Action Potential Duration In Myocytes Research Articles

K+ currents in ventricular cardiomyocytes of p.N98S-calmodulin mutant mice.

Missense mutations in calmodulin (CaM)-encoding genes are associated with life-threatening ventricular arrhythmia syndromes. Here, we investigated a role of cardiac K+ channel dysregulation in arrhythmogenic long QT syndrome (LQTS) using a knock-in mouse model heterozygous for a recurrent mutation (p.N98S) in the Calm1 gene (Calm1N98S/+). Single-cell patch-clamp technique and whole-heart optical voltage mapping were used to assess action potentials and whole-cell currents. Ventricular action potential duration (APD) at baseline was similar between genotypes. The β-adrenergic agonist isoproterenol prolonged APD in myocytes and isolated perfused hearts from Calm1N98S/+, but not wild-type (Calm1+/+), mice. Current density-voltage relationships for the small-conductance calcium-activated K+ (SK) current and the inward rectifier K+ current did not significantly differ between Calm1+/+ and Calm1N98S/+ ventricular cardiomyocytes ± isoproterenol. Peak densities of other voltage-gated K+ currents were significantly larger in Calm1N98S/+ versus Calm1+/+ cells at voltages ≥40 mV both without and with isoproterenol. Isoproterenol reduced outward KATP currents more in Calm1N98S/+ versus Calm1+/+ myocytes. Dialysis of Calm1+/+ cardiomyocytes with exogenous wild-type or N98S-CaM protein (5 μmol/l) via the pipette respectively increased and eliminated SK currents. The specific SK channel inhibitor apamin did not significantly alter APD of Calm1+/+ or Calm1N98S/+ hearts ± isoproterenol. Thus, dysregulation of SK or voltage-gated K+ channels does not contribute to the β-adrenergic-induced LQTS of Calm1N98S/+ mice, possibly because cardiomyocyte content of endogenous N98S-CaM and/or its affinity for CaM binding domains may be too low to modulate channel properties. The larger KATP current inhibition by isoproterenol may delay Calm1N98S/+ myocyte repolarization at low intracellular [ATP].

Spexin Diminishes Atrial Fibrillation Vulnerability by Acting on Galanin Receptor 2.

G protein-coupled receptors play a critical role in atrial fibrillation (AF). Spexin is a novel ligand of galanin receptors (GALRs). In this study, we investigated the regulation of spexin and GALRs on AF and the underlying mechanisms. Global spexin knockout (SPX-KO) and cardiomyocyte-specific GALRs knockout (GALR-cKO) mice underwent burst pacing electrical stimulation. Optical mapping was used to determine atrial conduction velocity and action potential duration. Atrial myocyte action potential duration and inward rectifying K+ current (IK1) were recorded using whole-cell patch clamps. Isolated cardiomyocytes were stained with Fluo-3/AM dye, and intracellular Ca2+ handling was examined by CCD camera. A mouse model of AF was established by Ang-II (angiotensin II) infusion. Spexin plasma levels in patients with AF were lower than those in subjects without AF, and knockout of spexin increased AF susceptibility in mice. In the atrium of SPX-KO mice, potassium inwardly rectifying channel subfamily J member 2 (KCNJ2) and sarcolipin (SLN) were upregulated; meanwhile, IK1 current was increased and Ca2+ handling was impaired in isolated atrial myocytes of SPX-KO mice. GALR2-cKO mice, but not GALR1-cKO and GALR3-cKO mice, had a higher incidence of AF, which was associated with higher IK1 current and intracellular Ca2+ overload. The phosphorylation level of CREB (cyclic AMP responsive element binding protein 1) was upregulated in atrial tissues of SPX-KO and GALR2-cKO mice. Chromatin immunoprecipitation confirmed the recruitment of p-CREB to the proximal promoter regions of KCNJ2 and SLN. Finally, spexin treatment suppressed CREB signaling, decreased IK1 current and decreased intracellular Ca2+ overload, which thus reduced the inducibility of AF in Ang-II-infused mice. Spexin reduces atrial fibrillation susceptibility by inhibiting CREB phosphorylation and thus downregulating KCNJ2 and SLN transcription by GALR2 receptor. The spexin/GALR2/CREB signaling pathway represents a novel therapeutic avenue in the development of agents against atrial fibrillation.

