct differences among the examined models. In all pathophysiological models, the aAP was significantly shorter than in the corresponding wild type mice. The different ion current components we measured various changes in potassium, sodium and calcium currents. The repolarizing TASK-1 potassium current was significantly increased in all models. The DCM model caused by an R636Q mutation in the RBM20 gene showed the most severe changes in cellular electrophysiology. Considering the detected atrial changes, we investigated the effects of SGLTi on atrial cardiomyocytes of the different models. When elevated concentrations of dapa-, empa- and sotagliflozin were administrated on atrial cardiomyocytes, a class-I-antiarrhythmic effect with reduced aAP inducibility and changes in the aAP duration could be observed. Conclusion Characterization of murine models of cardiac disease with respect to classification of AC subtypes revealed various electrical remodeling processes at the level of ion currents involved in aAP formation. A potent inductor of atrial proarrhythmogenity in the AC subtypes was an aAP shortening caused by an increase in repolarizing potassium currents, mediated especially by TASK-1. The most severe electrical remodeling in atria cardiomyocytes was observed in the RBM20-mutant model (DCM). SGLTi exhibit anti-arrhythmic effects in different murine disease models and may provide future treatment options for patients with AC.

PECULIARITIES OF CONNECTIVE TISSUE METABOLISM IN PATIENTS WITH POST-INFARCTION REMODELING AND REPERFUSION SYNDROME

Early restoration of coronary blood flow in acute myocardial infarction with elevation helps reduce the area of myocardial necrosis, prevent dilatation of the ventricular cavity, and reduce the development of arrhythmias and mortality. However, despite modern technologies in STEMI treatment, the development of reperfusion syndrome, in particular, arrhythmias, is possible after the restoration of blood flow in the infarct-related artery, which is a consequence of acute cellular, metabolic and local electrophysiological changes in the myocardium.
 The aim: is to investigate the long-term effects of reperfusion syndrome on the catabolic/anabolic activity of connective tissue and the processes of connective tissue framework formation in the post-infarction period in patients with myocardial infarction.
 Materials and methods. The study involved 156 patients with acute myocardial infarction with ST segment elevation (STEMI), and 15 apparently healthy individuals (control of laboratory parameters). The average age of the patients was (57.8±1.2) years. The percent of male patients prevailed (76 %).
 All MI patients were divided into two groups: Group I (98 people) – MI patients without clinical signs of reperfusion syndrome during revascularization, Group II (58 people) – MI patients with the signs of reperfusion syndrome in the form of rhythm and conductivity disturbances during revascularization of coronary arteries by percutaneous coronarography intervention (PCI) with prolonged stenting of the infarct-dependent coronary artery.
 Peculiarities of connective tissue metabolism were studied by the content of free and bound oxyproline, glycosaminoglycans, and antibodies to atypical collagens (Ig M and Ig G) in blood serum.
 Results. The recovery period after myocardial infarction showed an increase in collagen catabolism over collagen synthesizing processes in patients with MI who had reperfusion syndrome. The described patterns were also established by changes in glycosaminoglycans. The study showed an increase in the level of antibodies to atypical collagens. Particularly, the level of Ig M, which was within the norm (0.43±0.18) ng/ml, in patients with MI without reperfusion manifestations was three times higher (p<0.001), and in cases of combination of MI and reperfusion syndrome – the highest (p<0.001).
 Conclusions:
 
 Changes in collagen-synthesizing processes are observed in patients with MI, with a tendency to increase the processes of catabolism of the connective tissue matrix.
 The presence of reperfusion syndrome manifestations in MI patients after revascularization aggravates the processes of catabolism, even in the recovery period after MI.
 Increase in the activity of catabolic processes stimulates fibrosis and formation of rigid matrix and atypical collagen, to which antibodies are synthesized, thus blocking the pathological circuit of restructuring of the heart connective tissue framework, and accordingly its pathological remodeling.
 The obtained study results contribute to the identification of more sophisticated approaches to revascularization techniques to avoid negative effects of reperfusion syndrome, as well as new effective medications for collagen synthesis correction, which can increase the effectiveness of rehabilitative management of such

Circadian rhythmicity in murine blood: Electrical effects of malaria infection and anemia.

