Atrial Action Potential Research Articles

Putative binding sites for arachidonic acid on the human cardiac Kv 1.5 channel.

In human heart, the Kv 1.5 channel contributes to repolarization of atrial action potentials. This study examined the electrophysiological and molecular mechanisms underlying arachidonic acid (AA)-induced inhibition of the human Kv 1.5 (hKv 1.5) channel. Site-directed mutagenesis was conducted to mutate amino acids that reside within the pore domain of the hKv 1.5 channel. Whole-cell patch-clamp method was used to record membrane currents through wild type and mutant hKv 1.5 channels heterologously expressed in CHO cells. Computer docking simulation was conducted to predict the putative binding site(s) of AA in an open-state model of the Kv 1.5 channel. The hKv 1.5 current was minimally affected at the onset of depolarization but was progressively reduced during depolarization by the presence of AA, suggesting that AA acts as an open-channel blocker. AA itself affected the channel at extracellular sites independently of its metabolites and signalling pathways. The blocking effect of AA was attenuated at pH 8.0 but not at pH 6.4. The blocking action of AA developed rather rapidly by co-expression of Kv β1.3. The AA-induced block was significantly attenuated in H463C, T480A, R487V, I502A, I508A, V512A and V516A, but not in T462C, A501V and L510A mutants of the hKv 1.5 channel. Docking simulation predicted that H463, T480, R487, I508, V512 and V516 are potentially accessible for interaction with AA. AA itself interacts with multiple amino acids located in the pore domain of the hKv 1.5 channel. These findings may provide useful information for future development of selective blockers of hKv 1.5 channels.

The positive frequency-dependent electrophysiological effects of the IKur inhibitor XEN-D0103 are desirable for the treatment of atrial fibrillation

BackgroundSelective inhibitors of Kv1.5 channels are being developed for the treatment of atrial fibrillation (AF).ObjectivesThe purpose of this study was to investigate the effects of the highly selective Kv1.5 inhibitor XEN-D0103 on human atrial action potentials (APs) at high excitation rates and to assess safety.MethodsIntracellular APs (stimulation rates 1–5 Hz) were measured in right atrial trabeculae from patients in sinus rhythm (SR), chronic AF (cAF; AF of >6 months duration), and paroxysmal AF (pAF). The safety and tolerability of XEN-D0103 were tested in a double-blind, randomized, placebo-controlled phase 1 study.ResultsDepending on its concentration, XEN-D0103 elevated the plateau potential. At 1 Hz, XEN-D0103 (3 µM) shortened action potential duration at 90% repolarization (APD90) and effective refractory period (ERP) in SR preparations, but prolonged these parameters in cAF preparations. In SR and pAF preparations, the shortening effects on APD90 and ERP turned into prolongation at high rates. In cAF trabeculae, XEN-D0103 prolonged APD90 and ERP at 2 and 3 Hz. At high rates, more SR and pAF preparations failed to capture excitation in the presence of the drug than in its absence. XEN-D0103 (10 µM) did not significantly affect human ventricular APs. Even with plasma concentrations reaching 7000 ng/mL, XEN-D0103 did not increase ∆∆QTcF (QT interval corrected by the Fridericia formula) in the analysis of electrocardiograms of healthy volunteers, and no subjects receiving an active treatment had a QT or QTcF interval >450 ms, or increase in QTcF from baseline >30 ms.ConclusionAPD prolongation and suppression of APs by XEN-D0103 at high stimulation rates in SR and pAF tissue, but not cAF, could be of therapeutic benefit for reducing AF burden. This concept needs to be confirmed in clinical trials.

Vernakalant as a Novel Anti-Arrhythmic Agent for Converting of Atrial Fibrillation, Molecular Mechanism, Updated Clinical Efficacy, and Future Development

The rising incidence of atrial fibrillation (AF) has stimulated researches to identify novel therapeutic options for such most common and refractory cardiac arrhythmia in clinical practice. Rhythm control strategy is shown to be associated with a lower risk of progression to permanent AF and greater clinical benefit as compared with rate control. Remarkable progress has been witnessed in rhythm control strategy particularly along with the development of mapping and ablation technology, while still should pharmacological cardioversion serve as an integrated approach for the management of AF especially in the emergency department or centers not equipped with ablation professionals. Concerns regarding the safety and efficacy of existing conventional antiarrhythmic drugs (AADs) limit their clinical use. Vernakalant, with its relatively atrial selective antiarrhythmic profile, is developed as a novel AAD for pharmacological cardioversion of AF. Its mechanisms involve potassium and sodium channels blocking effects during atrial action potential. A series of clinical trials have demonstrated the rapid, efficacious and safe effect of vernakalant over placebo or conventional AADs in terminating recent-onset AF among patients with structurally normal or minimal heart disease; but current evidence does not show a superior role of vernakalant in treating long-duration AF or atrial flutter. More evidence with respect to comparisons of vernakalant with conventional AADs as well as their synergic effects is needed. Cost-effectiveness analyses of vernakalant applied in prospective and "real world" practice remain to be assessed.

