Action Potential Duration Measurements Research Articles

Modulation of Cardiac Ryanodine Receptors by Chloride Intracellular Ion Channel 2

Pathogenic variants of the Chloride intracellular ion channel 2 (CLIC2) are implicated in hypertrophic cardiomyopathy (HCM), a diverse heart condition leading to arrhythmic sudden cardiac death and heart failure. CLIC2 is a unique class of metamorphic protein, related to the enzymatic glutathione s-transferase family and form functional ion channel. Artificial bilayer studies have shown that CLIC2 negatively modulates the activity of cardiac ryanodine receptor 2 (RyR2), implicating the sarcoplasmic/endoplasmic reticulum (SR/ER) Ca2+ release. However, the subsequent consequences of RyR2 and CLIC2 interaction in cardiac physiology remains unknown. Therefore, we ablated clic2 in human induced pluripotent stem cell (hiPSC) line using the CRISPR-Cas9 system and performed cardiomyocyte differentiation. clic2−/− hiPSC-derived cardiomyocytes (hiPSC-CMs) showed altered electrophysiological properties leading to impaired excitation-contraction coupling. clic2−/− hiPSC-CMs showed an increase in beat rate and field potential duration by 143.49±5.96% and 162.43±5.82%, respectively, whereas conduction velocity and spike amplitude decreased by 43.81±2.22% and 43.32±3.54%, respectively compared to WT hiPSC-CMs. Additionally, action potential duration measurements of clic2−/− hiPSC-CMs showed a decrease in APD30, APD50, and APD90 by 74.42%, 65.19%, and 66.46%, respectively compared to WT hiPSC-CMs. Moreover, clic2−/− hiPSC-CMs showed altered Ca2+ handling, with a decrease in peak amplitude (57.07±2.25%) and duration (38.29±1.30%) compared to the WT hiPSC-CMs. The caffeine induced Ca2+ release in clic2−/− hiPSC-CMs, attributed to the SR/ER Ca2+ overload which decreased by 72.19±3.51% compared to WT hiPSC-CMs. Transcriptomics analysis of clic2−/− hiPSC-CMs revealed 223 transcripts with significant changes in expression levels (89 upregulated and 134 downregulated), implicating CLIC2 in hypertrophy cardiomyopathy, dilated cardiomyopathy, and other heart diseases. Furthermore, the protein expression of CLIC2 increases in end-stage failing human heart samples compared to the non-failing heart. In conclusion, our study sheds light on the novel role of CLIC2 in the regulation of cardiac (patho)physiology. National Institutes of Health (NIH) (HL133050, and HL157453); American Heart Association-Transformational Project Award; National Center for advancing translational sciences (TR004178, understudied proteins and druggable targets) (HS). Presidential Predoctoral Fellowship; Margaret T. Nishikawara Merit Scholarship Endowment in Physiology; Graduate School’s Alumni Grants for Graduate Research and Scholarship Program, The Ohio State University (SS). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Contribution of cytokine-mediated prolongation of QTc interval to the multi-hit theory of Torsade de Pointes

