Longer Action Potential Duration Research Articles

Sex differences in electrophysiology of the intact atria.

Atrial fibrillation (AF) is the most common cardiac arrhythmia and is associated with electrophysiological and calcium (Ca) handling remodeling. Though AF affects both sexes, there is disparity in the onset, presentation, treatment efficacy and outcomes of the disease: males develop AF earlier, but females experience abnormal symptoms, treatment side effects, and increased risk of mortality. Treatment success likely depends on the underlying arrhythmogenic substrate. However, sex-related differences in AF pathophysiology are currently unclear, complicated by sparse knowledge about how even the healthy atria differs between males and females. As such, this study aims to investigate sex differences in electrophysiology, Ca handling and arrhythmia susceptibility in the intact atria. Hearts were extracted from young (3-4 month) rabbits and optically mapped using voltage (RH237) and Ca-sensitive (Rhod-2 AM) indicators. Signals from the atria were isolated with minimal ventricular interference by covering all tissue beneath the coronary sulcus. Electrical stimulation was performed via a pacing electrode on the right atrium. Rapid pacing and premature stimuli protocols were performed to examine baseline electrophysiology and arrhythmia susceptibility that was further tested with pharmacological intervention with carbachol (0.5 µM). Females had longer action potential duration at 25% repolarization (APD25) but no difference in APD50, APD80 or Ca transient duration (CaTD50 or CaTD80) vs. males. There was similarly no difference in the pacing thresholds for emergence of alternans between sexes; however, females had steeper APD and CaTD restitution. Despite this, arrhythmia vulnerability was greater in males, with sustained reentry observed following application of carbachol and rapid pacing in 66% of animals. Our data suggests that males are more susceptible to reentrant arrhythmias due to innate differences in electrophysiology that may include autonomic modulation. Future work will focus on potential sex differences in the age-associated remodeling that predisposes to AF.

Calcium handling maturation and adaptation to increased substrate stiffness in human iPSC-derived cardiomyocytes: The impact of full-length dystrophin deficiency.

Cardiomyocytes differentiated from human induced Pluripotent Stem Cells (hiPSC- CMs) are a unique source for modelling inherited cardiomyopathies. In particular, the possibility of observing maturation processes in a simple culture dish opens novel perspectives in the study of early-disease defects caused by genetic mutations before the onset of clinical manifestations. For instance, calcium handling abnormalities are considered as a leading cause of cardiomyocyte dysfunction in several genetic-based dilated cardiomyopathies, including rare types such as Duchenne Muscular Dystrophy (DMD)-associated cardiomyopathy. To better define the maturation of calcium handling we simultaneously measured action potential and calcium transients (Ca-Ts) using fluorescent indicators at specific time points. We combined micropatterned substrates with long-term cultures to improve maturation of hiPSC-CMs (60, 75 or 90 days post-differentiation). Control-(hiPSC)-CMs displayed increased maturation over time (90 vs 60 days), with longer action potential duration (APD), increased Ca-T amplitude, faster Ca-T rise (time to peak) and Ca-T decay (RT50). The progressively increased contribution of the SR to Ca release (estimated by post-rest potentiation or Caffeine-induced Ca-Ts) appeared as the main determinant of the progressive rise of Ca-T amplitude during maturation. As an example of severe cardiomyopathy with early onset, we compared hiPSC-CMs generated from a DMD patient (DMD-ΔExon50) and a CRISPR-Cas9 genome edited cell line isogenic to the healthy control with deletion of a G base at position 263 of the DMD gene (c.263delG-CMs). In DMD-hiPSC-CMs, changes of Ca-Ts during maturation were less pronounced: indeed, DMD cells at 90 days showed reduced Ca-T amplitude and faster Ca-T rise and RT50, as compared with control hiPSC-CMs. Caffeine-Ca-T was reduced in amplitude and had a slower time course, suggesting lower SR calcium content and NCX function in DMD vs control cells. Nonetheless, the inotropic and lusitropic responses to forskolin were preserved. CRISPR-induced c.263delG-CM line recapitulated the same developmental calcium handling alterations observed in DMD-CMs. We then tested the effects of micropatterned substrates with higher stiffness. In control hiPSC-CMs, higher stiffness leads to higher amplitude of Ca-T with faster decay kinetics. In hiPSC-CMs lacking full-length dystrophin, however, stiffer substrates did not modify Ca-Ts but only led to higher SR Ca content. These findings highlighted the inability of dystrophin-deficient cardiomyocytes to adjust their calcium homeostasis in response to increases of extracellular matrix stiffness, which suggests a mechanism occurring during the physiological and pathological development (i.e. fibrosis).