Effects of Wild-Type and Mutant Forms of Atrial Natriuretic Peptide on Atrial Electrophysiology and Arrhythmogenesis.

Atrial natriuretic peptide (ANP) is a hormone with numerous beneficial cardiovascular effects. Recently, a mutation in the ANP gene, which results in the generation of a mutant form of ANP (mANP), was identified and shown to cause atrial fibrillation in people. The mechanism(s) through which mANP causes atrial fibrillation is unknown. Our objective was to compare the effects of wild-type ANP and mANP on atrial electrophysiology in mice and humans. Action potentials (APs), L-type Ca(2+) currents (ICa,L), and Na(+) current were recorded in atrial myocytes from wild-type or natriuretic peptide receptor C knockout (NPR-C(-/-)) mice. In mice, ANP and mANP (10-100 nmol/L) had opposing effects on atrial myocyte AP morphology and ICa,L. ANP increased AP upstroke velocity (Vmax), AP duration, and ICa,L similarly in wild-type and NPR-C(-/-) myocytes. In contrast, mANP decreased Vmax, AP duration, and ICa,L, and these effects were completely absent in NPR-C(-/-) myocytes. ANP and mANP also had opposing effects on ICa,L in human atrial myocytes. In contrast, neither ANP nor mANP had any effect on Na(+) current in mouse atrial myocytes. Optical mapping studies in mice demonstrate that ANP sped electric conduction in the atria, whereas mANP did the opposite and slowed atrial conduction. Atrial pacing in the presence of mANP induced arrhythmias in 62.5% of hearts, whereas treatment with ANP completely prevented the occurrence of arrhythmias. These findings provide mechanistic insight into how mANP causes atrial fibrillation and demonstrate that wild-type ANP is antiarrhythmic.

E3 ligase CHIP and Hsc70 regulate Kv1.5 protein expression and function in mammalian cells

Kv1.5 confers ultra-rapid delayed-rectifier potassium channel current (IKur) which contributes to repolarization of the atrial action potential. Kv1.5 proteins, degraded via the ubiquitin-proteasome pathway, decreased in some atrial fibrillation patients. Carboxyl-terminus heat shock cognate 70-interacting protein (CHIP), an E3 ubiquitin ligase, is known to ubiquitinate short-lived proteins. Here, we investigated the roles of CHIP in Kv1.5 degradation to provide insights into the mechanisms of Kv1.5 decreases and treatments targeting Kv1.5 for atrial fibrillation. Coexpression of CHIP with Kv1.5 in HEK293 cells increased Kv1.5 protein ubiquitination and decreased the protein level. Immunofluorescence revealed decreases of Kv1.5 proteins in the endoplasmic reticulum and on the cell membrane. A siRNA against CHIP suppressed Kv1.5 protein ubiquitination and increased its protein level. CHIP mutants, lacking either the N-terminal tetratricopeptide region domain or the C-terminal U-box domain, failed to exert these effects on Kv1.5 proteins. Immunoprecipitation showed that CHIP formed complexes with Kv1.5 proteins and heat shock cognate protein 70 (Hsc70). Effects of Hsc70 on Kv1.5 were similar to CHIP by altering interaction of CHIP with Kv1.5 protein. Coexpression of CHIP and Hsc70 with Kv1.5 additionally enhanced Kv1.5 ubiquitination. Kv1.5 currents were decreased by overexpression of CHIP or Hsc70 but were increased by knockdown of CHIP or Hsc70 in HEK 293 cells stably expressing Kv1.5. These effects of CHIP and Hsc70 were also observed on endogenous Kv1.5 in HL-1 mouse cardiomyocytes, decreasing IKur and prolonging action potential duration. These results indicate that CHIP decreases the Kv1.5 protein level and functional channel by facilitating its degradation in concert with chaperone Hsc70.

Regulation of Voltage-Gated K+ Channel Kv1.5 by the Janus Kinase JAK3.