Circadian rhythms are biological adaptations to the day-night cycle, whereby cells adapt to changes in the external environment or internal physiology according to the time of day. Whilst many cellular clock mechanisms involve gene expression feedback mechanisms, clocks operate even where gene expression is absent. For example, red blood cells (RBCs) do not have capacity for gene expression, and instead possess an electrophysiological oscillator where cytosolic potassium plays a key role in timekeeping. We examined murine blood under normal conditions as well as in two perturbed states, malaria infection and induced anemia, to assess changes in baseline cellular electrophysiology and its implications for the electrophysiological oscillator. Blood samples were analyzed at 4-h intervals over 2days by dielectrophoresis, and microscopic determination of parasitemia. We found that cytoplasmic conductivity (indicating the concentration of free ions in the cytoplasm and related to the membrane potential) exhibited circadian rhythmic behavior in all three cases (control, malaria and anemia). Compared to control samples, cytoplasm conductivity was decreased in the anemia group, whilst malaria-infected samples were in antiphase to control. Furthermore, we identified rhythmic behavior in membrane capacitance of malaria infected cells that was not replicated in the other samples. Finally, we reveal the historically famous rhythmicity of malaria parasite replication is in phase with cytoplasm conductivity. Our findings suggest the electrophysiological oscillator can impact on malaria parasite replication and/or is vulnerable to perturbation by rhythmic parasite activities.

TNNI3K delays atrioventricular conduction and reduces connexin-45 gap junctional coupling

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): The Dutch Research Council (NWO Talent Scheme) Introduction Cardiac conduction delay is the main substrate for triggering arrhythmias. Hence, prolongation of the PR interval on the electrocardiogram (ECG) is a strong predictor of atrial fibrillation, the most common cardiac arrhythmia. In previous research, cardiac troponin I-interacting kinase (TNNI3K) has been identified as a regulator of the PR interval. Various inbred mouse strains showed a correlation between Tnni3k expression levels and PR interval durations. Additionally, transgenic mice overexpressing hTNNI3K presented an extreme PR interval prolongation. Objective This study aims to unravel the mechanism underlying TNNI3K-driven PR interval prolongation. Methods ECG parameters were recorded in mice expressing physiological levels of Tnni3k (congenic), overexpressing hTNNI3K (TNNI3Ktg), or overexpressing kinase-dead hTNNI3K (TNNI3K-KDtg) and were compared to low-Tnni3k expressing DBA/2J control mice. Atrioventricular (AV) conduction was measured in Langendorff-perfused isolated hearts by electrical mapping. Cellular electrophysiology and conductance were measured using the (dual) patch-clamp technique. AV nodal collagen levels were identified by Pico Sirius Red staining. Candidate interactors were identified by immunoprecipitation of transfected HEK293A cell lysates. In stable HeLa-Connexin 45 (Cx45) cell lines expressing TNNI3K, Cx45 protein expression and phosphorylation levels were investigated by Western blot. Localisation of Cx45 was characterised by immunofluorescence. Results At six weeks of age, congenic and TNNI3Ktg mice show a 17% and 35% prolonged PR interval duration, respectively. Nonetheless, the PR interval of TNNI3K-KDtg mice did not change. Electrical mapping experiments on TNNI3Ktg mouse hearts show a corresponding AV conduction delay, which was neither driven by changes in cellular atrial cardiomyocyte electrophysiology nor driven by AV-nodal fibrosis. We next identified Cx45, a highly expressed connexin in the AV node, as a potential TNNI3K interactor. HeLa-Cx45 cells expressing TNNI3K demonstrated a reduced Cx45 conductance compared to controls without changes in kinetics. Moreover, we observed relatively decreased Cx45 phosphorylation and increased Cx45 intracellular accumulation. Conclusion We here present TNNI3K as a kinase-driven modulator of PR interval prolongation and AV conduction delay, which is independent of atrial electrophysiology and AV nodal fibrosis. We further identified Cx45 as a novel interactor of TNNI3K. The presence of TNNI3K reduces Cx45 gap junctional conductance and promotes intracellular Cx45 protein accumulation, which could explain the PR interval prolongation in vivo. Altogether, this study implies a crucial role for TNNI3K in AV nodal conduction.

