Action Potential Prolongation Research Articles

Dissolution of spiral wave's core using cardiac optogenetics.

Rotating spiral waves in the heart are associated with life-threatening cardiac arrhythmias such as ventricular tachycardia and fibrillation. These arrhythmias are treated by a process called defibrillation, which forces electrical resynchronization of the heart tissue by delivering a single global high-voltage shock directly to the heart. This method leads to immediate termination of spiral waves. However, this may not be the only mechanism underlying successful defibrillation, as certain scenarios have also been reported, where the arrhythmia terminated slowly, over a finite period of time. Here, we investigate the slow termination dynamics of an arrhythmia in optogenetically modified murine cardiac tissue both in silico and ex vivo during global illumination at low light intensities. Optical imaging of an intact mouse heart during a ventricular arrhythmia shows slow termination of the arrhythmia, which is due to action potential prolongation observed during the last rotation of the wave. Our numerical studies show that when the core of a spiral is illuminated, it begins to expand, pushing the spiral arm towards the inexcitable boundary of the domain, leading to termination of the spiral wave. We believe that these fundamental findings lead to a better understanding of arrhythmia dynamics during slow termination, which in turn has implications for the improvement and development of new cardiac defibrillation techniques.

P38γ/δ activation alters cardiac electrical activity and predisposes to ventricular arrhythmia.

Ventricular fibrillation (VF) is a leading immediate cause of sudden cardiac death. There is a strong association between aging and VF, although the mechanisms are unclear, limiting the availability of targeted therapeutic interventions. Here we found that the stress kinases p38γ and p38δ are activated in the ventricles of old mice and mice with genetic or drug-induced arrhythmogenic conditions. We discovered that, upon activation, p38γ and p38δ cooperatively increase the susceptibility to stress-induced VF. Mechanistically, our data indicate that activated p38γ and p38δ phosphorylate ryanodine receptor 2 (RyR2) disrupt Kv4.3 channel localization, promoting sarcoplasmic reticulum calcium leak, Ito current reduction and action potential duration prolongation. In turn, this led to aberrant intracellular calcium handling, premature ventricular complexes and enhanced susceptibility to VF. Blocking this pathway protected genetically modified animals from VF development and reduced the VF duration in aged animals. These results indicate that p38γ and p38δ are a potential therapeutic target for sustained VF prevention.

Abstract 12860: A Novel Cardiac Presentation of a Genetic Syndrome

Introduction: Cute Syndrome is caused by a missense mutation in SCN8-A which encodes voltage gated sodium channel alpha subunit 8 (Nav1.6). Gain of function mutations trigger epileptic encephalopathy, while loss of function mutations are associated with cerebellar ataxia and cognitive problems 1 . While these channels are essential for progression of action potentials across the cardiac circuit, SCN8-A mutations are not associated with arrhythmias. Meanwhile, SCN5-A mutations have been implicated in heart block, LQTS, Brugada syndrome, and ventricular fibrillation. Here we describe a case of heart block and sinus node dysfunction in a patient with SCN8-A mutation. Presentation: A 19 year old male with past medical history of SCN8A mutation admitted for characterization of seizures. He has had focal seizures since the age of 13 with multiple admissions for seizures. He had new staring spells with shaking of his hands and inability to walk multiple times a week. He was admitted for Long Term EEG and was noted to have two episodes of loss of consciousness with progressive bradycardia followed by sinus pauses. He urgently had a temporary pacemaker placed followed by placement of a PPM. He has remained seizure free since his PPM implantation. Discussion: Voltage gated Sodium channels are an integral part of the cardiac action potential. SCN8-A encodes Nav1.6. A mutation in this gene, primarily expressed in neurons but has also been seen in cardiac tissue, could cause arrhythmias. Human mutations have been associated with intellectual disabilities and seizure disorders 1,2,3 . Mouse models have shown that null mice have prolonged cardiac action potentials, suggesting role in cardiac arrythmias 4 . There have been no documented human cases of SCN8-A mutations with arrhythmias. Our patient's seizures were thought to be due to the neurological manifestations of his mutation. Evaluation of cardiac and EEG monitoring determined that sinus pauses preceded seizures, and he has been seizure free since his PPM. This case demonstrates the need for further research to characterize cardiac manifestations of SCN8-A mutations and patients with the mutation may benefit from further cardiac evaluation.

Human engineered cardiac tissue model of hypertrophic cardiomyopathy recapitulates key hallmarks of the disease and the effect of chronic mavacamten treatment.