The tyrosine kinase Janus kinase 3 (JAK3) participates in the regulation of cell proliferation and apoptosis. The kinase further influences ion channels and transport proteins. The present study explored whether JAK3 contributes to the regulation of the voltage-gated K(+) channel Kv1.5, which participates in the regulation of diverse functions including atrial cardiac action potential and tumor cell proliferation. To this end, cRNA encoding Kv1.5 was injected into Xenopus oocytes with or without additional injection of cRNA encoding wild-type JAK3, constitutively active (A568V)JAK3, or inactive (K851A)JAK3. Voltage-gated K(+) channel activity was measured utilizing dual electrode voltage clamp, and Kv1.5 channel protein abundance in the cell membrane was quantified utilizing chemiluminescence of Kv1.5 containing an extracellular hemagglutinin epitope (Kv1.5-HA). As a result, Kv1.5 activity and Kv1.5-HA protein abundance were significantly decreased by wild-type JAK3 and (A568V)JAK3, but not by (K851A)JAK3. Inhibition of Kv1.5 protein insertion into the cell membrane by brefeldin A (5 μM) resulted in a decline of the voltage-gated current, which was similar in the absence and presence of (A568V)JAK3, suggesting that (A568V)JAK3 did not accelerate Kv1.5 protein retrieval from the cell membrane. A 24 h treatment with ouabain (100 µM) significantly decreased the voltage-gated current in oocytes expressing Kv1.5 without or with (A568V)JAK3 and dissipated the difference between oocytes expressing Kv1.5 alone and oocytes expressing Kv1.5 with (A568V)JAK3. In conclusion, JAK3 contributes to the regulation of membrane Kv1.5 protein abundance and activity, an effect sensitive to ouabain and thus possibly involving Na(+)/K(+) ATPase activity.

168 Atrial arrhythmias and electrical remodelling after endurance training in a mouse model of arrhythmogenic right ventricular cardiomyopathy

<h3>Background and purpose</h3> Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited disorder that causes ventricular arrhythmias and sudden death. The disease manifests predominantly in the right heart and is more common in endurance athletes. ARVC patients also show increased atrial arrhythmias. Loss-of-function mutations in desmosomal proteins such as plakoglobin are associated with ARVC, and endurance training exacerbates ventricular arrhythmias and right ventricular enlargement in mice with reduced expression of plakoglobin (Plako^+/–). Chronic intensive endurance training has recently been observed to increase susceptibility to atrial arrhythmias in rats. We therefore tested whether standardised endurance training affects atrial electrophysiology or increases in mice with heterozygous plakoglobin deficiency (Plako^+/–). <h3>Methods</h3> Plako^+/– and WT mice group swim-trained for 5 d/week over 8 weeks, gradually increased from 2 to 90 min/d (average swim times 50h). Atrial size and function was assessed by echocardiography. Left atrial (LA) monophasic action potentials (MAPs) were recorded from Langendorff-perfused hearts. Action potential durations (APDs) were measured during RA pacing at 100ms paced cycle lengths (CL). Atrial arrhythmias were provoked by programmed stimulation (S1-S2). Atrial arrhythmias were defined as fast irregular atrial activity &gt;1s). Transmembrane action potentials (TAPs) were recorded at 100ms CL from isolated, superfused LA using floating glass microelectrodes. All continuous parameters are expressed as mean±SEM. <h3>Results</h3> Swim-training increased atrial arrhythmia susceptibility in Plako^+/–but not WT mice (Plako^+/– sedentary: 1/9 vs trained: 9/17 hearts),(WT sedentary: 3/11 hearts vs trained: 1/11 hearts). Training induced mild leftventricular hypertrophy in both genotypes (LV mass increase 6–14%, p &lt; 0.05). As shown previously, training also increased right ventricular size in Plako^+/– vs. trained WT, e.g. Parasternal RV diameter Plako^+/– (1.79 ± 0.03 mm) vs. WT (1.66 ± 0.02 mm) after training, p &lt; 0.05. Training increased LA size as assessed in echocardiography in both genotypes versus sedentary (LA size WT: 3.11 ± 0.14 sedentary vs 3.79 ± 0.17 mm² trained, p &lt; 0.05; Plako^+/–: 3.23 ± 0.1 mm² sedentary vs 4.14 ± 0.23 mm² trained, p &lt; 0.05). LA APD from MAPs tended to be shorter in trained WT and were significantly shorter in trained Plako^+/- mice (Figure 2). RA APD90s measured from TAPs were also shorter after training (WT: 22 ± 1 sedentary vs 19 ± 1 ms trained; Plako^+/- 24 ± 1 sedentary vs 18 ± 1 ms trained). <h3>Conclusion</h3> Our observations suggest that endurance training shortens atrial action potential duration and increases left atrial size in both WT and plakoglobin deficient atria. Endurance training increased atrial arrhythmia susceptibility in mice with a deficiency in the mechanical cell-cell contact protein plakoglobin.