Development an ovine myocardial infarction- induced heart failure model and characterisation of altered calcium homeostasis mechanisms

Abstract Introduction Ventricular arrhythmias (VA) and heart failure (HF) are the major complications following myocardial infarction (MI). In both conditions, there is a key role for perturbed calcium homeostasis of which the underlying mechanisms remain unclear. A preclinical model that faithfully presents most of the features of MI-induced HF has been lacking. The complexity of this syndrome means that animal modelling is difficult. As the hearts of large animals share many electrophysiological similarities to humans, ovine modelling of cardiac diseases could better reflect human pathologies than in small mammals. Question Is it possible to develop a clinically relevant ovine model with moderate cardiac dysfunction following myocardial infarction? Methods MI was induced in sheep by inflating an angioplasty balloon distal to the second diagonal branch of the left anterior descending artery for 90 min. Cardiac function was monitored for 20 weeks using electrocardiography (ECG), echocardiography, blood biochemical analysis, and subjective signs of cardiac deterioration (lethargy, dyspnoea, and cough). 20 weeks post-MI, the animals were humanely killed and single left ventricular myocytes were isolated from the infarct border zone. Changes in cellular electrophysiology and intracellular calcium concentration were monitored using whole-cell patch technique in voltage-clamp mode and the calcium sensitive fluorescent indicator Fura-2 (K5 salt). Results By using minimally invasive procedures, we obtained a survival rate of 80% (n=15). During surgery, our data show clinical features of ischaemia, including changes in the ECG features (elevation of the ST and T segment, left bundle branch block and/or pathological Q waves) and elevation of the cardiac biomarker such as troponin I. Following MI, we observed a decline in ejection fraction (−25±3%, p<0.0001) and an increase in whole animal arrhythmias (incidence of VA 72 hours post-MI, ∼70%). On cardiac removal apico-septal transmural necrosis / scarring was evident. Importantly, the L-type calcium current (ICaL) was decreased in MI cells compared to healthy cells (−1.87±0.73 pA/pF, p<0.05), but isoprenaline had no effet on ICaL (0.48±1.2 pA/pF, p=0.70) in MI cells. Moreover, the amplitude of the systolic calcium transient (−0.33±0.1 F/F0, p<0.05) and the sarcoplasmic reticulum calcium content (−23±7 μmol/L, p<0.01) were also decreased. The shortening velocity of the sarcomere was also decreased in MI cells (−0.55±0.18 μm/s, p<0.01). Conclusion We successfully established an ovine MI model using minimally invasive procedure which displays a moderately impaired cardiac function, reduced contractility, and pro-arrhythmic electrophysiological remodelling. Future analysis will examine the role of the L-type calcium channel with respect to the excitation-contraction coupling process and myocyte contractility and how we can improve therapeutic strategies towards VA and HF. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): British Heart Foundation

Untangling a Web: Basic Mechanisms of the Complex Interactions Between Sleep, Circadian Rhythms, and Epilepsy

Seizures have sleep–wake and circadian patterns in various epilepsies and, in turn, disrupt sleep and circadian rhythms. The resultant sleep deprivation (SD) is an exacerbating factor for seizures that sets up a vicious cycle that can potentially lead to disease progression and even to epilepsy-related mortality. A variety of cellular or network electrophysiological changes and changes in expression of clock-controlled genes or other transcription factors underlie sleep–wake and circadian distribution of seizures, as well as the disruptions seen in both. A broad understanding of these mechanisms may help in designing better treatments to prevent SD-induced seizure exacerbation, disrupt the vicious cycle of disease progression, and reduce epilepsy-related mortality.