The interplay of sex-specific electrophysiology and repolarization heterogeneity governs ventricular arrhythmia sustainability in women

Abstract Background The role of sex-specific electrophysiology in the development of lethal ventricular arrhythmia is poorly understood. Since female ventricles have a longer action potential duration (APD) than male ventricles, we hypothesized that female hearts are more vulnerable to sustained ventricular arrhythmia in the presence of known arrhythmogenic substrates. Purpose To determine if prolonged APD in females leads to sustained ventricular arrhythmia in the presence of left ventricular (LV) hypertrophy, diffuse fibrosis, and repolarization heterogeneity. Methods Magnetic resonance imaging (MRI) data were used to develop computational biventricular models of two aortic stenosis patients (male, 54 years; female, 64 years) before (LV hypertrophy) and 3 months after (regressed LV hypertrophy) aortic valve replacement (AVR). These patients were chosen since they exhibit reduced LV mass, less fibrosis, and no ventricular arrhythmias after AVR. The models were assigned sex-specific cellular electrophysiology and an apicobasal repolarization gradient derived from human data. Simulations of apical pacing with a cycle length (CL) of 750 ms were performed while APD at 90% repolarization (APD90) and repolarization time (activation time + APD90) were calculated. Reentrant arrhythmia was induced by rapidly pacing the LV with a 2 mm wide apicobasal line electrode at CL≤265 ms. The simulations were repeated in the presence of diffuse myocardial fibrosis derived from the MRI patient data, and repeated again with an apicobasal repolarization gradient reduced by ≈35%. Ventricular mass was calculated as the product of model volume and myocardial density. Results In hypertrophic ventricles with default apicobasal repolarization gradient, rapid pacing led to reentry that lasted longer in the female (>25 s) than in the male (1.0 s) ventricles. The duration of reentry increased in the presence of fibrosis in the male ventricles (male: 6.6 s; female >25 s) and decreased in the female ventricles when the apicobasal repolarization gradient was reduced (Figure). Sustained reentry (>25 s) was not observed in the ventricles with regressed hypertrophy, regardless of the presence of fibrosis or repolarization gradient. Average APD90 was longer in the female ventricles both before (male: 267±35 ms; female: 318±34 ms) and after (male: 272±35 ms; female: 328±38 ms) AVR. The female heart had lower myocardial mass than the male heart both before (280 vs. 406 g) and after AVR (227 vs. 310 g). Conclusion Longer APD and steeper apicobasal repolarization gradients in hypertrophic female ventricles resulted in sustained arrhythmia, despite their lower mass when compared to males. The absence of sustained reentry in the post-AVR models indicates a potential electrophysiological benefit of AVR and encourages future clinical studies to quantify sex-specific differences in spatial repolarization heterogeneity. Funding Acknowledgement Type of funding sources: Public grant(s) – EU funding. Main funding source(s): European Union's Horizon 2020 research and innovation program under the ERA-NET co-fund action No. 680969 with ANR (ERA-CVD SICVALVES) and Agence Nationale de la Recherche

Disrupted CaV1.2 selectivity causes overlapping long QT and Brugada syndrome phenotypes in the CACNA1C-E1115K iPS cell model

A missense mutation in the α1c subunit of voltage-gated L-type Ca2+ channel-coding CACNA1C-E1115K, located in the Ca2+ selectivity site, causes a variety of arrhythmogenic phenotypes. We aimed to investigate the electrophysiological features and pathophysiological mechanisms of CACNA1C-E1115K in patient-specific induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CMs). We generated iPSCs from a patient carrying heterozygous CACNA1C-E1115K with overlapping phenotypes of long QT syndrome, Brugada syndrome, and mild cardiac dysfunction. Electrophysiological properties were investigated using iPSC-CMs. We used iPSCs from a healthy individual and an isogenic iPSC line corrected using CRISPR-Cas9-mediated gene editing as controls. A mathematical E1115K-CM model was developed using a human ventricular cell model. Patch-clamp analysis revealed that E1115K-iPSC-CMs exhibited reduced peak Ca2+ current density and impaired Ca2+ selectivity with an increased permeability to monovalent cations. Consequently, E1115K-iPSC-CMs showed decreased action potential plateau amplitude, longer action potential duration (APD), and a higher frequency of early afterdepolarization compared with controls. In optical recordings examining the antiarrhythmic drug effect, late Na+ channel current (INaL) inhibitors (mexiletine and GS-458967) shortened APDs specifically in E1115K-iPSC-CMs. The AP-clamp using a voltage command obtained from E1115K-iPSC-CMs with lower action potential plateau amplitude and longer APD confirmed the upregulation of INaL. An in silico study recapitulated the invitro electrophysiological properties. Our iPSC-based analysis in CACNA1C-E1115K with disrupted CaV1.2 selectivity demonstrated that the aberrant currents through the mutant channels carried by monovalent cations resulted in specific action potential changes, which increased endogenous INaL, thereby synergistically contributing to the arrhythmogenic phenotype.