BackgroundTorsade de pointes is a potentially lethal polymorphic ventricular tachyarrhythmia that can occur in the setting of long QT syndrome (LQTS). LQTS is multi-hit in nature and multiple factors combine their effects leading to increased arrhythmic risk. While hypokalemia and multiple medications are accounted for in LQTS, the arrhythmogenic role of systemic inflammation is increasingly recognized but often overlooked. We tested the hypothesis that the inflammatory cytokine interleukin(IL)-6 will significantly increase the incidence of arrhythmia when combined with other pro-arrhythmic conditions (hypokalemia and the psychotropic medication, quetiapine). MethodsGuinea pigs were injected intraperitoneally with IL-6/soluble IL-6 receptor and QT changes were measured in vivo. Subsequently, hearts were cannulated via Langendorff perfusion for ex vivo optical mapping measurements of action potential duration (APD90) and arrhythmia inducibility. Computer simulations (MATLAB) were performed to investigate IKr inhibition at varying IL-6 and quetiapine concentrations. ResultsIL-6 prolonged QTc in vivo guinea pigs from 306.74 ± 7.19 ms to 332.60 ± 8.75 ms (n = 8, p = .0021). Optical mapping on isolated hearts demonstrated APD prolongation in IL-6- vs saline groups (3Hz APD90:179.67 ± 2.47 ms vs 153.5 ± 7.86 ms, p = .0357). When hypokalemia was introduced, the APD90 increased to 195.8 ± 5.02 ms[IL-6] and 174.57 ± 10.7 ms[saline] (p = .2797), and when quetiapine was added to hypokalemia to 207.67 ± 3.03 ms[IL-6] and 191.37 ± 9.49 ms[saline] (p = .2449). After the addition of hypokalemia ± quetiapine, arrhythmia was induced in 75% of IL-6-treated hearts (n = 8), while in none of the control hearts (n = 6). Computer simulations demonstrated spontaneous depolarizations at ∼83% aggregate IKr inhibition. ConclusionsOur experimental observations strongly suggest that controlling inflammation, specifically IL-6, could be a viable and important route for reducing QT prolongation and arrhythmia incidence in the clinical setting.

Comparative analysis of the cardiomyocyte differentiation potential of induced pluripotent stem cells reprogrammed from human atrial or ventricular fibroblasts.

Background: We had shown that cardiomyocytes (CMs) were more efficiently differentiated from human induced pluripotent stem cells (hiPSCs) when the hiPSCs were reprogrammed from cardiac fibroblasts rather than dermal fibroblasts or blood mononuclear cells. Here, we continued to investigate the relationship between somatic-cell lineage and hiPSC-CM production by comparing the yield and functional properties of CMs differentiated from iPSCs reprogrammed from human atrial or ventricular cardiac fibroblasts (AiPSC or ViPSC, respectively). Methods: Atrial and ventricular heart tissues were obtained from the same patient, reprogrammed into AiPSCs or ViPSCs, and then differentiated into CMs (AiPSC-CMs or ViPSC-CMs, respectively) via established protocols. Results: The time-course of expression for pluripotency genes (OCT4, NANOG, and SOX2), the early mesodermal marker Brachyury, the cardiac mesodermal markers MESP1 and Gata4, and the cardiovascular progenitor-cell transcription factor NKX2.5 were broadly similar in AiPSC-CMs and ViPSC-CMs during the differentiation protocol. Flow-cytometry analyses of cardiac troponin T expression also indicated that purity of the two differentiated hiPSC-CM populations (AiPSC-CMs: 88.23% ± 4.69%, ViPSC-CMs: 90.25% ± 4.99%) was equivalent. While the field-potential durations were significantly longer in ViPSC-CMs than in AiPSC-CMs, measurements of action potential duration, beat period, spike amplitude, conduction velocity, and peak calcium-transient amplitude did not differ significantly between the two hiPSC-CM populations. Yet, our cardiac-origin iPSC-CM showed higher ADP and conduction velocity than previously reported iPSC-CM derived from non-cardiac tissues. Transcriptomic data comparing iPSC and iPSC-CMs showed similar gene expression profiles between AiPSC-CMs and ViPSC-CMs with significant differences when compared to iPSC-CM derived from other tissues. This analysis also pointed to several genes involved in electrophysiology processes responsible for the physiological differences observed between cardiac and non-cardiac-derived cardiomyocytes. Conclusion: AiPSC and ViPSC were differentiated into CMs with equal efficiency. Detected differences in electrophysiological properties, calcium handling activity, and transcription profiles between cardiac and non-cardiac derived cardiomyocytes demonstrated that 1) tissue of origin matters to generate a better-featured iPSC-CMs, 2) the sublocation within the cardiac tissue has marginal effects on the differentiation process.