Cellular Stress p38MAPK Activation Decrease NaV1.5 Current Density and Contributes to the Develop of Arrhythmia in Elderly

Rationale: During progressive aging, atrial and ventricular conduction velocities are slower which has been associated with combined NaV1.5 and Cx43 functional disruption and fibrosis, consequently resulting in greater vulnerability of aged hearts to develop arrhythmias. The p38MAPK proteins are members of the mitogen-activated protein kinase family. They are expressed in the heart and activated during stress responses. However, it is unknown whether p38MAPK regulates functional expression of cardiac NaV1.5 channels or if p38MAPK hyperactivation increases arrhythmia susceptibility in aged hearts. Hypothesis: The human cardiac NaV1.5 channel exhibits a p38MAPK-dependent modulation and in aged hearts p38MAPK hyperactivation contributes to development of arrhythmia susceptibility in elderly. Methods and Results: Using a multidisciplinary approach based on biochemical techniques, together with electrophysiological and cell imaging experiments the data show that with aging, stress-associated p38MAPK activation increases, hearts present cellular electrophysiological changes, and old mice had increased susceptibility to develop arrhythmias. Moreover, activation of p38MAPK decrease INa current density in transiently transfected HEK-293T cells with NaV1.5 and dV/dtmax in hiPSC-CMs. Furthermore, p38MAPK activation does not change voltage dependence of activation or inactivation or recovery from inactivation of NaV1.5 channel neither affected total NaV1.5 protein expression. Finally, pretreatment with a specific inhibitor of p38MAPK prevented the INa and dV/dtmax decrease in HEK-293T cells and hiPSC-CMs, respectively. Conclusions Altogether the results raise the possibility that p38MAPK signaling contributes to age-dependent NaV1.5 channel dysregulation, and consequently increases the susceptibility to develop arrhythmias. It may be possible to modulate p38MAPK activity to treat or prevent arrhythmias in the elderly population.

ATP increases [Ca2+ ]i and activates a Ca2+ -dependent Cl- current in rat ventricular fibroblasts.

What is the central question of this study? Although electrophysiological and biophysical characteristics of heart fibroblasts have been studied in detail, their responses to prominent paracrine agents in the myocardium have not been addressed adequately. Our experiments characterize changes in cellular electrophysiology and intracellular calcium in response to ATP. What is the main finding and its importance? In rat ventricular fibroblasts maintained in cell culture, we find that ATP activates a specific subset of Ca2+ -activated Cl- channels as a consequence of binding to P2Y purinoceptors and then activating phospholipaseC. This response is not dependent on [Ca2+ ]o but requires an increase in [Ca2+ ]i and is modulated by the type of nucleotide that is the purinergic agonist. Effects of ATP on enzymatically isolated rat ventricular fibroblasts maintained in short-term (36-72h) cell culture were examined. Immunocytochemical staining of these cells revealed that a fibroblast, as opposed to a myofibroblast, phenotype was predominant. ATP, ADP or uridine 5'-triphosphate (UTP) all produced large increases in [Ca2+ ]i . Voltage-clamp studies (amphotericin-perforated patch) showed that ATP (1-100μm) activated an outwardly rectifying current, with a reversal potential very close to the Nernst potential for Cl- . In contrast, ADP was much less effective, and UTP produced no detectable current. The non-selective Cl- channel blockers niflumic acid, DIDS and NPPB (each at 100μm), blocked the responses to 100μm ATP. An agonist for P2Y purinoceptors, 2-MTATP, activated a very similar outwardly rectifying C1- current. The P2Y receptor antagonists, suramin and PPADS (100μm each), significantly inhibited the Cl- current produced by 100μm ATP. ATP was able to activate this Cl- current when [Ca2+ ]o was removed, but not when [Ca2+ ]i was buffered with BAPTA-AM. In the presence of the phospholipaseC inhibitor U73122, this Cl- current could not be activated. PCR analysis revealed strong signals for a number of P2Y purinoceptors and for the Ca2+ -activated Cl- channel, TMEM16F (also denoted ANO6). In summary, these results demonstrate that activation of P2Y receptors by ATP causes a phospholipaseC-dependent increase in [Ca2+ ]i , followed by activation of a Ca2+ -dependent Cl- current in rat ventricular fibroblasts.

Increased susceptibility of spontaneously hypertensive rats to ventricular tachyarrhythmias in early hypertension.