Introduction: The development of patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) offers an opportunity to study genotype-phenotype correlation of hypertrophic cardiomyopathy (HCM), one of the most common inherited cardiac diseases. However, immaturity of the iPSC-CMs and the lack of a multicellular composition pose concerns over its faithfulness in disease modeling and its utility in developing mechanism-specific treatment. Methods: The Biowire platform was used to generate 3D engineered cardiac tissues (ECTs) using HCM patient-derived iPSC-CMs carrying a β-myosin mutation (MYH7-R403Q) and its isogenic control (WT), withal ECTs contained healthy human cardiac fibroblasts. ECTs were subjected to electro-mechanical maturation for 6weeks before being used in HCM phenotype studies. Results: Both WT and R403Q ECTs exhibited mature cardiac phenotypes, including a lack of automaticity and a ventricular-like action potential (AP) with a resting membrane potential < -75mV. Compared to WT, R403Q ECTs demonstrated many HCM-associated pathological changes including increased tissue size and cell volume, shortened sarcomere length and disorganized sarcomere structure. In functional assays, R403Q ECTs showed increased twitch amplitude, slower contractile kinetics, a less pronounced force-frequency relationship, a smaller post-rest potentiation, prolonged AP durations, and slower Ca2+ transient decay time. Finally, we observed downregulation of calcium handling genes and upregulation of NPPB in R403Q vs. WT ECTs. In an HCM phenotype prevention experiment, ECTs were treated for 5-weeks with 250nM mavacamten or a vehicle control. We found that chronic mavacamten treatment of R403Q ECTs: (i) shortened relaxation time, (ii) reduced APD90 prolongation, (iii) upregulated ADRB2, ATP2A2, RYR2, and CACNA1C, (iv) decreased B-type natriuretic peptide (BNP) mRNA and protein expression levels, and (v) increased sarcomere length and reduced sarcomere disarray. Discussion: Taken together, we demonstrated R403Q ECTs generated in the Biowire platform recapitulated many cardiac hypertrophy phenotypes and that chronic mavacamten treatment prevented much of the pathology. This demonstrates that the Biowire ECTs are well-suited to phenotypic-based drug discovery in a human-relevant disease model.

Tricyclic hydrocarbon fluorene attenuates ventricular ionic currents and pressure development in the navaga cod

The release of polycyclic aromatic hydrocarbons (PAHs) into the environment due to oil and diesel fuel spills is a serious threat to Arctic fish populations. PAHs produce multiple toxic effects in fish, but disturbance of electrical and contractile activity of the heart seems to be the most negative effect. Our study focused on the effects of fluorene, a tricyclic PAH resembling the well-investigated tricyclic phenanthrene, on major ionic currents and action potential (AP) waveform in isolated ventricular myocytes and on contractile activity in isolated whole hearts of polar navaga cod (Eleginus nawaga). Among the studied currents, the repolarizing rapid delayed rectifier K+ current IKr demonstrated the highest sensitivity to fluorene with IC50 of 0.54 μM. The depolarizing inward currents, INa and ICaL, were inhibited with 10 μM fluorene by 20.2 ± 2.8 % and 27.9 ± 8.4 %, respectively, thereby being much less sensitive to fluorene than IKr. Inward rectifier IK1 current was insensitive to fluorene (up to 10 μM). While 3 μM fluorene prolonged APs, 10 μM also slowed the AP upstroke. Resting membrane potential was not affected by any tested concentrations. In isolated heart experiments 10 μM fluorene caused modest depression of ventricular contractile activity. Thus, we have demonstrated that fluorene, a tricyclic PAH present in high quantities in crude oil, strongly impacts electrical activity with only slight effects on contractile activity in the heart of the polar fish, the navaga cod.

Lumbar V3 interneurons provide direct excitatory synaptic input onto thoracic sympathetic preganglionic neurons, linking locomotor, and autonomic spinal systems.