Chronic Volume Overload Caused by Abdominal Aorto-Venocaval Shunt Provides Arrhythmogenic Substrates in the Rat Atrium.

Atrial enlargement is thought to provide arrhythmogenic substrates, leading to the induction of atrial fibrillation (AF). In this study, we investigated the anatomical, molecular biological, and electrophysiological characteristics of remodeled atria in an animal model with chronic volume overload. We used rats that underwent abdominal aorto-venocaval shunt (AVS) surgery. In the in vivo studies, marked changes in electrocardiogram parameters, such as the P-wave duration, PR interval, and QRS width, as well as prolongation of the atrial effective refractory period were observed 12 weeks after the creation of AVS (AVS-12W), which were undetected at 8 weeks postoperative (AVS-8W) despite obvious atrial and ventricular enlargement. Moreover, the duration of AF induced by burst pacing in the AVS-12W rats was significantly longer than that in the Sham and AVS-8W rats. In the isolated atria, a longer action potential duration at 90% repolarization was detected in the AVS-12W rats compared with that in the Sham group. The mRNA levels of the Kv and Kir channels in the right atrium were mostly upregulated in the AVS-8W rats but were downregulated in the AVS-12W rats. These results show that chronic volume overload caused by abdominal AVS provides arrhythmogenic substrates in the rat atrium. The difference in gene expression in the right atrium between the AVS-8W and AVS-12W rats may partly explain the acquisition of arrhythmogenicity.

Fibroblast Growth Factor 1 Reduces Pulmonary Vein and Atrium Arrhythmogenesis via Modification of Oxidative Stress and Sodium/Calcium Homeostasis.

RationaleAtrial fibrillation is a critical health burden. Targeting calcium (Ca2+) dysregulation and oxidative stress are potential upstream therapeutic strategies. Fibroblast growth factor (FGF) 1 can modulate Ca2+ homeostasis and has antioxidant activity. The aim of this study was to investigate whether FGF1 has anti-arrhythmic potential through modulating Ca2+ homeostasis and antioxidant activity of pulmonary vein (PV) and left atrium (LA) myocytes.MethodsPatch clamp, western blotting, confocal microscopy, cellular and mitochondrial oxidative stress studies were performed in isolated rabbit PV and LA myocytes treated with or without FGF1 (1 and 10 ng/mL). Conventional microelectrodes were used to record electrical activity in isolated rabbit PV and LA tissue preparations with and without FGF1 (3 μg/kg, i.v.).ResultsFGF1-treated rabbits had a slower heart rate than that observed in controls. PV and LA tissues in FGF1-treated rabbits had slower beating rates and longer action potential duration than those observed in controls. Isoproterenol (1 μM)-treated PV and LA tissues in the FGF1-treated rabbits showed less changes in the increased beating rate and a lower incidence of tachypacing (20 Hz)-induced burst firing than those observed in controls. FGF1 (10 ng/mL)-treated PV and LA myocytes had less oxidative stress and Ca2+ transient than those observed in controls. Compared to controls, FGF1 (10 ng/mL) decreased INa−L in PV myocytes and lowered Ito, IKr−tail in LA myocytes. Protein kinase C (PKC)ε inhibition abolished the effects of FGF1 on the ionic currents of LA and PV myocytes.ConclusionFGF1 changes PV and LA electrophysiological characteristics possibly via modulating oxidative stress, Na+/Ca2+ homeostasis, and the PKCε pathway.