NaV1.6 dysregulation within myocardial T-tubules by D96V calmodulin enhances proarrhythmic sodium and calcium mishandling.

Calmodulin (CaM) plays critical roles in cardiomyocytes, regulating Na+ (NaV) and L-type Ca2+ channels (LTCCs). LTCC dysregulation by mutant CaMs has been implicated in action potential duration (APD) prolongation and arrhythmogenic long QT (LQT) syndrome. Intriguingly, D96V-CaM prolongs APD more than other LQT-associated CaMs despite inducing comparable levels of LTCC dysfunction, suggesting dysregulation of other depolarizing channels. Here, we provide evidence implicating NaV dysregulation within transverse (T) tubules in D96V-CaM-associated arrhythmias. D96V-CaM induced a proarrhythmic late Na+ current (INa) by impairing inactivation of NaV1.6, but not the predominant cardiac NaV isoform NaV1.5. We investigated arrhythmia mechanisms using mice with cardiac-specific expression of D96V-CaM (cD96V). Super-resolution microscopy revealed close proximity of NaV1.6 and RyR2 within T-tubules. NaV1.6 density within these regions increased in cD96V relative to WT mice. Consistent with NaV1.6 dysregulation by D96V-CaM in these regions, we observed increased late NaV activity in T-tubules. The resulting late INa promoted aberrant Ca2+ release and prolonged APD in myocytes, leading to LQT and ventricular tachycardia in vivo. Cardiac-specific NaV1.6 KO protected cD96V mice from increased T-tubular late NaV activity and its arrhythmogenic consequences. In summary, we demonstrate that D96V-CaM promoted arrhythmias by dysregulating LTCCs and NaV1.6 within T-tubules and thereby facilitating aberrant Ca2+ release.

Data-driven, patient-specific simulations of long-term heart rate variability and cardiac alternans formation.

Individuals suffering from chronic heart failure (CHF) have a greater risk for death mediated by ventricular arrhythmias, which may be related to changes in heart rate. Heart rate constantly fluctuates from beat-to-beat on a timescale ranging from seconds to hours under physiological conditions, termed heart rate variability (HRV). Individuals with CHF have reduced HRV and faster heart rates, both of which influence the formation of pro-arrhythmic alternans, a beat-to-beat alternation in the action potential duration (APD) and intracellular calcium (Ca) levels. It is unclear how long-term changes in heart rate influence alternans and arrhythmia formation. We obtained sequences of RR intervals (time between successive R waves in an electrocardiogram) from individuals with normal sinus rhythm (NSR) and CHF from a Physionet database. These RR-sequences are used to define the pacing period for a discrete-time coupled map model of APD and intracellular Ca cycling for a single cardiac myocyte, modified to account for human heart rates and pathological Ca-remodeling in CHF. These modifications include fitting human ventricular myocyte APD restitution parameters, including a new term for Ca leak from the sarcoplasmic reticulum (SR), and scaling SR Ca-related parameters. Importantly, this minimal model enables simulation of long-term trends, on the order of 24 hours, in large populations. We find that the alternations in APD and Ca vary over time, such that there are periods with both small and large beat-to-beat differences in both populations. However, the CHF population tends to have a larger alternations magnitude, demonstrating that CHF-associated heart rates promotes more pro-arrhythmic phenomena. Future work will extend this methodology to determine what statistical properties govern the CHF RR-sequences, and further to test to what extent pro-arrhythmic alternans depends on CHF-related changes in heart rate or remodeling.