Hypertension is a risk factor for sudden cardiac death caused by ventricular tachycardia and fibrillation (VT/VF). We hypothesized that, in early hypertension, the susceptibility to stress-induced VT/VF increases. We compared the susceptibility of 5- to 6-month-old male spontaneously hypertensive rats (SHR) and age/sex-matched normotensive rats (NR) to VT/VF during challenge with oxidative stress (H2 O2 ; 0.15 mmol l(-1) ). We found that only SHR hearts exhibited left ventricular fibrosis and hypertrophy. H2 O2 promoted VT in all 30 SHR but none of the NR hearts. In 33% of SHR cases, focal VT degenerated to VF within 3 s. Simultaneous voltage-calcium optical mapping of Langendorff-perfused SHR hearts revealed that H2 O2 -induced VT/VF arose spontaneously from focal activations at the base and mid left ventricular epicardium. Microelectrode recording of SHR hearts showed that VT was initiated by early afterdepolarization (EAD)-mediated triggered activity. However, despite the increased susceptibility of SHR hearts to VT/VF, patch clamped isolated SHR ventricular myocytes developed EADs and triggered activity to the same extent as NR ventricular myocytes, except with larger EAD amplitude. During the early stages of hypertension, when challenged with oxidative stress, SHR hearts showed an increased ventricular arrhythmogenicity that stems primarily from tissue remodelling (hypertrophy, fibrosis) rather than cellular electrophysiological changes. Our findings highlight the need for early hypertension treatment to minimize myocardial fibrosis, ventricular hypertrophy, and arrhythmias.

Electrophysiological Changes Correlated with Temperature Increases Induced by High-Intensity Focused Ultrasound Ablation

To gain better understanding of the detailed mechanisms of high-intensity focused ultrasound (HIFU) ablation for cardiac arrhythmias, we investigated how the cellular electrophysiological (EP) changes were correlated with temperature increases and thermal dose (cumulative equivalent minutes [CEM43]) during HIFU application using Langendorff-perfused rabbit hearts. Employing voltage-sensitive dye di-4-ANEPPS, we measured the EP and temperature during HIFU using simultaneous optical mapping and infrared imaging. Both action potential amplitude (APA) and action potential duration at 50% repolarization (APD50) decreased with temperature increases, and APD50 was more thermally sensitive than APA. EP and tissue changes were irreversible when HIFU-induced temperature increased above 52.3 ± 1.4°C and log10(CEM43) above 2.16 ± 0.51 (n = 5), but were reversible when temperature was below 50.1 ± 0.8°C and log10(CEM43) below –0.9 ± 0.3 (n = 9). EP and temperature/thermal dose changes were spatially correlated with HIFU-induced tissue necrosis surrounded by a transition zone.

Oxidation‐ and CaMKII‐Mediated Sarcoplasmic Reticulum Ca2+ Leak Triggers Atrial Fibrillation in Aging

Advanced age is a well-recognized predisposition to atrial fibrillation (AF). However, the cellular electrophysiological changes that underlie the heightened susceptibility to AF in aged individuals remain poorly understood. Sarcoplasmic reticulum (SR) Ca(2+) leak that results from posttranslational modification of type 2 ryanodine receptor channels (RyR2) has been implicated in arrhythmogenesis. We hypothesize that aging alters atrial myocytes Ca(2+) homeostasis and RyR2 function, which create a substrate for AF initiation. We examined the susceptibility to AF in aged (24 months) and young adult (4-5 months) mice using an intraesophageal atrial electrical stimulation protocol. Aged mice showed significant higher AF induction rate (43.3%, n = 30) than young adults (8.8%, n = 34, P < 0.01). In accordance with these in vivo findings, significantly increased diastolic SR Ca(2+) leak and arrhythmogenic Ca(2+) activities with reduced SR Ca(2+) content were observed in aged atrial myocytes. Western blot showed RyR2 oxidation and phosphorylation at Ser2814 (Ca(2+) /calmodulin-dependent protein kinase II [CaMKII] site), but not phosphorylation at Ser2808 (protein kinase A [PKA] and CaMKII site), were increased in aged atrial myocytes. The selective CaMKII inhibitor (KN-93), as well as the antioxidant reagent (DTT) reversed the diastolic Ca(2+) leak and the frequency of spontaneous Ca(2+) transients in aged atrial myocytes, whereas PKA inhibition with H-89 was ineffective. Aging increases both the oxidation and CaMKII-phosphorylation of RyR2, which result in diastolic SR Ca(2+) leak and facilitate AF initiation. These results contribute to the electrophysiological remodeling of aged atria and suggest a therapeutic strategy for AF treatment in aging.