PO-629-02 NEW EVIDENCE TO CHALLENGE CLINGEN'S “DISPUTED EVIDENCE” DESIGNATION FOR AKAP9 AS A BONA FIDE SUSCEPTIBILITY GENE FOR CONGENITAL LONG QT SYNDROME

A-kinase-anchoring protein-9 (AKAP9) mediates the phosphorylation state of the KCNQ1-encoded Kv7.1 potassium channel. Loss-of-function (LOF) variants in KCNQ1 cause type 1 long QT syndrome (LQT1). In 2007, we published data suggesting AKAP9-S1570L as a novel LQTS-causative variant and AKAP9 as a novel LQTS-susceptibility gene. However, in a recent re-appraisal of LQTS genes, the Clinical Genome Resource (ClinGen) demoted AKAP9 to a “disputed evidence” gene due to apparent lack of sufficient evidence to support causation of LQTS. To characterize the electrophysiology of a LQTS patient-derived induced pluripotent stem cell-cardiomyocyte (iPSC-CM) AKAP9-S1570L model and its corresponding CRISPR/Cas9 gene-edited/variant-corrected isogenic control (IC). iPSC-CMs were generated from an AKAP9-S1570L positive female diagnosed with LQTS at 14 years of age with a QTc > 480 ms, history of syncope, a LQT1-suggestive treadmill stress test, and a positive family history of QT-prolongation. Standard whole-cell patch clamp technique was performed to measure the action potential duration (APD) of both the AKAP9-S1570L and IC iPSC-CMs. To determine if KCNQ1 contributes to the APD prolongation of the variant iPSC-CMs, the KCNQ1 channel specific activator, ML277 was used in additional APD measurements. APD50 (457 ± 21) and APD90 (549 ± 22 ms) were significantly prolonged in the AKAP9-S1570L iPSC-CMs compared to the IC iPSC-CMs (APD50, 335 ± 15 ms; APD90, 413 ± 16 ms; p<0.05). Addition of ML277 resulted in a greater degree of APD shortening in AKAP9-S1570L iPSC-CMs (APD50 shortening, 349 ± 40 ms (p<0.05); APD90 shortening, 397 ± 42 ms (p<0.05) than in IC iPSC-CMs (APD50 shortening, 219 ± 31 ms; APD90 shortening, 242 ± 33 ms). This indicates KCNQ1 LOF may contribute to the mechanism underlying APD prolongation in the patient-specific AKAP9-S1570L iPSC-CMs, consistent with our previously published data. Using patient specific iPSC-CMs and isogenic controls, we provide new evidence bolstering AKAP9-S1570L as the pathogenic LQTS-causative variant responsible for this patient’s LQTS. Whether ClinGen deems this sufficient evidence to rescue AKAP9 from genetic purgatory and re-classify it as a sufficient evidence LQTS-susceptibility gene, remains to be seen.

Combination of Sildenafil and Ba2+ at a Low Concentration Show a Significant Synergistic Inhibition of Inward Rectifier Potassium Current Resulting in Action Potential Prolongation.

Sildenafil (Viagra) is a vasodilator mainly used in the treatment of erectile dysfunction. Atrial or ventricular fibrillation may rarely occur as a side effect during sildenafil therapy. Although changes in inward rectifier potassium currents including I K1 are known to contribute to the pathogenesis of fibrillation, the effect of sildenafil on I K1 has not been studied. In experiments, Ba2+ is used as a specific inhibitor of I K1 at high concentrations (usually 100 µM). Being an environmental contaminant, it is also present in the human body; Ba2+ plasmatic concentrations up to 1.5 µM are usually reported in the general population. This study was primarily aimed to investigate changes of I K1 induced by sildenafil in a wide range of concentrations (0.1–100 µM). Additionally, the effect of combination of sildenafil and Ba2+ at selected clinically-relevant concentrations was tested, at 0.1 µM both on I K1 and on the action potential duration (APD). Experiments were performed by the whole-cell patch-clamp technique on enzymatically isolated rat ventricular cardiomyocytes, mostly at 23°C with the exception of APD measurements which were performed at 37°C as well. Sildenafil caused a significant, reversible, and concentration-dependent inhibition of I K1 that did not differ at −50 and −110 mV. Simultaneous application of sildenafil and Ba2+ at 0.1 µM revealed a massive inhibition of both inward and outward components of I K1 (this synergy was missing at other tested combinations). The combined effect at 0.1 µM (45.7 ± 5.7 and 43.0 ± 6.9% inhibition at −50 and −110 mV, respectively) was significantly higher than a simple sum of almost negligible effects of the individual substances and it led to a significant prolongation of APD at both 23 and 37°C. To our knowledge, similar potentiation of the drug-channel interaction has not been described. The observed massive inhibition of I K1 induced by a combined action of the vasodilator sildenafil and environmental contaminant Ba2+ at a low concentration and resulting in a significant APD prolongation may contribute to the genesis of arrhythmias observed in some patients treated with sildenafil.