Age-related reductions in the excitability of phasic dorsal root ganglion neurons innervating the urinary bladder in female rats

Previous studies have revealed an impairment in bladder sensory transduction in aged animals. To examine the contributions of electrical property changes of bladder primary afferents to this impairment, we compared the electrical properties of dorsal root ganglion (DRG) neurons innervating the bladder among young (3 months), middle-aged (12 months), and old (24 months) female rats. The DRG neurons were labeled using axonal tracing techniques. Whole-cell current-clamp recordings of small and medium-sized neurons were performed to assess their passive and active properties. Two patterns of firing were identified based on responses to super-threshold stimuli (1.5, 2.0, 2.5, and 3.0 × rheobase): tonic neurons fired more action potentials (APs), whereas phasic neurons fired only one AP at the onset of stimulus. Tonic neurons were smaller and had a slower rate of AP rise, longer AP duration, more depolarized voltage threshold, and greater rheobase than phasic neurons. In phasic neurons, there was an age-associated increase in voltage threshold and an increase of rheobase (P < 0.05), suggesting an age-related decrease in excitability. In addition, both middle-aged and old rats had longer AP durations and slower rates of AP rise than young rats (P < 0.05). In tonic neurons, old rats had a greater AP overshoot and greater rate of AP rise, but no age-associated changes were identified in any other electrical properties. Our results suggest that the electrical properties of tonic and phasic bladder afferents are differentially altered with aging. A decrease in excitability may contribute to age-related reductions in bladder sensory function.

Study of the Characteristics of the Pulmonary Vein and Superior Vena Cava of Rabbits

Background: This study aims to (1) investigate the characteristics of the action potential and triggering activity of cardiomyocytes in the pulmonary vein (PV) and superior vena cava (SVC) of rabbits and (2) study the features of cation currents in cardiomyocytes in rabbit PV and SVC-inward rectifier potassium current (IK1), transient outward potassium current (Ito), and non-selective cation currents (INSCC). Methods: The standard glass microelectrode and whole-cell patch-clamp techniques were used to record the action potential and various currents in the above cells. Results: (1) Cardiomyocytes in either PV or SVC had longer action potential durations than in the adjacent atrium, and spontaneous early after depolarization (EAD) could occur in both PV and SVC under normal physiological conditions. (2) The action potential in PV cardiomyocytes had a relative refractory period but did not have an absolute refractory period, and this characteristic enabled a premature beat that triggered a second plateau response, which led to EAD. (3) INSCC was found for the first time in the PV, SVC, and atria. (4) The current intensity of IK1, Ito, and INSCC was significantly lower in the PV and SVC than in the left and right atria, and the difference in the current intensity in INSCC could influence the action potential. Conclusions: PV and SVC can both initiate and maintain AF, but PV is the primary ectopic foci in initiating AF. The present study found that the second plateau response was easily induced in cardiomyocytes in PA shortly after depolarization. This was a specific characteristic of the action potential of PV. In addition, we preliminarily analyzed the differences in the main outward currents and noted a voltage-dependent INSCC in both PV and SVC rabbits’ cardiomyocytes. Furthermore, the current intensities of IK1, Ito, and INSCC were significantly lower in the PV and SVC than in the left and right atria, and the difference in the current intensity of INSCC influenced the action potential. The different permeability of INSCC for cations at different phases may play a role in inducing EAD.

Abstract 209: iPSC Derived Cardiomyocytes Reproduce Divergent Phenotypes Caused by a LQTS Type-1 Likely Pathogenic Mutation

The purpose of this work was to investigate if iPSC derived cardiomyocytes would reproduce the divergent phenotypes observed in a proband and mother that carried the same likely-pathogenic variant in KCNQ1 gene. A 15 years old proband had syncope and cardiac arrest while swimming being diagnosed with LQTS based on a 12 lead ECG. The mother’s ECG showed no abnormalities. Genomic DNA from blood of daughter and mother was extracted using standard procedures. All coding exons and flanking intronic sequences of KCNQ1 (NM_000218) genes were amplified by polymerase chain reaction. Direct sequencing was performed on a 3500XL Genetic Analyzer. Data was analyzed with Geneious software for identification of mutations. For iPSC generation CD71+ CD36+ erythroblasts were reprogrammed using CytoTune™-iPS 2.0 Sendai Reprogramming Kit on irradiated mouse embryonic fibroblast feeder layers. Differentiation into cardiomyocytes was accomplished using the protocol described by Lian et al. (2012). Electrophysiological recordings were obtained from cardiomyocytes after 30–45 days of differentiation. Cells were transferred to acrylic plates pretreated with Matrigel and kept in culture for 3 days before microelectrode action potential (AP) were registered. The ECG recordings showed a corrected QT interval (QTc) of 516 ms for the daughter and 436 ms for the mother. Sequencing showed that both daughter and mother had a c.1763C&gt;T mutation resulting in p.Ile588Thr substitution. iPSC generated from daughter, mother and an unrelated control displayed normal karyotype and expressed pluripotent genes, besides spontaneously differentiating into cells of the three germ layers. Differentiation into cardiomyocytes was ascertained by immunofluorescence and flow cytometry for troponin T. In all differentiations more than 70% of cells were cardiomyocytes. Action potentials recordings showed that ventricular cardiomyocytes from the daughter exhibited significantly longer action potential durations at 90% repolarization than the mother’s or the unrelated control’s cardiomyocytes. Cardiomyocytes derived from iPS cells reproduced the clinical phenotypes exhibited by daughter and mother.