Although sympathetic autonomic systems are activated in parallel with locomotion, the neural mechanisms mediating this coordination are incompletely understood. Sympathetic preganglionic neurons (SPNs), primarily located in the intermediate laminae of thoracic and upper lumbar segments (T1-L2), increase activation of tissues and organs that provide homeostatic and metabolic support during movement and exercise. Recent evidence suggests integration between locomotor and autonomic nuclei occurs within the brainstem, initiating both descending locomotor and sympathetic activation commands. However, both locomotor and sympathetic autonomic spinal systems can be activated independent of supraspinal input, in part due to a distributed network involving propriospinal neurons. Whether an intraspinal mechanism exists to coordinate activation of these systems is unknown. We hypothesized that ascending spinal neurons located in the lumbar region provide synaptic input to thoracic SPNs. Here, we demonstrate that synaptic contacts from locomotor-related V3 interneurons (INs) are present in all thoracic laminae. Injection of an anterograde tracer into lumbar segments demonstrated that 8-20% of glutamatergic input onto SPNs originated from lumbar V3 INs and displayed a somatotopographical organization of synaptic input. Whole cell patch clamp recording in SPNs demonstrated prolonged depolarizations or action potentials in response to optical activation of either lumbar V3 INs in spinal cord preparations or in response to optical activation of V3 terminals in thoracic slice preparations. This work demonstrates a direct intraspinal connection between lumbar locomotor and thoracic sympathetic networks and suggests communication between motor and autonomic systems may be a general function of the spinal cord.

Abstract P1041: Multiplexed Cardiotoxicity And Efficacy Screening Using Kinetic Image Cytometry Of Human Induced Pluripotent Stem Cell-derived Cardiomyocytes

Traditionally, cardiotoxicity testing used low-throughput animal models which may not fully recapitulate human physiology. Stem cell technology enables screening in a more physiologically-relevant model system in less time and with less cost, but most current high-throughput screening modalities rely on whole-well or whole-field averaged data, which obfuscate the responses of individual hiPSC-cardiomyocytes (CMs). By measuring individual hiPSC-CMs, heterogenous responses that would otherwise be “averaged out” can be quantified, thus improving sensitivity. We previously published that the single-cell Kinetic Image Cytometry (KIC) methods developed at Vala Sciences predicted arrhythmogenic liability with an accuracy of 90% using intracellular Ca 2+ kinetics as a readout. In this study, we expand upon this methodology by loading metabolically matured hiPSC-CMs with Cal-520 (a green Ca 2+ dye) and BeRST-1 (a far red membrane voltage dye), acquiring interleaved movies of Ca 2+ and membrane potential at 60 Hz (30 Hz per channel). We then created image analysis algorithms to measure Ca 2+ transient (CT) and action potential (AP) kinetics, and to derive contraction measurements from those movies. To validate our approach, we exposed cells to isoproterenol, nifedipine, dofetilide, and butadiene 2-monoxime (BDM) at various concentrations. Isoproterenol increased beat rate, decreased both AP and CT durations, increased CT magnitude, and elicited a lusitropic effect. Nifedipine increased beat rate, decreased AP and CT durations, and reduced CT magnitude and contraction distance. Dofetilide reduced beat rate, prolonged APs and CTs, and increased contraction distance. BDM had multiple inhibitor effects on APs and CTs, and strongly reduced contraction. Thus, this multiplexed assay can quantify multiple effects of drugs on APs, CTs, and contraction, providing an unprecedented suite of information relevant to cardiosafety and efficacy.

Abstract P3039: The Biophysical Action Of Microrna-1 Is Critical To Maintaining The Homeostasis Of The Heart

MicroRNAs (miRs) are evolutionarily-conserved small noncoding RNAs (~22 nucleotides) and are involved in most biological events through the classical mechanism of RNA interference (RNAi). Recently, we discovered a novel action of miR to biophysically modulate the function of bound proteins. miR1, the most predominant miR in the heart, physically binds to the inward rectifier potassium channel Kir2.1, directly represses the I K1 current, and biophysically modulates cardiac electrophysiology. An arrhythmia-associated human single nucleotide polymorphism of miR1 (hSNP14A/G) specifically disrupts biophysical modulation while retaining the RNAi function. However, the physiological significance of miR’s biophysical action on the heart remains unknown. To study this, we created hSNP14A/G single nucleotide mutation on miR1-1 and miR1-2 by using CRISPR/Cas9 technology and successfully generated 14G-mutated transgenic homozygous mice (14G-Homo, miR1-1 14G/G :miR1-2 14G/G ), in which the mutated miR1 maintains the RNAi while with a specific deletion of the biophysical action. We found that 14G-Homo mice had high mortality with impaired heart pumping function and some mice developed heart failure at 6 months of age. The whole transcriptome RNA-sequencing assays revealed cardiac electrical remodeling in 14G-Homo hearts. Consistently, 14G-Homo hearts had prolonged QRS and QT intervals with slower electrical conduction through the ventricle, and 14G-Homo mice are susceptible to arrhythmia. Our patch clamping showed that 14G-Homo cardiomyocytes had significantly prolonged action potential with bigger I K1 and L-type Ca 2+ current (I Ca,L ) than WT cells. We also performed a CLIP RNA pulldown-mass spectrometry assay and identified more ion channel proteins that interact with miR1. From the validation, miR1 biophysically suppresses I Ca,L via directly binding to Cavβ2, the β subunit of the voltage-gated Ca 2+ channel. We conclude that miR1 biophysically modulates the function of the heart, which is critical to maintaining heart homeostasis. Our discovery expands the biological significance of miR biology and broadens its implication of RNA medicine development for cardiovascular diseases.