Electrophysiologic and molecular characteristics of cardiomyocytes after heavy ion irradiation in the frame of the ESA IBER-10 program

Since essentially no information is available on the effects of high linear energy transfer (LET) radiation on the heart, an assessment of possible late effects on the cardiovascular system is important with respect to the planning of manned long-term space missions [ 1, 2]. In order to examine if and to what extent heart muscle cells are affected by an exposure to heavy ions, primary avian cardiomyocytes were isolated, cultured in vitro and exposed at GSI (Darmstadt) to different ion species (carbon, titanium and nickel). Investigation of electrophysiologic radiation effects was performed on cells grown on a microelectrode array (MEA) allowing the monitoring of beat rate, spike shape, field action potential duration and signal conduction pathways across the electrode array. Data were analysed using the DrCell software [ 3]. In parallel, immunohistological stainings were conducted to examine cell cycle progression, DNA-damage repair and apoptosis.Our preliminary data indicate that primary cardiac cells possess a high robustness toward ionizing radiation as cultures receiving doses of up to 7 Gy still show unaltered electrophysiologic activity. This is exemplarily shown in Fig. 1, where the beat rate of cells irradiated with 2 and 7 Gy carbon ions has been plotted. Figure 1 also illustrates that within each treatment group, inter-sample variations are quite large and thus do not allow detection of subtle changes in cellular electrophysiology, probably due to the embryonic character of the cells.Interestingly, in terms of the formation of double-strand breaks (DSBs) and cell cycle progression delay, the cultures showed a dose-dependent reduction in proliferation and DSB accumulation.Taken together, our electrophysiologic studies indicate that the large variations within the same treatment group of avian embryonic cardiomyocytes obscure the detection of radiation effects. To account for this problem, we will use in further studies human cardiomyocytes differentiated from iPS cells, as these cells resemble more closely the condition of an adult human heart and do not undergo developmental changes as embryonic cell cultures do. Fig. 1.Normalized beat rates measured in samples before and after exposure to carbon ions. (spread-out Bragg peak, LET: 75 keV/µm). Average beat rates range from 22 to 53 beats/min.

Age‐related changes in cellular electrophysiology and calcium handling for atrial fibrillation

This study was to investigate whether or not the dysfunction of atrial repolarization and abnormality of the intracellular Ca2+ handling protein was augmented with ageing. Four groups of dogs were studied, adult and aged dogs in sinus rhythm (SR) and atrial fibrillation (AF) induced by rapid atrial pacing. We used whole cell patch clamp recording techniques to measure L-type Ca2+ current in cardiomyocytes dispersed from the left atria. Expressions of the Ca2+ handling protein were measured by real-time quantitative reverse transcription-polymerase chain reaction and Western blot methods. Cardiomyocytes from old atria showed longer action potential (AP) duration to 90% repolarization, lower AP plateau potential and peak L-type Ca2+ current densities at both age groups in SR. AF led to a higher maximum diastolic potential, an increase of amplitude of phase 0, decreases of AP duration to 90% repolarization, plateau potential and peak L-type Ca2+ current densities. Compared to the adult group, mRNA and protein expressions of the L-type calcium channel a1c were decreased, whereas expressions of calcium adenosine triphosphatase were increased in the aged group. Compared to SR group, expressions of Ca2+ handling protein except for phospholamban were significantly decreased in both age groups with AF. We conclude that these ageing-induced electrophysiological and molecular changes showed that general pathophysiological adaptations might provide a substrate conducive to AF.