Isolated superfused rats atrial model for the investigation of atrial fibrillation mechanisms and treatment

Abstract Background One of the major barriers to an improved mechanistic understanding of atrial fibrillation (AF), and thus in the pipeline of drug development, has been a lack of appropriate tissue models, especially in small animals. Aim We propose an advanced anatomical ex-vivo model based on rat atria for acute assessment of AF susceptibility. This novel model could yield a better understanding of arrhythmia mechanisms as well as the development of potential therapeutic strategies for the prevention or termination of atrial arrhythmias. Methods Wistar rats atria (N=25) were isolated, flattened and pinned to a custom-made silicon plate. Atria were superfused with an oxygenized Tyrode's solution. Tissues were then loaded with a voltage-sensitive dye and mapped using a high-resolution optical mapping system. AF was induced with 1uM carbamylcholine (N=23) coupled with pacing maneuvers and treated with 30uM Vernakalant (N=10) or 10uM Flecainide (N=10). Finally, the feasibility of a new ablation technique (electroporation) was evaluated. Results Optical mapping results suggested that the superfusion procedure led to a fast atrial recovery. Sinus activity was conserved for all atria for a long period. All the anatomical landmarks were clearly visualized. The acquired optical signals were analyzed during sinus rhythm and pacing, which allowed the creation of detailed activation maps and measurements of action potential duration (APD) and conduction velocity (CV) at different pacing rates. The resulting APD restitution curves revealed electrical excitation at high pacing rates (cycle length between 50ms and 300ms) with a relatively flattened curve. AF was successfully induced and optically mapping confirmed the presence of reentrant activity. AF was successfully treated using Vernacalant and Flecainide. Finally, we demonstrated the feasibility of a new ablation approach (electroporation) for creation of a continuous linear lesion serving as a functional block. Conclusion The isolated superfused atria model, coupled with voltage-sensitive dyes, can be utilized for long-term high-resolution functional imaging of the atria during sinus rhythm, pacing and arrhythmogenic activity. This allows the study of the atrial electrophysiological properties, the mechanisms involved in AF initiation, perpetuation, and termination as well as the study of drug and new ablation modalities. Funding Acknowledgement Type of funding sources: Public grant(s) – EU funding. Main funding source(s): European Research Council (ERC) Spontaneous activation of isolated atria

Global vs local control of cardiac alternans in a 1D numerical model of human ventricular tissue.