Primary cardiac manifestation of autosomal dominant polycystic kidney disease revealed by patient induced pluripotent stem cell-derived cardiomyocytes.

BackgroundMutations in PKD1 or PKD2 gene lead to autosomal dominant polycystic kidney disease (ADPKD). The mechanism of ADPKD progression and its link to increased cardiovascular mortality is still elusive.MethodsWe differentiated ADPKD patient induced pluripotent stem cells (iPSCs) to cardiomyocytes (CMs). The electrophysiological properties at the cellular level were analyzed by calcium imaging and whole cell patch clamping.FindingsThe ADPKD patient iPSC-CMs had decreased sarcoplasmic reticulum calcium content compared with Control-CMs. Spontaneous action potential of the PKD2 mutation line-derived CMs demonstrated slower beating rate and longer action potential duration. The PKD1 mutation line-derived CMs showed a comparable dose-dependent shortening of phase II repolarization with the Control-CMs, but a significant increase in beating frequency in response to L-type calcium channel blocker. The PKD1-mutant iPSC-CMs also showed a relatively unstable baseline as a greater percentage of cells exhibited delayed afterdepolarizations (DADs). Both the ADPKD patient iPSC-CMs showed more β-adrenergic agonist-elicited DADs compared with Control-CMs.InterpretationCharacterization of ADPKD patient iPSC-CMs provides new insights into the increased clinical risk of arrhythmias, and the results enable disease modeling and drug screening for cardiac manifestations of ADPKD.FundMinistry of Science and Technology, National Health Research Institutes, Academia Sinica Program for Technology Supporting Platform Axis Scheme, Thematic Research Program and Summit Research Program, and Kaohsiung Medical University Hospital, Taiwan.

Endotoxin modulates the electrophysiological characteristics of human embryonic stem cell‐differentiated cardiomyocytes

Gram‐negative bacteria‐induced infections result in fever, arrhythmia, and even death. Lipopolysaccharide (LPS), a constituent of bacteria, leads to an inflammatory response under sepsis and increase arrhythmogenesis. This study analyzed the effects on human embryonic stem cell‐differentiated cardiomyocytes (HIPSC‐CMs) exposed to LPS. A whole cell patch clamp was used to record the action potential (AP) and ionic currents with or without different doses of LPS in HIPSC‐CMs. Compared with the control, a different dose (0.04, 0.2, 1, and 5 µg/ml) of LPS‐treated HIPSC‐CMs resulted in a longer AP duration. The IC50 of sodium channel current was 1.254 µg/ml, L‐type calcium channel current was 5 µg/ml, and Ik channel currents were 1.254 µg/ml. LPS‐treated HIPSC‐CMs showed a lower sodium channel current, L‐type calcium channel current, and Ik channel currents. Furthermore, the expressions of Nav1.5 were decreased, and L‐Ca, Kv11.1, and Kv7.1 were increased in LPS‐treated HIPSC‐CMs. LPS‐induced arrhythmogenesis was related to the electrophysiological characteristics of sodium channel current, L‐type calcium channel current, and Ik channel currents. These results suggest a potential mechanism of cardiomyocyte injury in endotoxemia.

Transverse cardiac slicing and optical imaging for analysis of transmural gradients in membrane potential and Ca2+ transients in murine heart.