Carbon monoxide effects on human ventricle action potential assessed by mathematical simulations

Carbon monoxide (CO) that is produced in a number of different mammalian tissues is now known to have significant effects on the cardiovascular system. These include: (i) vasodilation, (ii) changes in heart rate and strength of contractions, and (iii) modulation of autonomic nervous system input to both the primary pacemaker and the working myocardium. Excessive CO in the environment is toxic and can initiate or mediate life threatening cardiac rhythm disturbances. Recent reports link these ventricular arrhythmias to an increase in the slowly inactivating, or “late” component of the Na+ current in the mammalian heart. The main goal of this paper is to explore the basis of this pro-arrhythmic capability of CO by incorporating changes in CO-induced ion channel activity with intracellular signaling pathways in the mammalian heart. To do this, a quite well-documented mathematical model of the action potential and intracellular calcium transient in the human ventricular myocyte has been employed. In silico iterations based on this model provide a useful first step in illustrating the cellular electrophysiological consequences of CO that have been reported from mammalian heart experiments. Specifically, when the Grandi et al. model of the human ventricular action potential is utilized, and after the Na+ and Ca2+ currents in a single myocyte are modified based on the experimental literature, early after-depolarization (EAD) rhythm disturbances appear, and important elements of the underlying causes of these EADs are revealed/illustrated. Our modified mathematical model of the human ventricular action potential also provides a convenient digital platform for designing future experimental work and relating these changes in cellular cardiac electrophysiology to emerging clinical and epidemiological data on CO toxicity.

Characterization of atrial histopathological and electrophysiological changes in a mouse model of aging

The detailed mechanisms of age-related atrial structural and electrophysiological changes remain elusive. Small animal models have recently been used for the investigation of atrial tachyarrhythmia. In this study, we investigated the hypothesis that atrial structural and electrical characterization with aging provides a substrate for atrial fibrillation using a mouse model of aging. Male Kunming mice aged2 (young), 12(middle-aged) and 24 months (aged) were used in this study. A surface electrocardiogram and sinus node recovery time (SNRT) were recorded at baseline. Atrial fibrillation (AF) inducibility and duration were measured by a transesophageal electrode catheter. Collagen content was assessed by the collagen volume fraction. Whole cell configuration using the patch clamp technique was performed for the transient outward potassium (Ito) and ultra-rapid delayed rectifier potassium (Ikur) currents. P-wave duration, SNRT and rate-corrected SNRT were longer in the aged group than in the remaining 2groups, paralleled by inducibility significantly being increased in the aged group. The right atrium had significantly higher levels of fibrosis than the left atrium in all the groups (P<0.05), whereas the extent of fibrosis in the left atrium had a higher positive correlation with age relative to the right atrium (P<0.05). Moreover, in old age, the dispersion of left relative to right atrium repolarization and augmented Ito currents contributed to vulnerability to AF. Nevertheless, Ikur currents in the atrial myocytes showed no age-related changes. The present study demonstrates that in addition to the structural alterations, aging can also cause integrative and cellular electrophysiological changes in a mouse model of aging, facilitating AF initiation and maintenance.

Myocardial Notch Signaling Reprograms Cardiomyoctes to a Conduction‐Like Phenotype

Notch signaling has previously been shown to play an essential role in regulating cell fate decisions in many systems. We hypothesized that Notch may be involved in directing the progressive lineage restriction of cardiomyocytes into specialized conduction cells. In murine hearts where Notch signaling is activated within the myocardium from early development onwards, Notch promotes a conduction‐like phenotype as evidenced by ectopic expression of conduction system‐specific genes, as well as cell autonomous changes in cellular electrophysiology. Activation of Notch in newborn ventricular cardiomyocytes from Contactin2‐EGFP transgenic mice, which specifically labels conduction cells, demonstrates Notch‐induced changes in “chamber” ventricular cardiomyocytes consistent with a phenotypic switch to Purkinje‐like cells, including transcriptional changes, action potential prolongation and hyperpolarization of the resting membrane potential. Taken together, this suggests that Notch can instruct the differentiation of cardiac progenitors into specialized conduction‐like cells and that this plasticity remains in late‐stage cardiomyocytes, which has potential implications for engineering of specialized cardiovascular tissues.Grant Funding Source: Career Award for Medical Scientists Burroughs Wellcome Fund and K08 HL107449‐01 to SR, U01 HL100405‐1 to JAE