Cytoskeletal disarray increases arrhythmogenic vulnerability during sympathetic stimulation in a model of hypertrophic cardiomyopathy

Familial hypertrophic cardiomyopathy (FHC) patients are advised to avoid strenuous exercise due to increased risk of arrhythmias. Mice expressing the human FHC-causing mutation R403Q in the myosin heavy chain gene (MYH6) recapitulate the human phenotype, including cytoskeletal disarray and increased arrhythmia susceptibility. Following in vivo administration of isoproterenol, mutant mice exhibited tachyarrhythmias, poor recovery and fatigue. Arrhythmias were attenuated with the β-blocker atenolol and protein kinase A inhibitor PKI. Mutant cardiac myocytes had significantly prolonged action potentials and triggered automaticity due to reduced repolarization reserve and connexin 43 expression. Isoproterenol shortened cycle length, and escalated electrical instability. Surprisingly isoproterenol did not increase CaV1.2 current. We found alterations in CaV1.2-β1 adrenergic receptor colocalization assessed using super-resolution nanoscopy, and increased CaV1.2 phosphorylation in mutant hearts. Our results reveal for the first time that altered ion channel expression, co-localization and β-adrenergic receptor signaling associated with myocyte disarray contribute to electrical instability in the R403Q mutant heart.

A Common East Asian aldehyde dehydrogenase 2*2 variant promotes ventricular arrhythmia with chronic light-to-moderate alcohol use in mice

Chronic heavy alcohol use is associated with lethal arrhythmias. Whether common East Asian-specific aldehyde dehydrogenase deficiency (ALDH2*2) contributes to arrhythmogenesis caused by low level alcohol use remains unclear. Here we show 59 habitual alcohol users carrying ALDH2 rs671 have longer QT interval (corrected) and higher ventricular tachyarrhythmia events compared with 137 ALDH2 wild-type (Wt) habitual alcohol users and 57 alcohol non-users. Notably, we observe QT prolongation and a higher risk of premature ventricular contractions among human ALDH2 variants showing habitual light-to-moderate alcohol consumption. We recapitulate a human electrophysiological QT prolongation phenotype using a mouse ALDH2*2 knock-in (KI) model treated with 4% ethanol, which shows markedly reduced total amount of connexin43 albeit increased lateralization accompanied by markedly downregulated sarcolemmal Nav1.5, Kv1.4 and Kv4.2 expressions compared to EtOH-treated Wt mice. Whole-cell patch-clamps reveal a more pronounced action potential prolongation in EtOH-treated ALDH2*2 KI mice. By programmed electrical stimulation, rotors are only provokable in EtOH-treated ALDH2*2 KI mice along with higher number and duration of ventricular arrhythmia episodes. The present research helps formulate safe alcohol drinking guideline for ALDH2 deficient population and develop novel protective agents for these subjects.

The Junctophilin-2 Mutation p.(Thr161Lys) Is Associated with Hypertrophic Cardiomyopathy Using Patient-Specific iPS Cardiomyocytes and Demonstrates Prolonged Action Potential and Increased Arrhythmogenicity.