MicroRNA-365 regulates human cardiac action potential duration

Abnormalities of ventricular action potential cause malignant cardiac arrhythmias and sudden cardiac death. Here, we aim to identify microRNAs that regulate the human cardiac action potential and ask whether their manipulation allows for therapeutic modulation of action potential abnormalities. Quantitative analysis of the microRNA targetomes in human cardiac myocytes identifies miR-365 as a primary microRNA to regulate repolarizing ion channels. Action potential recordings in patient-specific induced pluripotent stem cell-derived cardiac myocytes show that elevation of miR-365 significantly prolongs action potential duration in myocytes derived from a Short-QT syndrome patient, whereas specific inhibition of miR-365 normalizes pathologically prolonged action potential in Long-QT syndrome myocytes. Transcriptome analyses in these cells at bulk and single-cell level corroborate the key cardiac repolarizing channels as direct targets of miR-365, together with functionally synergistic regulation of additional action potential-regulating genes by this microRNA. Whole-cell patch-clamp experiments confirm miR-365-dependent regulation of repolarizing ionic current Iks. Finally, refractory period measurements in human myocardial slices substantiate the regulatory effect of miR-365 on action potential in adult human myocardial tissue. Our results delineate miR-365 to regulate human cardiac action potential duration by targeting key factors of cardiac repolarization.

Are Interactions between Epicardial Adipose Tissue, Cardiac Fibroblasts and Cardiac Myocytes Instrumental in Atrial Fibrosis and Atrial Fibrillation?

Atrial fibrillation is very common among the elderly and/or obese. While myocardial fibrosis is associated with atrial fibrillation, the exact mechanisms within atrial myocytes and surrounding non-myocytes are not fully understood. This review considers the potential roles of myocardial fibroblasts and myofibroblasts in fibrosis and modulating myocyte electrophysiology through electrotonic interactions. Coupling with (myo)fibroblasts in vitro and in silico prolonged myocyte action potential duration and caused resting depolarization; an optogenetic study has verified in vivo that fibroblasts depolarized when coupled myocytes produced action potentials. This review also introduces another non-myocyte which may modulate both myocardial (myo)fibroblasts and myocytes: epicardial adipose tissue. Epicardial adipocytes are in intimate contact with myocytes and (myo)fibroblasts and may infiltrate the myocardium. Adipocytes secrete numerous adipokines which modulate (myo)fibroblast and myocyte physiology. These adipokines are protective in healthy hearts, preventing inflammation and fibrosis. However, adipokines secreted from adipocytes may switch to pro-inflammatory and pro-fibrotic, associated with reactive oxygen species generation. Pro-fibrotic adipokines stimulate myofibroblast differentiation, causing pronounced fibrosis in the epicardial adipose tissue and the myocardium. Adipose tissue also influences myocyte electrophysiology, via the adipokines and/or through electrotonic interactions. Deeper understanding of the interactions between myocytes and non-myocytes is important to understand and manage atrial fibrillation.

Decreased bioavailability of hydrogen sulfide links vascular endothelium and atrial remodeling in atrial fibrillation

Oxidative stress drives the pathogenesis of atrial fibrillation (AF), the most common arrhythmia. In the cardiovascular system, cystathionine γ-lyase (CSE) serves as the primary enzyme producing hydrogen sulfide (H2S), a mammalian gasotransmitter that reduces oxidative stress. Using a case control study design in patients with and without AF and a mouse model of CSE knockout (CSE-KO), we evaluated the role of H2S in the etiology of AF. Patients with AF (n = 51) had significantly reduced plasma acid labile sulfide levels compared to patients without AF (n = 65). In addition, patients with persistent AF (n = 25) showed lower plasma free sulfide levels compared to patients with paroxysmal AF (n = 26). Consistent with an important role for H2S in AF, CSE-KO mice had decreased atrial sulfide levels, increased atrial superoxide levels, and enhanced propensity for induced persistent AF compared to wild type (WT) mice. Rescuing H2S signaling in CSE-KO mice by Diallyl trisulfide (DATS) supplementation or reconstitution with endothelial cell specific CSE over-expression significantly reduced atrial superoxide, increased sulfide levels, and lowered AF inducibility. Lastly, low H2S levels in CSE KO mice was associated with atrial electrical remodeling including longer effective refractory periods, slower conduction velocity, increased myocyte calcium sparks, and increased myocyte action potential duration that were reversed by DATS supplementation or endothelial CSE overexpression. Our findings demonstrate an important role of CSE and H2S bioavailability in regulating electrical remodeling and susceptibility to AF.