Cardiac alternans is a proarrhythmic state in which the action potential duration (APD) of cardiac myocytes alternate between long and short values and often occurs under conditions of rapid pacing of cardiac tissue. In the ventricles, alternans is especially dangerous due to the life-threatening risk of developing arrhythmias, such as ventricular fibrillation. Alternans can be formed in periodically paced tissue as a result of pacing itself. Recently, it has been demonstrated that this pacing-induced alternans can be prevented by performing constant diastolic interval (DI) pacing, in which DI is independent of APD. However, constant DI pacing is difficult to implement in experimental settings since it requires the real-time measurement of APD. A more practical way was proposed based on electrocardiograms (ECGs), which give an indirect measure of the global DI relaxation period through the TR interval assessment. Previously, we demonstrated that constant TR pacing prevented alternans formation in isolated Langendorff-perfused rabbit hearts. However, the efficacy of "local" constant DI pacing vs "global" constant TR pacing in preventing alternans formation has never been investigated. Thus, the purpose of this study was to implement an ECG-based constant TR pacing in a 1D numerical model of human ventricular tissue and to compare the dynamical behavior of cardiac tissue with that resulted from a constant DI pacing. The results showed that both constant TR and constant DI pacing prevented the onset of alternans until lower basic cycle length when compared to periodic pacing. For longer cable lengths, constant TR pacing was shown to exhibit greater control on alternans than constant DI pacing.

Clinical Trial in a Dish: Personalized Stem Cell–Derived Cardiomyocyte Assay Compared With Clinical Trial Results for Two QT‐Prolonging Drugs

Induced pluripotent stem cells (iPSCs) have shown promise in investigating donor‐specific phenotypes and pathologies. The iPSC‐derived cardiomyocytes (iPSC‐CMs) could potentially be utilized in personalized cardiotoxicity studies, assessing individual proarrhythmic risk. However, it is unclear how closely iPSC‐CMs derived from healthy subjects can recapitulate a range of responses to drugs. It is well known that QT‐prolonging drugs induce subject‐specific clinical response and that all healthy subjects do not necessarily develop arrhythmias or exhibit similar amounts of QT prolongation. We previously reported this variability in a study of four human ether‐a‐go‐go‐related gene (hERG) potassium channel–blocking drugs in which each subject underwent intensive pharmacokinetic and pharmacodynamic sampling such that subjects had 15 time‐matched plasma drug concentration and electrocardiogram measurements throughout 24 hours after dosing in a phase I clinical research unit. In this study, iPSC‐CMs were generated from those subjects. Their drug‐concentration‐dependent QT prolongation response from the clinic was compared with in vitro drug‐concentration‐dependent action potential duration (APD) prolongation response to the same two hERG‐blocking drugs, dofetilide and moxifloxacin. Comparative results showed no significant correlation between the subject‐specific APD response slopes and clinical QT response slopes to either moxifloxacin (P = 0.75) or dofetilide (P = 0.69). Similarly, no significant correlation was found between baseline QT and baseline APD measurements (P = 0.93). This result advances our current understanding of subject‐specific iPSC‐CMs and facilitates discussion into factors obscuring correlation and considerations for future studies of subject‐specific phenotypes in iPSC‐CMs.

Detachable glass microelectrodes for recording action potentials in active moving organs.

Here, we describe new detachable floating glass micropipette electrode devices that provide targeted action potential recordings in active moving organs without requiring constant mechanical constraint or pharmacological inhibition of tissue motion. The technology is based on the concept of a glass micropipette electrode that is held firmly during cell targeting and intracellular insertion, after which a 100-µg glass microelectrode, a "microdevice," is gently released to remain within the moving organ. The microdevices provide long-term recordings of action potentials, even during millimeter-scale movement of tissue in which the device is embedded. We demonstrate two different glass micropipette electrode holding and detachment designs appropriate for the heart (sharp glass microdevices for cardiac myocytes in rats, guinea pigs, and humans) and the brain (patch glass microdevices for neurons in rats). We explain how microdevices enable measurements of multiple cells within a moving organ that are typically difficult with other technologies. Using sharp microdevices, action potential duration was monitored continuously for 15 min in unconstrained perfused hearts during global ischemia-reperfusion, providing beat-to-beat measurements of changes in action potential duration. Action potentials from neurons in the hippocampus of anesthetized rats were measured with patch microdevices, which provided stable base potentials during long-term recordings. Our results demonstrate that detachable microdevices are an elegant and robust tool to record electrical activity with high temporal resolution and cellular level localization without disturbing the physiological working conditions of the organ.NEW & NOTEWORTHY Cellular action potential measurements within tissue using glass micropipette electrodes usually require tissue immobilization, potentially influencing the physiological relevance of the measurement. Here, we addressed this limitation with novel 100-µg detachable glass microelectrodes that can be precisely positioned to provide long-term measurements of action potential duration during unconstrained tissue movement.