Hypertrophic cardiomyopathy (HCM) is one of the most common genetic cardiac diseases; it is primarily caused by mutations in sarcomeric genes. However, HCM is also associated with mutations in non-sarcomeric proteins and a Finnish founder mutation for HCM in non-sarcomeric protein junctophilin-2 (JPH2) has been identified. This study aimed at assessing the issue of modelling the rare Finnish founder mutation in cardiomyocytes (CMs) differentiated from iPSCs; therefore, presenting the same cardiac abnormalities observed in the patients. To explore the abnormal functions in JPH2-HCM, skin fibroblasts from a Finnish patient with JPH2 p.(Thr161Lys) were reprogrammed into iPSCs and further differentiated into CMs. As a control line, an isogenic counterpart was generated using the CRISPR/Cas9 genome editing method. Finally, iPSC-CMs were evaluated for the morphological and functional characteristics associated with JPH2 mutation. JPH2-hiPSC-CMs displayed key HCM hallmarks (cellular hypertrophy, multi-nucleation, sarcomeric disarray). Moreover, JPH2-hiPSC-CMs exhibit a higher degree of arrhythmia and longer action potential duration associated with slower inactivation of calcium channels. Functional evaluation supported clinical observations, with differences in beating characteristics when compared with isogenic-hiPSC-CMs. Thus, the iPSC-derived, disease-specific cardiomyocytes could serve as a translationally relevant platform to study genetic cardiac diseases.

A novel workflow for advanced mapping of repolarization dispersion from endocardial unipolar electrograms in patients with coved-type brugada syndrome

Abstract Funding Acknowledgements Type of funding sources: None. Background Accentuation of the action potential notch in the epicardium causes the prolongation of the epicardial action potentials in the right ventricular outflow tract (RVOT) and is the basis for arrhythmogenesis in patients affected by Brugada syndrome (BrS). Activation-recovery interval (ARI) approximates the action potential duration and may be used to study repolarization dispersion in BrS. Purpose Our aim was to investigate repolarization dispersion within the RVOT in spontaneous coved-type ECG BrS patients and in controls, evaluating also the relation between repolarization dispersion and conduction abnormalities to better define the electro-anatomical substrate of the disease. Methods BrS probands (n=13) and control subjects (n=4) underwent endocardial mapping of RV with the CARTO 3 system. BrS patients were also studied with programmed ventricular stimulation (PVS). Data was exported from the CARTO system and converted into MatLab format using OpenEP. A specific region of interest (ROI) including the sub-pulmonary RVOT and entire RV free wall was then selected using Paraview. With an automated algorithm, we measured ARI and ARIc (corrected using the "Bazett formula") for each point of the ROI, which were then interpolated to obtain ARI maps. We also acquired right ventricular activation time (RVAT) and maps for the assessment of slow conduction zones. By dividing RVAT color scale in 5 ms steps (isoSteps), isochronal activation areas were identified in the ROI. Results Out of the 13 BrS subjects, 4 had VT or VF inducible during PVS (PVS+) while 9 did not (PVS-). One patient had appropriate ICD shocks during follow-up. BrS patients had higher mean ARI and ARIc compared to controls (288.5±22.2 vs 251.5±8.4 ms, p&amp;lt;0.001; 312.0±30.4 vs 281.8±10.3 ms, p=0.023). ARI was found to be higher in PVS+ than in PVS- patients (294.8±27.1 vs 285.8±20.1 ms, p=0.5), as well as ARIc (327.7±49.9 vs 304.6±16.4 ms, p=0.49) although these differences were not significant. BrS patients showed a longer RVAT (106.0±21.4 vs 74.5±5.5 ms, p=0.003), and increased zones of crowding, as expressed by isochronal steps (13.85±2.94 vs 9.25±1.26, p=0.01). Notably, a strong correlation was found between ARIc and RVAT (Pearson R coefficient = 0.83, p&amp;lt;0.05), independently from the results of PVS (PVS+ R=0.99, p=0.01; PVS- R=0.83, p=0.06). Conclusions We introduced a novel workflow for the electrical substrate characterisation of subjects with BrS phenotype. Our preliminary data show the presence of repolarization dispersion along with conduction slowing in the RVOT of BrS patients compared to controls. In our population, repolarization dispersion and impairment of RVOT conduction were strongly related especially in inducible BrS patients.

Association between Systole, Diastole, Sex, Age, Blood, and Urine Profile in Older Adults with Hypertension

The main cardiovascular changes associated with aging included decreased heart rates, oxygen extraction, arterial stiffening, vasoconstriction, systolic blood pressure elevation, myocardial thickening, reduced diastolic filling rate, rhythmic rate changes, and action potential prolongation. Multifactorial risk can include the pathology of the diseased. This study aims to ascertain the relationship between systole, diastole, sex, age, blood, and urine profiles in an elderly with Hypertension. The logistic regression study found three significant associations between diastole systolic and leucocyte sedimentation with Hypertension. For the net study, various hormones are another pathological marker that can be included in hypertension screening

Ivabradine for controlling heart rate in permanent atrial fibrillation: A translational clinical trial