Key points A robust cardiac slicing approach was developed for optical mapping of transmural gradients in transmembrane potential (V m) and intracellular Ca2+ transient (CaT) of murine heart.Significant transmural gradients in V m and CaT were observed in the left ventricle.Frequency‐dependent action potentials and CaT alternans were observed in all ventricular regions with rapid pacing, with significantly greater incidence in the endocardium than epicardium.The observations demonstrate the feasibility of our new approach to cardiac slicing for systematic analysis of intrinsic transmural and regional gradients in V m and CaT. Transmural and regional gradients in membrane potential and Ca2+ transient in the murine heart are largely unexplored. Here, we developed and validated a robust approach which combines transverse ultra‐thin cardiac slices and high resolution optical mapping to enable systematic analysis of transmural and regional gradients in transmembrane potential (V m) and intracellular Ca2+ transient (CaT) across the entire murine ventricles. The voltage dye RH237 or Ca2+ dye Rhod‐2 AM were loaded through the coronary circulation using a Langendorff perfusion system. Short‐axis slices (300 μm thick) were prepared from the entire ventricles (from the apex to the base) by using a high‐precision vibratome. Action potentials (APs) and CaTs were recorded with optical mapping during steady‐state baseline and rapid pacing. Significant transmural gradients in V m and CaT were observed in the left ventricle, with longer AP duration (APD50 and APD75) and CaT duration (CaTD50 and CaTD75) in the endocardium compared with that in the epicardium. No significant regional gradients were observed along the apico‐basal axis of the left ventricle. Interventricular gradients were detected with significantly shorter APD50, APD75 and CaTD50 in the right ventricle compared with left ventricle and ventricular septum. During rapid pacing, AP and CaT alternans were observed in most ventricular regions, with significantly greater incidence in the endocardium in comparison with epicardium. In conclusion, these observations demonstrate the feasibility of our new approach to cardiac slicing for systematic analysis of intrinsic transmural and regional gradients in V m and CaT in murine ventricular tissue.

Roles of impaired intracellular calcium cycling in arrhythmogenicity of diabetic mouse model.

Diabetes mellitus is associated an increased risk of ventricular arrhythmias (VAs), but the underlying electrophysiological mechanisms are not fully explored. This study was aimed to test whether dynamic factors and Cai handling play roles in arrhythmogenesis of a diabetic animal model. We used 26 db/db type 2 diabetes mice and 28 control mice in this study. VA inducibility was evaluated in vivo under isoflurane general anesthesia. The intracellular Ca2+ (Cai ) and membrane voltage (Vm ) signals of the Langendorff-perfused mouse hearts were simultaneously recorded using the optical mapping technique. Action potential duration (APD), Cai dynamics conduction velocity (CV), and arrhythmogenic alternans were analyzed. Western blot was conducted to examine expressions of calcium handling and associated ion channels proteins. The diabetic db/db mice showed significantly increased VA inducibility and severity. Longer APD and Cai transient duration and slower Cai decay and CV in the db/db mice than these in the control ones were observed. Dynamic pacing showed increased incidence of spatially discordant alternans leading to more VA inducibility in the db/db mice. Western blot analyses revealed increased phosphorylated-Ca2+ /calmodulin-dependent protein kinase II protein expression and decreased ryanodine receptor protein expression, which probably underlay the molecular mechanisms of enhanced arrhythmogenicity in db/db mice. The type 2 diabetic mouse hearts show impaired repolarization, Cai handling homeostasis, and cardiac conduction reserve, leading to vulnerability of spatially discordant alternans development and induction of VA. Altered Cai -handling protein expressions probably underlie the molecular mechanisms of arrhythmogenicity in the type 2 diabetes animal model.

Human electronegative low-density lipoprotein modulates cardiac repolarization via LOX-1-mediated alteration of sarcolemmal ion channels

Dyslipidemia is associated with greater risk of ventricular tachyarrhythmias in patients with cardiovascular diseases. We aimed to examine whether the most electronegative subfraction of low-density lipoprotein (LDL), L5, is correlated with QTc prolongation in patients with coronary artery disease (CAD) and investigate the effects of human L5 on the electrophysiological properties of cardiomyocytes in relation to the lectin-like oxidized LDL receptor (LOX-1). L5 was isolated from the plasma of 40 patients with angiography documented CAD and 13 patients with no CAD to correlate the QTc interval respectively. The mean concentration of L5 was higher and correlated with QTc in patients with CAD compared to controls. To examine the direct effect of L5 on QTc, mice were intravenously injected with L5 or L1. L5-injected wild-type but not LOX-1−/− mice showed longer QTc compared to L1-injected animals in vivo with corresponding longer action potential duration (APD) in cardiomyocytes incubated with L5 in vitro. The APD prolongation was mediated by an increase of L-type calcium current and a decrease of transient outward potassium current. We show that L5 was positively correlated with QTc prolongation in patients with ischemic heart disease. L5 can modulate cardiac repolarization via LOX-1-mediated alteration sarcolemmal ionic currents.

Sex and regional differences in rabbit right ventricular L-type calcium current levels and mathematical modelling of arrhythmia vulnerability.