169 PITX2C deficiency augments the anti-arrhythmic properties of flecainide: results in a mouse model and validation in a human atrium simulation study

Background and objectives Polymorphisms close to the PITX2 gene on chromosome 4q25 are associated with incident and recurrent atrial fibrillation (AF). Carriers of variant rs10033464 respond relatively well to antiarrhythmic drug therapy with sodium channel blockers. To test whether altered atrial PITX2 mRNA expression alters the electrophysiological effect of sodium channel blockers, we studied the effect of flecainide (Flec) in hearts of mice with heterozygous Pitx2c gene deletion, a model for reduced Pitx2 mRNA expression and AF susceptibility. Methods We assessed the effect of 1µM Flec on left atrial (LA) monophasic, transmembrane and optical action potentials in Pitx2c +/– mice and their wild type (WT) littermates. We measured conduction velocity (CV), action potential duration (APD), AP amplitude (APA), maximum upstroke velocity (dV/dt max ) and resting membrane potential (RMP) during atrial pacing at 80–120 ms fixed-rate cycle lengths (CL); and effective refractory periods (ERP) with arrhythmia inducibility during programmed (S2) stimulation. Post-repolarisation refractoriness (PRR) was defined as the difference between ERP and APD90. We assessed whether the electrophysiological changes associated with reduced Pitx2c mRNA expression alters the response to sodium current (I Na ) block using the Courtemanche et al . human atrial model. Taqman low density array analysis was also used to measure changes in 20 ion channel, and related, genes of interest. Results Flec abolished arrhythmias in Pitx2c +/– (6/18 base vs 0/15 Flec, p +/– atria compared with WT (Pitx2c +/– 14 ± 2 ms, n = 13 vs WT 7 ± 2 ms, n = 10; p +/– 21 ± 3 ms, n = 6 vs WT 6 ± 4 ms, n = 6, p +/– atria had a slightly more positive resting membrane potential (WT –70 ± 0.7 mV, n = 30 cells vs PITX2c +/– –68 ± 0.7 mV, n = 32 cells, p +/– 73 ± 1.3 mV, n = 32). Flec had no effect on the RMP but reduced CV, APA and dV/dt max in both genotypes to a similar extent. Hearts with PRR were not susceptible to arrhythmias (0/17). As with the isolated hearts, I Na block prolonged PRR in the Pitx2c deficiency simulation (500 ms CL: reference model 30ms; Pitx2c deficiency model 42 ms) and had no differential effects of I Na on RMP, CV, APD, or dV/dt max . The human atrial model thus qualitatively reproduced the experimental findings. mRNA expression of Kcnk5 and Kcna6 was altered in Pitx2c +/– left atria. Conclusion Flecainide suppresses atrial arrhythmias in mice with reduced Pitx2 mRNA expression. We propose that this genotype-specific antiarrhythmic effect can be explained by a more positive resting membrane potential and higher post-repolarisation refractoriness. These data suggest that flecainide may be effective for rhythm control in carriers of genetic variants on chromosome 4q25.

Generation of highly purified human cardiomyocytes from peripheral blood mononuclear cell-derived induced pluripotent stem cells.

Induced pluripotent stem (iPS) cells have an enormous potential for physiological studies. A novel protocol was developed combining the derivation of iPS from peripheral blood with an optimized directed differentiation to cardiomyocytes and a subsequent metabolic selection. The human iPS cells were retrovirally dedifferentiated from activated T cells. The subsequent optimized directed differentiation protocol yielded 30-45% cardiomyocytes at day 16 of differentiation. The derived cardiomyocytes expressed appropriate structural markers like cardiac troponin T, α-actinin and myosin light chain 2 (MLC2V). In a subsequent metabolic selection with lactate, the cardiomyocytes content could be increased to more than 90%. Loss of cardiomyocytes during metabolic selection were less than 50%, whereas alternative surface antibody-based selection procedures resulted in loss of up to 80% of cardiomyocytes. Electrophysiological characterization confirmed the typical cardiac features and the presence of ventricular, atrial and nodal-like action potentials within the derived cardiomyocyte population. Our combined and optimized protocol is highly robust and applicable for scalable cardiac differentiation. It provides a simple and cost-efficient method without expensive equipment for generating large numbers of highly purified, functional cardiomyocytes. It will further enhance the applicability of iPS cell-derived cardiomyocytes for disease modeling, drug discovery, and regenerative medicine.