Pharmacological options for rate control in atrial fibrillation are scarce. Ivabradine was postulated to reduce the ventricular rate in this setting. The objectives of this study were to evaluate the mechanism of inhibition of atrioventricular conduction produced by ivabradine and to determine its efficacy and safety in atrial fibrillation. The effects of ivabradine on atrioventricular node and ventricular cells were studied by invitro whole-cell patch-clamp experiments and mathematical simulation of human action potentials. In parallel, a multicenter, randomized, open-label, phase III clinical trial compared ivabradine with digoxin for uncontrolled permanent atrial fibrillation despite β-blocker or calcium channel blocker treatment. Ivabradine 1 μM inhibited "funny" current and rapidly activating delayed rectifier potassium channel current by 28.9% and 22.8%, respectively (P < .05). The sodium channel current and L-type calcium channel current were reduced only at 10 μM. Ivabradine slowed the firing frequency of a modeled human atrioventricular node action potential by 10.6% and induced a minimal prolongation of ventricular action potential. Thirty-five (51.5%) patients were randomized to ivabradine and 33 (49.5%) to digoxin. The mean daytime heart rate decreased by 11.6 beats/min (-11.5%) in the ivabradine arm (P = .02) vs 19.6 (-20.6%) in the digoxin arm (P < .001), although the noninferiority margin of efficacy was not met (Z = -1.95; P = .97). The primary safety end point occurred in 3 patients (8.6%) on ivabradine and in 8 (24.2%) on digoxin (P = .10). Ivabradine produced a moderate rate reduction in patients with permanent atrial fibrillation. The inhibition of funny current in the atrioventricular node seems to be the main mechanism responsible for this reduction. Compared with digoxin, ivabradine was less effective, was better tolerated, and had a similar rate of serious adverse events.

Remodeled connexin 43 hemichannels alter cardiac excitability and promote arrhythmias.

Connexin-43 (Cx43) is the most abundant protein forming gap junction channels (GJCs) in cardiac ventricles. In multiple cardiac pathologies, including hypertrophy and heart failure, Cx43 is found remodeled at the lateral side of the intercalated discs of ventricular cardiomyocytes. Remodeling of Cx43 has been long linked to spontaneous ventricular arrhythmia, yet the mechanisms by which arrhythmias develop are still debated. Using a model of dystrophic cardiomyopathy, we previously showed that remodeled Cx43 function as aberrant hemichannels (non-forming GJCs) that alter cardiomyocyte excitability and, consequently, promote arrhythmias. Here, we aim to evaluate if opening of remodeled Cx43 can serve as a general mechanism to alter cardiac excitability independent of cellular dysfunction associated with a particular cardiomyopathy. To address this issue, we used a genetically modified Cx43 knock-in mouse (S3A) that promotes cardiac remodeling of Cx43 protein without apparent cardiac dysfunction. Importantly, when S3A mice were subjected to cardiac stress using the β-adrenergic agonist isoproterenol (Iso), they displayed acute and severe arrhythmias, which were not observed in WT mice. Pretreatment of S3A mice with the Cx43 hemichannel blocker, Gap19, prevented Iso-induced abnormal electrocardiographic behavior. At the cellular level, when compared with WT, Iso-treated S3A cardiomyocytes showed increased membrane permeability, greater plasma membrane depolarization, and Ca2+ overload, which likely caused prolonged action potentials, delayed after depolarizations, and triggered activity. All these cellular dysfunctions were also prevented by Cx43 hemichannel blockers. Our results support the notion that opening of remodeled Cx43 hemichannels, regardless of the type of cardiomyopathy, is sufficient to mediate cardiac-stress-induced arrhythmogenicity.

Optogenetic manipulation of cardiac repolarization gradients using sub-threshold illumination.