Role of ATP Sensitive Potassium Channel in Exercise-Training Mediated Adaptations in Ventricular Repolarization

Regular exercise-training is known to reduce the incidences and severity of ischemic heart disease, but the mechanistic nature is unknown. We showed that regular exercise shortened the left ventricular action potential duration (APD) at high heart rate (HR), which would prevent Ca2+ overload and/or inadequate filling under conditions of metabolic stress. Here we test the hypothesis that exercise-training induced APD shortening at high HR is due to the up-regulation of ATP sensitive potassium channel (KATP). Female and male Sprague-Dawley rats were randomly assigned to voluntary wheel running or control groups. After 6-8 weeks training, cardiac myocytes were isolated from the apex and base regions of the left ventricle with collagenase–protease dispersion technique. APD was measured with glass micro-electrode, current-voltage relationship was recorded with discontinuous single electrode voltage clamp, and the expression level of KATP channel pore-forming subunit Kir6.2 and regulatory subunits SUR2A were determined by Western blots. At 1Hz, KATP activator pinacidil (100 µM) shortened APD in myocytes from both sexes and, the shortening was significantly greater in exercise-trained compared to sedentary groups. The KATP inhibitor glibenclamide (2 µM) prolonged the 10Hz APD more in exercise-trained than control rats. Regular exercise enhanced KATP outward current density in apex myocytes from both sexes; in base, trained females exhibited larger KATP inward current than controls. In both sexes, Kir6.2 expression was elevated by wheel-running in apex but not base region. Exercise-training increased SUR2A density in base region of both sexes, and eliminated its reginal difference in females. In conclusion, exercise-training induced APD shortening under energy demanding conditions (e.g. high HR) is caused at least in part by KATP up-regulation, an adaptation that reduces energy requirements for ion homeostasis and maintains a diastolic interval adequate for myocardial relaxation.

Enhanced basal late sodium current appears to underlie the age-related prolongation of action potential duration in guinea pig ventricular myocytes.

Aging hearts have prolonged QT interval and are vulnerable to oxidative stress. Because the QT interval indirectly reflects the action potential duration (APD), we examined the hypotheses that 1) the APD of ventricular myocytes increases with age; 2) the age-related prolongation of APD is due to an enhancement of basal late Na+ current (INaL); and 3) inhibition of INaL may protect aging hearts from arrhythmogenic effects of hydrogen peroxide (H2O2). Experiments were performed on ventricular myocytes isolated from (young) 1-mo- and (old) 1-yr-old guinea pigs (GPs). The APD of myocytes from old GPs was significantly longer than that from young GPs and was shortened by the INaL inhibitors GS967 and tetrodotoxin. The magnitude of INaL was significantly larger in myocytes from old than from young GPs. The CaMKII inhibitors KN-93 and AIP and the NaV1.5-channel blocker methanethiosulfonate ethylammonium blocked the INaL. There were no significant differences between myocytes from young and old GPs in L-type Ca2+ current and the rapidly and slowly activating delayed rectifier K+ currents, although the inward rectifier K+ current was slightly decreased in myocytes from old GPs. H2O2 induced more early afterdepolarizations in myocytes from old than from young GPs. The effect of H2O2 was attenuated by GS967. The results suggest that 1) the APD of myocytes from old GPs is prolonged, 2) a CaMKII-mediated increase in NaV1.5-channel INaL is responsible for the prolongation of APD, and 3) inhibition of INaL may be beneficial for maintaining electrical stability under oxidative stress in myocytes of old GPs.NEW & NOTEWORTHY The action potential duration is significantly longer in ventricular myocytes from old than from young guinea pigs, which may explain, at the cellular level, the increase in QT interval with age. A CaMKII-mediated enhancement of NaV1.5-channel late current is responsible for the age-related prolongation of action potential duration. The enhanced basal late sodium current may predispose cardiac myocytes of old animals to oxidative stress and arrhythmogenesis.

Ventricular action potential adaptation to regular exercise: role of β-adrenergic and KATP channel function.