What is the central question of this study? Regional variations of ventricular L-type calcium current (ICa-L ) amplitude may underlie the increased arrhythmia risk in adult females. Current amplitude variations have been described for the left ventricle but not for the right ventricle. What is the main finding and its importance? Adult female rabbit right ventricular base myocytes exhibit elevated ICa-L compared with female apex or male myocytes. Oestrogen upregulated ICa-L in cultured female myocytes. Mathematical simulations modelling long QT syndrome type 2 demonstrated that elevated ICa-L prolonged action potentials and induced early after-depolarizations. Thus, ventricular arrhythmias in adult females may be associated with an oestrogen-induced upregulation of ICa-L . Previous studies have shown that adult rabbit left ventricular myocytes exhibit sex and regional differences in L-type calcium current (ICa-L ) levels that contribute to increased female susceptibility to arrhythmogenic early after-depolarizations (EADs). We used patch-clamp recordings from isolated adult male and female rabbit right ventricular myocytes to determine apex-base differences in ICa-L density and used mathematical modelling to examine the contribution of ICa-L to EAD formation. Current density measured at 0mV in female base myocytes was 67% higher than in male base myocytes and 55% higher than in female apex myocytes. No differences were observed between male and female apex myocytes, between male apex and base myocytes, or in the voltage dependences of ICa-L activation or inactivation. The role of oestrogen was investigated using cultured adult female right ventricular base myocytes. After 2days, 17β-estradiol (1nm) produced a 65% increase in ICa-L density compared with untreated control myocytes, suggesting an oestrogen-induced upregulation of ICa-L . Action potential simulations using a modified Luo-Rudy cardiomyocyte model showed that increased ICa-L density, at the level observed in female base myocytes, resulted in longer duration action potentials, and when combined with a 50% reduction of the rapidly inactivating delayed rectifier potassium current conductance to model long QT syndrome type 2, the action potential was accompanied by one or more EADs. Thus, we found higher levels of ICa-L in adult female right ventricle base myocytes and the upregulation of this current by oestrogen. Simulations of long QT syndrome type 2 showed that elevated ICa-L contributed to genesis of EADs.

Reduced expression of HCN channels in the sinoatrial node of streptozotocin-induced diabetic rats.

Diabetes mellitus (DM) is associated with an electrical remodeling of the heart, increasing the risk of arrhythmias. However, knowledge of electrical remodeling in the sinoatrial node (SAN) by DM is limited. We investigated the expression of HCN channel isoforms, HCN1-HCN4, in SAN from streptozotocin (STZ)-induced diabetic rats and the age-matched controls. We found that the STZ-induced diabetic rats have a lower intrinsic heart rate, a lengthened sinoatrial conduction time, and rate-corrected maximal sinoatrial node recovery time in vivo as well as a longer cycle length (CL) in vitro, as compared with the control. Optical mapping of the SAN demonstrated an inferior leading pacemaker site, reduced SAN conduction velocity and diastolic depolarization slope, and a longer action potential duration in the STZ-induced diabetic rats than in the control. The transcripts and proteins of HCN2 and HCN4 in diabetic SAN were reduced. Specific blockade of HCN channels by 3 μmol/L ivabradine significantly prolonged the CL of a Langendorff heart by 18% in the diabetic rats and 26% in the control. The reduced expression of HCN channel isoforms in the SAN of the STZ-induced diabetic rat may be an important contributor to the reduced SAN function in DM.

The Effect of Substrate Stiffness on Cardiomyocyte Action Potentials.

The stiffness of myocardial tissue changes significantly at birth and during neonatal development, concurrent with significant changes in contractile and electrical maturation of cardiomyocytes. Previous studies by our group have shown that cardiomyocytes generate maximum contractile force when cultured on a substrate with a stiffness approximating native cardiac tissue. However, effects of substrate stiffness on the electrophysiology and ion currents in cardiomyocytes have not been fully characterized. In this study, neonatal rat ventricular myocytes were cultured on the surface of flat polyacrylamide hydrogels with elastic moduli ranging from 1 to 25 kPa. Using whole-cell patch clamping, action potentials and L-type calcium currents were recorded. Cardiomyocytes cultured on hydrogels with a 9 kPa elastic modulus, similar to that of native myocardium, had the longest action potential duration. Additionally, the voltage at maximum calcium flux significantly decreased in cardiomyocytes on hydrogels with an elastic modulus higher than 9 kPa, and the mean inactivation voltage decreased with increasing stiffness. Interestingly, the expression of the L-type calcium channel subunit α gene and channel localization did not change with stiffness. Substrate stiffness significantly affects action potential length and calcium flux in cultured neonatal rat cardiomyocytes in a manner that may be unrelated to calcium channel expression. These results may explain functional differences in cardiomyocytes resulting from changes in the elastic modulus of the extracellular matrix, as observed during embryonic development, in ischemic regions of the heart after myocardial infarction, and during dilated cardiomyopathy.