Introduction: Mechanisms underlying cardiac arrhythmias are typically driven by abnormalities in cardiac conduction and/or heterogeneities in repolarization time (RT) across the heart. While conduction slowing can be caused by either electrophysiological defects or physical blockade in cardiac tissue, RT heterogeneities are mainly related to action potential (AP) prolongation or abbreviation in specific areas of the heart. Importantly, the size of the area with altered RT and the difference between the short RT and long RT (RT gradient) have been identified as critical determinators of arrhythmogenicity. However, current experimental methods for manipulating RT gradient rely on the use of ion channel inhibitors, which lack spatial and temporal specificity and are commonly only partially reversible. Therefore, the conditions facilitating sustained arrhythmia upon the presence of RT heterogeneities and/or defects in cardiac conduction remain to be elucidated. Methods: We here employ an approach based on optogenetic stimulation in a low-intensity fashion (sub-threshold illumination), to selectively manipulate cardiac electrical activity in defined areas of the heart. Results: As previously described, subthreshold illumination is a robust tool able to prolong action potentials (AP), decrease upstroke velocity as well as slow cardiac conduction, in a fully reversible manner. By applying a patterned sub-threshold illumination in intact mouse hearts constitutively expressing the light-gated ion channel channelrhodopsin-2 (ChR2), we optically manipulate RT gradients and cardiac conduction across the heart in a spatially selective manner. Moreover, in a proof-of-concept assessment we found that in the presence of patterned sub-threshold illumination, mouse hearts were more susceptible to arrhythmias. Hence, this optogenetic-based approach may be able to mimic conduction slowing and RT heterogeneities present in pathophysiological conditions.

Multichannel modulation of depolarizing and repolarizing ion currents increases the positive rate‐dependent action potential prolongation

Prolongation of the action potential duration (APD) could prevent reentrant arrhythmias if prolongation occurs at the fast excitation rates of tachycardia with minimal prolongation at slow excitation rates (i.e., if prolongation is positive rate-dependent). APD prolongation by current anti-arrhythmic agents is either reverse (larger APD prolongation at slow rates than at fast rates) or neutral (similar APD prolongation at slow and fast rates), which may not result in an effective anti-arrhythmic action. In this report we show that, in computer models of the human ventricular action potential, the combined modulation of both depolarizing and repolarizing ion currents results in a stronger positive rate-dependent APD prolongation than modulation of repolarizing potassium currents. A robust positive rate-dependent APD prolongation correlates with an acceleration of phase 2 repolarization and a deceleration of phase 3 repolarization, which leads to a triangulation of the action potential. A positive rate-dependent APD prolongation decreases the repolarization reserve with respect to control, which can be managed by interventions that prolong APD at fast excitation rates and shorten APD at slow excitation rates. For both computer models of the action potential, ICaL and IK1 are the most important ion currents to achieve a positive rate-dependent APD prolongation. In conclusion, multichannel modulation of depolarizing and repolarizing ion currents, with ion channel activators and blockers, results in a robust APD prolongation at fast excitation rates, which should be anti-arrhythmic, while minimizing APD prolongation at slow heart rates, which should reduce pro-arrhythmic risks.

Patient-specific induced pluripotent stem cell properties implicate Ca2+-homeostasis in clinical arrhythmia associated with combined heterozygous RYR2 and SCN10A variants.

We illustrate use of induced pluripotent stem cells (iPSCs) as platforms for investigating cardiomyocyte phenotypes in a human family pedigree exemplified by novel heterozygous RYR2-A1855D and SCN10A-Q1362H variants occurring alone and in combination. The proband, a four-month-old boy, presented with polymorphic ventricular tachycardia. Genetic tests revealed double novel heterozygous RYR2-A1855D and SCN10A-Q1362H variants inherited from his father (F) and mother (M), respectively. His father showed ventricular premature beats; his mother was asymptomatic. Molecular biological characterizations demonstrated greater TNNT2 messenger RNA (mRNA) expression in the iPSCs-induced cardiomyocytes (iPS-CMs) than in the iPSCs. Cardiac troponin Ts became progressively organized but cytoplasmic RYR2 and SCN10A aggregations occurred in the iPS-CMs. Proband-specific iPS-CMs showed decreased RYR2 and SCN10A mRNA expression. The RYR2-A1855D variant resulted in premature spontaneous sarcoplasmic reticular Ca2+ transients, Ca2+ oscillations and increased action potential durations. SCN10A-Q1362H did not confer any specific phenotype. However, the combined heterozygous RYR2-A1855D and SCN10A-Q1362H variants in the proband iPS-CMs resulted in accentuated Ca2+ homeostasis disorders, action potential prolongation and susceptibility to early afterdepolarizations at high stimulus frequencies. These findings attribute the clinical phenotype in the proband to effects of the heterozygous RYR2 variant exacerbated by heterozygous SCN10A modification. This article is part of the theme issue 'The heartbeat: its molecular basis and physiological mechanisms'.