Regular exercise training is known to affect the action potential duration (APD) and improve heart function, but involvement of β-adrenergic receptor (β-AR) subtypes and/or the ATP-sensitive K+ (KATP) channel is unknown. To address this, female and male Sprague-Dawley rats were randomly assigned to voluntary wheel-running or control groups; they were anesthetized after 6-8 wk of training, and myocytes were isolated. Exercise training significantly increased APD of apex and base myocytes at 1 Hz and decreased APD at 10 Hz. Ca2+ transient durations reflected the changes in APD, while Ca2+ transient amplitudes were unaffected by wheel running. The nonselective β-AR agonist isoproterenol shortened the myocyte APD, an effect reduced by wheel running. The isoproterenol-induced shortening of APD was largely reversed by the selective β1-AR blocker atenolol, but not the β2-AR blocker ICI 118,551, providing evidence that wheel running reduced the sensitivity of the β1-AR. At 10 Hz, the KATP channel inhibitor glibenclamide prolonged the myocyte APD more in exercise-trained than control rats, implicating a role for this channel in the exercise-induced APD shortening at 10 Hz. A novel finding of this work was the dual importance of altered β1-AR responsiveness and KATP channel function in the training-induced regulation of APD. Of physiological importance to the beating heart, the reduced response to adrenergic agonists would enhance cardiac contractility at resting rates, where sympathetic drive is low, by prolonging APD and Ca2+ influx; during exercise, an increase in KATP channel activity would shorten APD and, thus, protect the heart against Ca2+ overload or inadequate filling.NEW & NOTEWORTHY Our data demonstrated that regular exercise prolonged the action potential and Ca2+ transient durations in myocytes isolated from apex and base regions at 1-Hz and shortened both at 10-Hz stimulation. Novel findings were that wheel running shifted the β-adrenergic receptor agonist dose-response curve rightward compared with controls by reducing β1-adrenergic receptor responsiveness and that, at the high activation rate, myocytes from trained animals showed higher KATP channel function.

The novel late Na+ current inhibitor, GS-6615 (eleclazine) and its anti-arrhythmic effects in rabbit isolated heart preparations.

Enhanced late Na+ current (late INa ) in the myocardium is pro-arrhythmic. Inhibition of this current is a promising strategy to stabilize ventricular repolarization and suppress arrhythmias. Here, we describe GS-6615, a selective inhibitor of late INa , already in clinical development for the treatment of long QT syndrome 3 (LQT3). The effects of GS-6615 to inhibit late INa , versus other ion currents to shorten the ventricular action potential duration (APD), monophasic APD (MAPD) and QT interval, and decrease to the incidence of ventricular arrhythmias was determined in rabbit cardiac preparations. To mimic the electrical phenotype of LQT3, late INa was increased using the sea anemone toxin (ATX-II). GS-6615 inhibited ATX-II enhanced late INa in ventricular myocytes (IC50 =0.7μM), shortened the ATX-II induced prolongation of APD, MAPD, QT interval, and decreased spatiotemporal dispersion of repolarization and ventricular arrhythmias. Inhibition by GS-6615 of ATX-II enhanced late INa was strongly correlated with shortening of myocyte APD and isolated heart MAPD (R2 =0.94 and 0.98 respectively). In contrast to flecainide, GS-6615 had the minimal effects on peak INa . GS-6615 did not decrease the maximal upstroke velocity of the action potential (Vmax) nor widen QRS intervals. GS-6615 was a selective inhibitor of late INa , stabilizes the ventricular repolarization and suppresses arrhythmias in a model of LQT3. The concentrations at which the electrophysiological effects of GS-6615 were observed are comparable to plasma levels associated with QTc shortening in patients with LQT3, indicating that these effects are clinically relevant.

Inhibition of late Na+ current, a novel target to improve diastolic function and electrical abnormalities in Dahl salt-sensitive rats.