Abstract 9743: Profibrillatory Atrial Autonomic and Cellular Electrophysiological Remodelling with Endurance Training

Background: Endurance exercise (Ex) increases AF risk, but underlying mechanisms are unknown. Here, we studied Ex induced autonomic and cellular electrophysiological changes controlling AF risk. Methods: Ex rats (daily treadmill training X 16 wks) were compared to matched sedentary (Sed) controls. Parasympathetic and sympathetic tone were assessed from heart rate (HR) changes caused by atropine and propranolol respectively. Baroreflex sensitivity was studied by HR responses to phenylephrine-induced BP changes. Ion currents (I KACh , I to , I CaL ) were measured in atrial myocytes by patch clamp. Atrial refractory period (ARP) was measured in vivo with transvenous electrode catheters. mRNA expression was quantified by qPCR. Results: Atrial myocytes were hypertrophied in Ex (129±8.7 pF vs 73±5 pF Sed, p&lt;0.001). Parasympathetic tone was significantly enhanced in Ex rats by 83%, with no changes in sympathetic tone. Reflex vagal sensitivity was enhanced (60% increase in HR-reduction vs BP-increase slope with phenylephrine boluses). Post-synaptic responses were also increased, as shown by significant increases in I KACh induced by 100 μ M acetylcholine, ACh (Fig. A). mRNA expression of beta-adrenergic (β1, β2) and muscarinic (M2, M3) receptors and associated G proteins did not differ between groups. Regulatory G signaling proteins (RGS, Fig. B), which decrease ACh responses, were downregulated in Ex. Balanced downregulation of voltage gated K + and Ca 2+ current subunits (Fig. C) and currents (Fig. D) left ARP unchanged in the absence of ACh. AF inducibility was enhanced in Ex rats, an effect that was abolished by atropine. Conclusions: Ex promotes AF by enhancing vagal tone through pre (baroreflex) and post (RGS, I KACh ) synaptic mechanisms. Ion channel expression changes in a balanced way, leaving repolarization unaffected in the absence of ACh.

The Autonomic Nervous System in Cardiac Electrophysiology

The autonomic nervous system plays an integral role in the modulation of normal cardiac electrophysiology. This is achieved via a complex network of pre- and postganglionic sympathetic and parasympathetic fibers that synapse on extrinsic and intrinsic cardiac ganglia and ultimately directly innervate cardiac myocytes. Alterations in autonomic tone may induce changes in local cellular electrophysiology that may manifest clinically in a number of ways, ranging from changes in heart rate to changes in heart rhythm. These relationships between autonomic tone and the evolution of cardiac dysrhythmias are areas of evolving research, with increasing evidence for a key role for autonomic ganglia in the pathogenesis of atrial fibrillation and sympathetic nerves in the predilection toward ventricular tachycardia in areas of myocardial scar. In this review, we highlight what is known about the anatomy and physiology of the cardiac autonomic nervous system, the evidence supporting the relationship of autonomic tone to clinically significant arrhythmias, and a variety of mechanisms (eg, direct ion channel effects) and diagnostic tools that exist to help define this relationship. Further emphasized are potential future avenues of research needed to elucidate the relationship between changes in normal autonomic tone and the pathogenesis of cardiac arrhythmias.

Chronic Single Nephrectomy Prolongs Action Potential Duration by Inhibiting Transient Outward Currents in Left Ventricular Myocytes

Chronic kidney disease (CKD) may contribute to left ventricular hypertrophy, fibrosis, diastolic dysfunction, and impaired coronary flow reserve, which ultimately lead to an increased risk for adverse cardiovascular events. We hypothesized that cardiovascular remodeling is a continuous process starting from the early stage of CKD and sought to explore the cellular electrophysiologic changes in left ventricular myocytes after single nephrectomy (SNx) in rats.