CE-452779-1 IVABRADINE FOR CONTROLLING HEART RATE IN PERMANENT ATRIAL FIBRILLATION: RESULTS OF THE TRANSLATIONAL CLINICAL TRIAL BRAKE-AF

Ivabradine has been postulated to reduce the ventricular rate in atrial fibrillation. However, the mechanisms responsible for that reduction and its performance as rate-control drug are not known To evaluate the mechanism of inhibition of atrioventricular conduction produced by ivabradine and to determine its efficacy and safety compared with digoxin in permanent atrial fibrillation The effects of ivabradine in atrioventricular nodal and ventricular cells were studied by in-vitro whole-cell patch-clamp experiments . Changes in If, INa, ICaL, ICaT, IKs, IKr, and IK1 conductances were integrated in mathematical simulations of nodal and ventricular action potentials. In parallel, a multicenter, randomized, open-label, phase III clinical trial compared ivabradine (5mg BID increased to 7.5mg BID after one month) versus digoxin (0.10-0.25mg QD adjusted by serum levels) for uncontrolled permanent atrial fibrillation despite beta-blockers or calcium channel blockers. Primary efficacy and safety endpoints were the reduction of the mean daytime heart rate at 3 months and a combination of syncope, clinically significant bradycardia or serious drug-related adverse events, respectively. The primary efficacy endpoint was evaluated by intention to treat using the O'Brien–Fleming method. Safety endpoints were evaluated with χ2 or Fisher’s exact tests Ivabradine 1 μM inhibited If and IKr by 28.9% and 22.8% (P<0.05) respectively. INa and ICaL were also reduced but only at 10 μM. Ivabradine slowed the firing frequency of a modelled human AV nodal action potential by 10.6% and induced a minimal prolongation of the ventricular action potential. The clinical trial randomized 35 patients to ivabradine and 33 to digoxin. Mean daytime heart rate decreased by 11.6 bpm (-11.5%) in the ivabradine arm (p=0.02) Vs 19.6 (-20.6%) in the digoxin arm (p<0.001) however, the non-inferiority margin of efficacy was not met (Z:-1.95; p=0.97). Primary safety endpoint occurred in 3 (8.6%) patients on ivabradine and in 8 (24.2%) on digoxin (p=0.10). Therapeutic adherence was lower in the digoxin arm (78.8% vs. 97.1%; p=0.02) Ivabradine produced a discrete heart rate reduction in patients with permanent atrial fibrillation. Compared with digoxin, ivabradine was less effective, better tolerated and had a similar rate of serious adverse events.The inhibition of If in the AV node seems the main mechanism responsible for the ivabradine-induced heart rate reduction in atrial fibrillation.

Feedback contributions to excitation-contraction coupling in native functioning striated muscle.

Skeletal and cardiac muscle excitation-contraction coupling commences with Nav1.4/Nav1.5-mediated, surface and transverse (T-) tubular, action potential generation. This initiates feedforward, allosteric or Ca2+-mediated, T-sarcoplasmic reticular (SR) junctional, voltage sensor-Cav1.1/Cav1.2 and ryanodine receptor-RyR1/RyR2 interaction. We review recent structural, physiological and translational studies on possible feedback actions of the resulting SR Ca2+ release on Nav1.4/Nav1.5 function in native muscle. Finite-element modelling predicted potentially regulatory T-SR junctional [Ca2+]TSR domains. Nav1.4/Nav1.5, III-IV linker and C-terminal domain structures included Ca2+ and/or calmodulin-binding sites whose mutations corresponded to specific clinical conditions. Loose-patch-clamped native murine skeletal muscle fibres and cardiomyocytes showed reduced Na+ currents (INa) following SR Ca2+ release induced by the Epac and direct RyR1/RyR2 activators, 8-(4-chlorophenylthio)adenosine-3',5'-cyclic monophosphate and caffeine, abrogated by the RyR inhibitor dantrolene. Conversely, dantrolene and the Ca2+-ATPase inhibitor cyclopiazonic acid increased INa. Experimental, catecholaminergic polymorphic ventricular tachycardic RyR2-P2328S and metabolically deficient Pgc1β-/- cardiomyocytes also showed reduced INa accompanying [Ca2+]i abnormalities rescued by dantrolene- and flecainide-mediated RyR block. Finally, hydroxychloroquine challenge implicated action potential (AP) prolongation in slowing AP conduction through modifying Ca2+ transients. The corresponding tissue/organ preparations each showed pro-arrhythmic, slowed AP upstrokes and conduction velocities. We finally extend discussion of possible Ca2+-mediated effects to further, Ca2+, K+ and Cl-, channel types. This article is part of the theme issue 'The heartbeat: its molecular basis and physiological mechanisms'.