Late Na(+) current (INaL) is enhanced in myocytes of animals with chronic heart failure and patients with hypertrophic cardiomyopathy. To define the role of INaL in diastolic heart failure, the effects of GS-458967 (GS-967), a potent INaL inhibitor on mechanical and electrical abnormalities, were determined in an animal model of diastolic dysfunction. Dahl salt-sensitive (DSS) rats fed a high-salt (HS) diet for 8 wk, compared with a normal salt (NS) diet, had increased left ventricular (LV) mass (1,257 ± 96 vs. 891 ± 34 mg) and diastolic dysfunction [isovolumic relaxation time (IVRT): 26.8 ± 0.5 vs. 18.9 ± 0.2 ms; early transmitral flow velocity/early mitral annulus velocity (E/E') ratio: 25.5 ± 1.9 vs. 14.9 ± 0.9]. INaL in LV myocytes from HS rats was significantly increased to 0.41 ± 0.02 from 0.14 ± 0.02 pA/pF in NS rats. The action potential duration (APD) was prolonged to 136 ± 12 from 68 ± 9 ms in NS rats. QTc intervals were longer in HS vs. NS rats (267 ± 8 vs. 212 ± 2 ms). Acute and chronic treatment with GS-967 decreased the enhanced INaL to 0.24 ± 0.01 and 0.17 ± 0.02 pA/pF, respectively, vs. 0.41 ± 0.02 pA/pF in the HS group. Chronic treatment with GS-967 dose-dependently reduced LV mass, the increases in E/E' ratio, and the prolongation of IVRT by 27, 27, and 20%, respectively, at the 1.0 mg·kg(-1)·day(-1) dose without affecting blood pressure or LV systolic function. The prolonged APDs in myocytes and QTc of HS rats were significantly reduced with GS-967 treatment. These results indicate that INaL is a significant contributor to the LV diastolic dysfunction, hypertrophy, and repolarization abnormalities and thus, inhibition of this current is a promising therapeutic target for diastolic heart failure.

Effects of docosahexaenoic acid on atrial physiological parameter and TASK-1 protein expression in atrial fibrillation rats

To investigate the effect of docosahexaenoic acid (DHA) on atrial physiological parameter and its related mechanisms in atrial fibrillation rats. Eighty Sprague-Dawley (SD) rats which were sensitive to acetylcholine-calcium chloride mixture were randomly divided into four groups:control (CTL), control treated with DHA (DHA), atrial fibrillation (AF) and atrial fibrillation treated with DHA (DHA+AF). The duration of atrial fibrillation was measured. The atrial myocyte action potential duration (APD) and TWIK-related acid-sensitive K+ channels-1 (TASK-1) currents were recorded by whole-cell patch-clamp technique. The expression of TASK-1 at protein level in atrial tissue was detected by Western blot method. Atrial fibrillation of the rats was induced by acetylcholine-calcium chloride mixture, and the duration time of atrial fibrillation was increased with the drug-induced time prolonged. Compared with control group, the time of atrial myocyte action potential duration at 50% repolarization (APD50) and at 90% repolarization (APD90) were significantly shorten in AF group, TASK-1 current density and TASK-1 protein expression were increased (P<0.05). Compared with AF group, the duration of atrial fibrillation was decreased,the time of atrial myocyte APD50 and APD90 were prolonged, TASK-1 current density and protein expression were significantly reduced in DHA+AF group (P<0.05). DHA can prolong the atrial myocyte APD in atrial fibrillation rats, which may be related to down-regulation of TASK-1 protein expression and decreasing TASK-1 current density in atrial tissue.

Carbon monoxide inhibits inward rectifier potassium channels in cardiomyocytes.

Reperfusion-induced ventricular fibrillation (VF) severely threatens the lives of post-myocardial infarction patients. Carbon monoxide (CO)--produced by haem oxygenase in cardiomyocytes--has been reported to prevent VF through an unknown mechanism of action. Here, we report that CO prolongs action potential duration (APD) by inhibiting a subset of inward-rectifying potassium (Kir) channels. We show that CO blocks Kir2.2 and Kir2.3 but not Kir2.1 channels in both cardiomyocytes and HEK-293 cells transfected with Kir. CO directly inhibits Kir2.3 by interfering with its interaction with the second messenger phosphatidylinositol (4,5)-bisphosphate (PIP2). As the inhibition of Kir2.2 and Kir2.3 by CO prolongs APD in myocytes, cardiac Kir2.2 and Kir2.3 are promising targets for the prevention of reperfusion-induced VF.