Flecainide exerts paradoxical effects on sodium currents and atrial arrhythmia in murine RyR2-P2328S hearts.

AimsCardiac ryanodine receptor mutations are associated with catecholaminergic polymorphic ventricular tachycardia (CPVT), and some, including RyR2-P2328S, also predispose to atrial fibrillation. Recent work associates reduced atrial Nav1.5 currents in homozygous RyR2-P2328S (RyR2S/S) mice with slowed conduction and increased arrhythmogenicity. Yet clinically, and in murine models, the Nav1.5 blocker flecainide reduces ventricular arrhythmogenicity in CPVT. We aimed to determine whether, and how, flecainide influences atrial arrhythmogenicity in RyR2S/S mice and their wild-type (WT) littermates.MethodsWe explored effects of 1 μm flecainide on WT and RyR2S/S atria. Arrhythmic incidence, action potential (AP) conduction velocity (CV), atrial effective refractory period (AERP) and AP wavelength (λ = CV × AERP) were measured using multi-electrode array recordings in Langendorff-perfused hearts; Na+ currents (INa) were recorded using loose patch clamping of superfused atria.ResultsRyR2S/S showed more frequent atrial arrhythmias, slower CV, reduced INa and unchanged AERP compared to WT. Flecainide was anti-arrhythmic in RyR2S/S but pro-arrhythmic in WT. It increased INa in RyR2S/S atria, whereas it reduced INa as expected in WT. It increased AERP while sparing CV in RyR2S/S, but reduced CV while sparing AERP in WT. Thus, RyR2S/S hearts have low λ relative to WT; flecainide then increases λ in RyR2S/S but decreases λ in WT.ConclusionsFlecainide (1 μm) rescues the RyR2-P2328S atrial arrhythmogenic phenotype by restoring compromised INa and λ, changes recently attributed to increased sarcoplasmic reticular Ca2+ release. This contrasts with the increased arrhythmic incidence and reduced INa and λ with flecainide in WT.

Influence of Zoledronic Acid on Atrial Electrophysiological Parameters and Electrocardiographic Measurements.

Our objective was to determine effects of zoledronic acid (ZA) on atrial electrophysiological parameters and electrocardiographic measurements. Ex vivo perfusion study: Isolated guinea pig hearts were perfused with modified Krebs-Henseleit (K-H) buffer with or without ZA 0.07 mg/kg/L (each n = 6). In ZA-perfused hearts, atrial action potential at 90% repolarization (APD90 ) decreased more from baseline than in controls (-23.2% ± -5.1% vs. -2.1% ± -8.1%, P < 0 .0001), as did APD30 (-28.8% ± -3.8% vs. -2.1% ± -2.1%, P < 0.0001). In vivo dose-response study: Guinea pigs underwent intraperitoneal injections every 2 weeks in 1 of 4 groups (each n = 8): ZA 0.007 mg/kg (low-dose), ZA 0.07 mg/kg (medium-dose), ZA 0.7 mg/kg (high-dose), or placebo. Hearts were excised at 8 weeks and perfused with modified K-H. Atrial effective refractory period (ERP) was lower with medium- and high-dose ZA versus placebo (P = 0.004). Atrial APD30 was lower with high-dose ZA versus placebo, low and medium doses (P < 0.001). Canine ECG study: Mature female beagles received intravenous ZA 0.067 mg/kg or saline (placebo; each n = 6) every 2 weeks for 12 weeks. P wave dispersion was greater in the ZA group (7.7 ± 3.7 vs. 3.4 ± 2.6 ms, P = 0.04). There were no significant differences in P wave index, maximum or minimum P wave duration, or PR interval. ZA shortens left atrial APD and ERP and increases P wave dispersion.

Interaction of DPP10a with Kv4.3 channel complex results in a sustained current component of human transient outward current Ito.