Nighttime instabilities of neurophysiological, cardiovascular, and respiratory activity: integrative modeling and preliminary results

Unstable (cyclical alternating pattern, or CAP) sleep is associated with surges of sympathetic nervous system activity, increased blood pressure and vasoconstriction, heightened baroreflex sensitivity, and unstable heart rhythm and breathing. In susceptible persons, CAP sleep provokes clinically significant events, including hypertensive crises, sleep-disordered breathing, and cardiac arrhythmias. Here we explore the neurophysiology of CAP sleep and its impact on cardiovascular and respiratory functions. We show that: (i) an increase in neurophysiological recovery rate can explain the emergence of slow, self-sustained, hypersynchronized A1 CAP-sleep pattern and its transition to the faster A2-A3 CAP-sleep patterns; (ii) in a two-dimensional, continuous model of cardiac tissue with heterogeneous action potential duration (APD) distribution, heart rate accelerations during CAP sleep may encounter incompletely recovered electrical excitability in cell clusters with longer APD. If the interaction between short cycle length and incomplete, spatially heterogeneous repolarization persists over multiple cycles, irregularities and asymmetry of depolarization front may accumulate and ultimately lead to a conduction block, retrograde conduction, breakup of activation waves, reentrant activity, and arrhythmias; and (iii) these modeling results are consistent with the nighttime data obtained from patients with structural heart disease (N=13) that show clusters of atrial and ventricular premature beats occurring during the periods of unstable heart rhythm and respiration that accompany CAP sleep. In these patients, CAP sleep is also accompanied by delayed adaptation of QT intervals and T-wave alternans.

Manifestation of automaticity in the pulmonary-vein myocardium of rats with abdominal aorto-venocaval shunt

Effect of abdominal aorto-venocaval shunt (AVS) on the automaticity of the pulmonary-vein myocardium was studied in the rat. Spontaneous electrical activity was observed in one third of the isolated pulmonary-vein preparations from the AVS rats, but scarcely in those from sham-operated rats; the activity was induced by tertiapin and suppressed by carbachol or chelation of intracellular Ca2+. The evoked action potentials in AVS rats had less negative resting membrane potential and longer action potential duration than those in sham-operated rats. These results suggest that the automaticity of the rat pulmonary-vein myocardium is manifested under chronic volume overload.

Ablation of androgen receptor gene triggers right ventricular outflow tract ventricular tachycardia

BackgroundSex hormones and calcium (Ca2+) regulation play roles in the pathophysiology of ventricular tachycardia from right ventricular outflow tract (RVOT). The purpose of this study was to evaluate whether androgen receptor knockout (ARKO) can increase RVOT arrhythmogenesis through modulating RVOT electrophysiology and Ca2+ homeostasis. MethodsConventional microelectrodes were used to study the action potential (AP) in RVOT tissues prepared from wild type (WT) and ARKO mice (aged 6–10months) before and after caffeine (1mM), isoproterenol (1μM), adenosine (10μM) and flecainide (5μM) administration. The Fluo-3 fluorescence Ca2+ imaging with confocal microscopy and western blots were used to investigate intracellular Ca2+ (Ca2+i) transients, Ca2+ sparks, and the expressions of ionic channel proteins in ARKO and WT RVOT myocytes. ResultsWe found that ARKO RVOTs (n=13) had longer AP duration, faster burst firing (5.4±0.7 vs. 3.4±0.7Hz, P<0.05), and higher incidence of early afterdepolarizations (82% vs. 8%, P<0.001) than WT RVOTs (n=11). Adenosine and flecainide can suppress caffeine- or isoproterenol-induced spontaneous rates and burst firing in WT RVOTs, but not in ARKO RVOTs. ARKO RVOT myocytes had a higher frequency (7.7±2.8 vs. 1.3±0.4spark/mm/s, P<0.05) and incidence (89% vs. 47%, P<0.05) of Ca2+ sparks, and greater expressions of Cav1.2, NCX, phosphorylated RyR (s2814), phosphorylated phospholamban (Thr17), CAMKII and GRK2 than WT RVOT myocytes. However, ARKO and WT RVOT myocytes exhibit similar Ca2+i transients and SR Ca2+ content, and less expression of calsequestrin. ConclusionsARKO changes RVOT electrophysiology and Ca2+ homeostasis with increased ventricular arrhythmogenesis.