NHE Isoform Switching and KChIP2 Upregulation in Aging Porcine Atria

Aging increases the risk of cardiac pathologies including atrial fibrillation and can alter myocardial responsiveness to therapeutic agents. Here, seeking molecular correlates of myocardial aging processes, we performed global “whole transcript” analysis of 25,388 genes using 572,667 probes to compare the left atrial (LA) transcriptomes of young adult (9 months old) versus elderly (10 years old) female swine. NHE2 (>9-fold) and KChIP2 (3.8-fold) exhibited the highest aging-related expression increases. Real-time qPCR recapitulated these findings and indicated a 50% decrease in LA NHE1, a twofold increase in right atrial KChIP2, but no significant changes for these transcripts in either ventricle. Notably, even in young adult pigs, NHE2 transcript was detectable and enriched in the atria over the ventricles. NHE1, the recognized cardiac isoform of the sodium hydrogen exchanger, has proven a compelling but clinically disappointing therapeutic target with respect to reperfusion arrhythmias. Our data challenge the dogma that NHE1 is alone in the myocardium and suggest that NHE2 could negatively impact the pharmacological responsiveness of atrial tissue to NHE1-specific inhibitors. KChIP2 is a cytosolic β subunit essential for generation of I to. The increased KChIP2 expression we observed with aging substantially shortened in silico atrial myocyte action potential duration, a predisposing factor in atrial fibrillation. Consistent with this, 4/5 elderly swine sustained pacing-induced AF≥15 s after cessation of stimulation, compared to 0/3 young swine. Our findings uncover potential molecular bases for increased arrhythmogenicity and reduced pharmacologic efficacy in the aging atrium, in a large animal model of human cardiac physiology.

Postnatal development of transmural gradients in expression of ion channels and Ca2+-handling proteins in the ventricle

Transmural gradients in myocyte action potential duration (APD) and Ca(2+)-handling proteins are argued to be important for both the normal functioning of the ventricle and arrhythmogenesis. In rabbit, the transmural gradient in APD (left ventricular wedge preparation) is minimal in the neonate. During postnatal development, APD increases both in the epicardium and the endocardium, but the prolongation is more substantial in the endocardium leading to a significant transmural gradient. We have investigated changes in the expression of ion channels and also Ca(2+)-handling proteins in the subepicardial and subendocardial layers of the left ventricular free wall in neonatal (2-7 days of age) and adult male (~6 months of age) New Zealand White rabbits using quantitative PCR and also, when possible, in situ hybridisation and immunohistochemistry. In the adult, there were significant and substantial transmural gradients in Ca(v)1.2, KChIP2, ERG, K(v)LQT1, K(ir)2.1, NCX1, SERCA2a and RyR2 at the mRNA and, in some cases, protein level-in every case the mRNA or protein was more abundant in the epicardium than the endocardium. Of the eight transmural gradients seen in the adult, only three were observed in the neonate and, in two of these cases, the gradients were smaller than those in the adult. However, in the neonate there were also transmural gradients not observed in the adult: in HCN4, Na(v)1.5, minK, K(ir)3.1 and Cx40 mRNAs - in every case the mRNA was more abundant in the endocardium than the epicardium. If the postnatal changes in ion channel mRNAs are used to predict changes in ionic conductances, mathematical modelling predicts the changes in APD observed experimentally. It is concluded that many of the well known transmural gradients in the ventricle develop postnatally.

An in silico canine cardiac midmyocardial action potential duration model as a tool for early drug safety assessment

Cell lines expressing ion channels (IC) and the advent of plate-based electrophysiology device have enabled a molecular understanding of the action potential (AP) as a means of early QT assessment. We sought to develop an in silico AP (isAP) model that provides an assessment of the effect of a compound on the myocyte AP duration (APD) using concentration-effect curve data from a panel of five ICs (hNav1.5, hCav1.2, hKv4.3/hKChIP2.2, hKv7.1/hminK, hKv11.1). A test set of 53 compounds was selected to cover a range of selective and mixed IC modulators that were tested for their effects on optically measured APD. A threshold of >10% change in APD at 90% repolarization (APD(90)) was used to signify an effect at the top test concentration. To capture the variations observed in left ventricular midmyocardial myocyte APD data from 19 different dogs, the isAP model was calibrated to produce an ensemble of 19 model variants that could capture the shape and form of the APs and also quantitatively replicate dofetilide- and diltiazem-induced APD(90) changes. Provided with IC panel data only, the isAP model was then used, blinded, to predict APD(90) changes greater than 10%. At a simulated concentration of 30 μM and based on a criterion that six of the variants had to agree, isAP prediction was scored as showing greater than 80% predictivity of compound activity. Thus, early in drug discovery, the isAP model allows integrating separate IC data and is amenable to the throughput required for use as a virtual screen.