The sustained component of the K(+) outward current in human atrial myocytes is believed to be due to the slowly inactivating ultra-rapid potassium current I Kur and not to the fast inactivating transient outward current Ito. Here we provide evidence for contribution of Ito to this late current due to the effects of dipeptidyl peptidase-like protein (DPP) 10 (DPP10a) interacting with Kv4.3 channels. We studied the late current component of Ito in human atrial myocytes and CHO cells co-expressing Kv4.3 or Kv4.3/KChIP2 (control) and DPP proteins using voltage-clamp technique and a pharmacological approach. A voltage dependent and slowly inactivating late current (43% of peak amplitude) could be observed in atrial myocytes. We found a similar current in CHO cells expressing Kv4.3/KChIP2 + DPP10a, but not in cells co-expressing Kv4.3 + DPP or Kv4.3/KChIP2 + DPP6-S. Assuming that DPP10a influences atrial Ito, we detected DPP10 expression of three alternatively spliced mRNAs, DPP10 protein and colocalization of Kv4.3 and DPP10 proteins in human atrial myocytes. DPP10a did not affect properties of expressed Kv1.5 excluding a contribution to the sustained IKur in atrial cells. To test for the contribution of Kv4-based Ito on sustained K(+) outward currents in human atrial myocytes, we used 4-AP to block IKur, in combination with Heteropoda toxin 2 to block Kv4 channels. We could clearly separate an Ito fraction of about 19% contributing to the late current in atrial myocytes. Thus, the interaction of DPP10a, expressed in human atrium, with Kv4.3 channels generates a sustained current component of Ito, which may affect late repolarization phase of atrial action potentials.

Electronic Expression of IK1 in Human Induced Pluripotent Stem Cell Derived Cardiocytes Reveals Atrial vs Ventricular Specific Properties

Cardiomyocytes derived from human induced pluripotent stem cells (hIPSC-CMs) provide a promising platform for understanding human cardiac pathology and physiology. HIPSC-CMs differentiate into a mixed population of cells composed of ventricular, atrial, and nodal cell types; cellular phenotype is generally distinguished by morphology of the action potential (APs). However, in hIPSC-CMs a very small inward rectifying potassium channel (IK1) relative to native cardiac cells causes a depolarized membrane potential which contributes to inconsistent AP properties and misidentification of cellular phenotype.We used Electronic expression of IK1 via dynamic clamp to restore the AP and resting membrane potential back to a physiological levels. This allowed for improved discrimination of atrial and ventricular cells based on AP morphology. Using standard patch clamp techniques, we compared the inward sodium currents (INa) of cells with atrial and ventricular AP morphologies. INa current densities in cells with atrial like APs were larger than ventricular-like APs (at −30 mV, in pA/pF: −71.22 ± 6.96 (n=5) vs. −46.25 ±5.03 (n=14), p < 0.05). Analysis of steady-state inactivation parameters of INa showed that cells with an atrial like AP had a more negative steady-state inactivation (V1/2: −81.84 ± 2.75 (n=5) vs. −74.72 ±0.8 (n=14), p<0.05). Cells with atrial APs had a larger Kv1.5-like component at +50 mV than ventricular APs (in pA/pF: 3.71 ± 0.55 (n=5) vs 1.00 ± 0.10 (n=16),P<0.05) but similar peak currents: (6.89 ± 0.50 (n=5) vs. 6.58 ± 0.67 (n=14),P=N.S). IK1 current density was more than 3x smaller in cells with atrial-like APs at −120 mV (in pA/pF: −0.71 ± 0.08 (n=5) vs. −3.45 ± 0.61 (n=14), p<0.05). These data suggest that differential AP morphology in hIPSC-CM has a similar electrophysiological basis to native cells.

SPAK and OSR1 Sensitivity of Voltage-Gated K+ Channel Kv1.5

SPS1-related proline/alanine-rich kinase (SPAK) and oxidative stress-responsive kinase 1 (OSR1) are potent regulators of several transporters and ion channels. The kinases are under regulation of with-no-K(Lys) (WNK) kinases. The present study explored whether SPAK and/or OSR1 modify the expression and/or activity of the voltage-gated K(+) channel Kv1.5, which participates in the regulation of diverse functions including atrial cardiac action potential and tumor cell proliferation. cRNA encoding Kv1.5 was injected into Xenopus oocytes with or without additional injection of cRNA encoding wild-type SPAK, constitutively active (T233E)SPAK, WNK insensitive (T233A)SPAK, catalytically inactive (D212A)SPAK, wild-type OSR1, constitutively active (T185E)OSR1, WNK insensitive (T185A)OSR1, and catalytically inactive (D164A)OSR1. Voltage-gated K(+) channel activity was quantified utilizing dual electrode voltage clamp and Kv1.5 channel protein abundance in the cell membrane utilizing chemiluminescence of Kv1.5 containing an extracellular hemagglutinin epitope (Kv1.5-HA). Kv1.5 activity and Kv1.5-HA protein abundance were significantly decreased by wild-type SPAK and (T233E)SPAK, but not by (T233A)SPAK and (D212A)SPAK. Similarly, Kv1.5 activity and Kv1.5-HA protein abundance were significantly down-regulated by wild-type OSR1 and (T185E)OSR1, but not by (T185A)OSR1 and (D164A)OSR1. Both, SPAK and OSR1 decrease cell membrane Kv1.5 protein abundance and activity.