Tetrahydrobiopterin depletion and NOS2 uncoupling contribute to heart failure-induced alterations in atrial electrophysiology.

Heart failure is a common antecedent to atrial fibrillation; both heart failure and atrial fibrillation are associated with increased myocardial oxidative stress. Chronic canine heart failure reduces atrial action potential duration and atrial refractoriness. We hypothesized that inducible nitric oxide synthase 2 (NOS2) contributes to atrial oxidative stress and electrophysiologic alterations. A 16-week canine tachypacing model of heart failure was used (n= 21). At 10 weeks, dogs were randomized to either placebo (n = 12) or active treatment (n = 9) with NOS cofactor, tetrahydrobiopterin (BH(4), 50 mg), and NOS substrate (L-arginine, 3 g) twice daily for 6 weeks. A group of matched controls (n = 7) was used for comparison. Heart failure increased atrial NOS2 and reduced atrial BH(4), while L-arginine was unchanged. Treatment reduced inducible atrial fibrillation and normalized the heart failure-induced shortening of the left atrial myocyte action potential duration. Treatment increased atrial [BH(4)] while [L-arginine] was unchanged. Treatment did not improve left ventricular function or dimensions. Heart failure-induced reductions in atrial [BH(4)] resulted in NOS uncoupling, as measured by NO and superoxide anion (O(2)(·-)) production, while BH(4) and L-arginine treatment normalized NO and O(2)(·-). Heart failure resulted in left atrial oxidative stress, which was attenuated by BH(4) and L-arginine treatment. Chronic non-ischaemic heart failure results in atrial oxidative stress and electrophysiologic abnormalities by depletion of BH(4) and uncoupling of NOS2. Modulation of NOS2 activity by repletion of BH(4) may be a safe and effective approach to reduce the frequency of atrial arrhythmias during heart failure.

Mitochondrial Reactive Oxygen Species Regulate the Cardiac Na + Channel

Background: Pyridine nucleotides regulate the cardiac Na+ current (INa) through PKC activation and reactive oxygen species (ROS), which is inhibited by NAD+ through PKA activation. Here, we investigated the source of ROS induced by elevated NADH and how it affected the cardiac Na+ channel (Nav1.5). Methods: HEK cells stably expressing Nav1.5 and rat neonatal ventricular myocytes were utilized. Effects on INa were assessed by whole-cell patch clamp recording. Also monitored were ROS generation, mitochondrial membrane potential (ΔΨm), and myocytes action potential (AP). Results: Decrease of INa (52±9%; P<0.01) induced by NADH (100 μM) in HEK cells was reversed by mitoTEMPO, rotenone, malonate, DIDS, PK11195 and 4’-chlorodiazepam. Antimycin A (20 μM) also decreased INa (51±4%, P<0.01), which was blocked by NAD+, forskolin, or rotenone. Inhibitors for complex IV, nitric oxide synthase, NADPH oxidases, xanthine oxidases, mitochondrial permeability transition pore, and mitochondrial ATP-sensitive K+ channel did not alter NADH effect on INa. Analogous results were observed in myocytes. Rotenone, mitoTEMPO, and 4’-chlorodiazepam also blocked the effect of a mutant A280V glycerol-3-phosphate dehydrogenase 1-like protein on reducing INa, indicating a mitochondrial role in Brugada syndrome. Fluorescent microscopy revealed that elevated NADH led to mitochondrial ROS generation but did not affect the mitochondrial ΔΨm. NADH treatment did not affect the resting potential and the AP duration of myocytes. Nevertheless, a decrease of the maximum upstroke velocity of action potential (68±12%, P<0.05) was observed. Conclusions: Altering the oxidized to reduced NAD(H) balance can activate mitochondrial ROS production from complex I/III and release from the mitochondrial inner membrane anion channel leading to reduced INa. This signaling cascade may help explain the link between altered metabolism, conduction block, and arrhythmic risk.

Validation of a voltage-sensitive dye-based method for assessing dog myocyte action potential duration

Validation of a voltage-sensitive dye-based method for assessing dog myocyte action potential duration