Calcium-activated potassium current modulates ventricular repolarization in chronic heart failure.

The role of IKCa in cardiac repolarization remains controversial and varies across species. The relevance of the current as a therapeutic target is therefore undefined. We examined the cellular electrophysiologic effects of IKCa blockade in controls, chronic heart failure (HF) and HF with sustained atrial fibrillation. We used perforated patch action potential recordings to maintain intrinsic calcium cycling. The IKCa blocker (apamin 100 nM) was used to examine the role of the current in atrial and ventricular myocytes. A canine tachypacing induced model of HF (1 and 4 months, n = 5 per group) was used, and compared to a group of 4 month HF with 6 weeks of superimposed atrial fibrillation (n = 7). A group of age-matched canine controls were used (n = 8). Human atrial and ventricular myocytes were isolated from explanted end-stage failing hearts which were obtained from transplant recipients, and studied in parallel. Atrial myocyte action potentials were unchanged by IKCa blockade in all of the groups studied. IKCa blockade did not affect ventricular myocyte repolarization in controls. HF caused prolongation of ventricular myocyte action potential repolarization. IKCa blockade caused further prolongation of ventricular repolarization in HF and also caused repolarization instability and early afterdepolarizations. SK2 and SK3 expression in the atria and SK3 in the ventricle were increased in canine heart failure. We conclude that during HF, IKCa blockade in ventricular myocytes results in cellular arrhythmias. Furthermore, our data suggest an important role for IKCa in the maintenance of ventricular repolarization stability during chronic heart failure. Our findings suggest that novel antiarrhythmic therapies should have safety and efficacy evaluated in both atria and ventricles.

Simultaneous mapping of membrane voltage and calcium in zebrafish heart in vivo reveals chamber-specific developmental transitions in ionic currents.

The cardiac action potential (AP) and the consequent cytosolic Ca2+ transient are key indicators of cardiac function. Natural developmental processes, as well as many drugs and pathologies change the waveform, propagation, or variability (between cells or over time) of these parameters. Here we apply a genetically encoded dual-function calcium and voltage reporter (CaViar) to study the development of the zebrafish heart in vivo between 1.5 and 4 days post fertilization (dpf). We developed a high-sensitivity spinning disk confocal microscope and associated software for simultaneous three-dimensional optical mapping of voltage and calcium. We produced a transgenic zebrafish line expressing CaViar under control of the heart-specific cmlc2 promoter, and applied ion channel blockers at a series of developmental stages to map the maturation of the action potential in vivo. Early in development, the AP initiated via a calcium current through L-type calcium channels. Between 90 and 102 h post fertilization (hpf), the ventricular AP switched to a sodium-driven upswing, while the atrial AP remained calcium driven. In the adult zebrafish heart, a sodium current drives the AP in both the atrium and ventricle. Simultaneous voltage and calcium imaging with genetically encoded reporters provides a new approach for monitoring cardiac development, and the effects of drugs on cardiac function.

Atrial Fibrillation and SCN5A Variants.

Although atrial fibrillation (AF) is clinically and genetically a highly heterogeneous disease, recent studies suggest that the arrhythmia may arise because of interactions between genetic and acquired risk factors - the so called "double-hit" hypothesis. Genome-wide association studies have identified common AF susceptibility loci, and linkage analysis and candidate gene approaches have identified mutations in genes that encode for cardiac ion channels and signaling proteins; however, most of the heritability of AF still remains unexplained. The voltage-dependent cardiac sodium channel, encoded by SCN5A, conducts the main cardiac inward sodium current (INa) and is responsible for the upstroke of the atrial action potential. Mutations in SCN5A, which encodes the α-subunit of the NaV1.5 channel, have been linked with increased susceptibility to not only AF but also ventricular arrhythmias (long QT syndrome, Brugada syndrome), progressive cardiac conduction disease, and overlap syndromes with mixed arrhythmia phenotypes. Over the last decade, functional characterization of SCN5A mutations by expressing the channel in heterologous expression systems and applying cellular electrophysiological techniques has not only advanced our understanding of molecular mechanisms of AF but also potentially identified a mechanism-based approach to treating this common and morbid condition.

Electrical excitability of the heart in a Chondrostei fish, the Siberian sturgeon (Acipenser baerii).

Sturgeon (family Acipenseridae) are regarded as living fossils due to their ancient origin and exceptionally slow evolution. To extend our knowledge of fish cardiac excitability to a Chondrostei fish, we examined electrophysiological phenotype of the Siberian sturgeon (Acipenser baerii) heart with recordings of epicardial ECG, intracellular action potentials (APs), and sarcolemmal ion currents. Epicardial ECG of A. baerii had the typical waveform of the vertebrate ECG with Q-T interval (average duration of ventricular AP) of 650±30 ms and an intrinsic heart rate of 45.5±5 beats min(-1) at 20°C. Similar to other fish species, atrial AP was shorter in duration (402±33 ms) than ventricular AP (585±40) (P<0.05) at 20°C. Densities of atrial and ventricular Na+ currents were similar (-47.6±4.5 and -53.2±5.1 pA/pF, respectively) and close to the typical values of teleost hearts. Two major K+ currents, the inward rectifier K+ current (IK1), and the delayed rectifier K+ current (IKr) were found under basal conditions in sturgeon cardiomyocytes. The atrial IKr (3.3±0.2 pA/pF) was about twice as large as the ventricular IKr (1.3±0.4 pA/pF) (P<0.05) conforming to the typical pattern of teleost cardiac IKr. Divergent from other fishes, the ventricular IK1 was remarkably small (-2.5±0.07 pA/pF) and not different from that of the atrial myocytes (-1.9±0.06 pA/pF) (P>0.05). Two ligand-gated K+ currents were also found: ACh-activated inward rectifier (IKACh) was present only in atrial cells, while ATP-sensitive K+ current (IKATP) was activated by a mitochondrial blocker, CCCP, in both atrial and ventricular cells. The most striking difference to other fishes appeared in Ca2+ currents (ICa). In atrial myocytes, ICa was predominated by nickel-sensitive and nifedipine-resistant T-type ICa, while ventricular myocytes had mainly nifedipine-sensitive and nickel-resistant L-type ICa. ICaT/ICaL ratio of the sturgeon atrial myocytes (2.42) is the highest value ever measured for a vertebrate species. In ventricular myocytes, ICaT/ICaL ratio was 0.09. With the exception of the large atrial ICaT and small ventricular IK1, electrical excitability of A. baerii heart is similar to that of teleost hearts.

Inter-subject variability in human atrial action potential in sinus rhythm versus chronic atrial fibrillation.

AimsHuman atrial electrophysiology exhibits high inter-subject variability in both sinus rhythm (SR) and chronic atrial fibrillation (cAF) patients. Variability is however rarely investigated in experimental and theoretical electrophysiological studies, thus hampering the understanding of its underlying causes but also its implications in explaining differences in the response to disease and treatment. In our study, we aim at investigating the ability of populations of human atrial cell models to capture the inter-subject variability in action potential (AP) recorded in 363 patients both under SR and cAF conditions.Methods and ResultsHuman AP recordings in atrial trabeculae (n = 469) from SR and cAF patients were used to calibrate populations of computational SR and cAF atrial AP models. Three populations of over 2000 sampled models were generated, based on three different human atrial AP models. Experimental calibration selected populations of AP models yielding AP with morphology and duration in range with experimental recordings. Populations using the three original models can mimic variability in experimental AP in both SR and cAF, with median conductance values in SR for most ionic currents deviating less than 30% from their original peak values. All cAF populations show similar variations in GK1, GKur and Gto, consistent with AF-related remodeling as reported in experiments. In all SR and cAF model populations, inter-subject variability in IK1 and INaK underlies variability in APD90, variability in IKur, ICaL and INaK modulates variability in APD50 and combined variability in Ito and IKur determines variability in APD20. The large variability in human atrial AP triangulation is mostly determined by IK1 and either INaK or INaCa depending on the model.ConclusionExperimentally-calibrated human atrial AP models populations mimic AP variability in SR and cAF patient recordings, and identify potential ionic determinants of inter-subject variability in human atrial AP duration and morphology in SR versus cAF.

An automated system using spatial oversampling for optical mapping in murine atria. Development and validation with monophasic and transmembrane action potentials

We developed and validated a new optical mapping system for quantification of electrical activation and repolarisation in murine atria. The system makes use of a novel 2nd generation complementary metal-oxide-semiconductor (CMOS) camera with deliberate oversampling to allow both assessment of electrical activation with high spatial and temporal resolution (128 × 2048 pixels) and reliable assessment of atrial murine repolarisation using post-processing of signals. Optical recordings were taken from isolated, superfused and electrically stimulated murine left atria. The system reliably describes activation sequences, identifies areas of functional block, and allows quantification of conduction velocities and vectors. Furthermore, the system records murine atrial action potentials with comparable duration to both monophasic and transmembrane action potentials in murine atria.