Early afterdepolarisation tendency as a simulated pro-arrhythmic risk indicator.

Drug-induced Torsades de Pointes (TdP) arrhythmia is of major interest in predictive toxicology. Drugs which cause TdP block the hERG cardiac potassium channel. However, not all drugs that block hERG cause TdP. As such, further understanding of the mechanistic route to TdP is needed. Early afterdepolarisations (EADs) are a cell-level phenomenon in which the membrane of a cardiac cell depolarises a second time before repolarisation, and EADs are seen in hearts during TdP. Therefore, we propose a method of predicting TdP using induced EADs combined with multiple ion channel block in simulations using biophysically-based mathematical models of human ventricular cell electrophysiology. EADs were induced in cardiac action potential models using interventions based on diseases that are known to cause EADs, including: increasing the conduction of the L-type calcium channel, decreasing the conduction of the hERG channel, and shifting the inactivation curve of the fast sodium channel. The threshold of intervention that was required to cause an EAD was used to classify drugs into clinical risk categories. The metric that used L-type calcium induced EADs was the most accurate of the EAD metrics at classifying drugs into the correct risk categories, and increased in accuracy when combined with action potential duration measurements. The EAD metrics were all more accurate than hERG block alone, but not as predictive as simpler measures such as simulated action potential duration. This may be because different routes to EADs represent risk well for different patient subgroups, something that is difficult to assess at present.

Refractoriness in human atria: Time and voltage dependence of sodium channel availability

BackgroundRefractoriness of cardiac cells limits maximum frequency of electrical activity and protects the heart from tonic contractions. Short refractory periods support major arrhythmogenic substrates and augmentation of refractoriness is therefore seen as a main mechanism of antiarrhythmic drugs. Cardiomyocyte excitability depends on availability of sodium channels, which involves both time- and voltage-dependent recovery from inactivation. This study therefore aims to characterise how sodium channel inactivation affects refractoriness in human atria. Methods and resultsSteady-state activation and inactivation parameters of sodium channels measured in vitro in isolated human atrial cardiomyocytes were used to parameterise a mathematical human atrial cell model. Action potential data were acquired from human atrial trabeculae of patients in either sinus rhythm or chronic atrial fibrillation. The ex vivo measurements of action potential duration, effective refractory period and resting membrane potential were well-replicated in simulations using this new in silico model. Notably, the voltage threshold potential at which refractoriness was observed was not different between sinus rhythm and chronic atrial fibrillation tissues and was neither affected by changes in frequency (1 vs. 3Hz). ConclusionsOur results suggest a preferentially voltage-dependent, rather than time-dependent, effect with respect to refractoriness at physiologically relevant rates in human atria. However, as the resting membrane potential is hyperpolarized in chronic atrial fibrillation, the voltage-dependence of excitability dominates, profoundly increasing the risk for arrhythmia re-initiation and maintenance in fibrillating atria. Our results thereby highlight resting membrane potential as a potential target in pharmacological management of chronic atrial fibrillation.

Coupling of ventricular action potential duration and local strain patterns during reverse remodeling in responders and nonresponders to cardiac resynchronization therapy

The high risk of ventricular arrhythmias in patients with heart failure remains despite the benefit of cardiac resynchronization therapy (CRT). An electromechanical interaction between regional myocardial strain patterns and the electrophysiological substrate is thought to be important. We investigated the in vivo relation between left ventricular activation recovery interval (ARI), as a surrogate measure of action potential duration (APD), and local myocardial strain patterns in responders and nonresponders to CRT. ARIs were recorded from the left ventricular epicardium in 20 patients with CRT 6 weeks and 6 months post implantation. Two-dimensional speckle tracking echocardiography was performed at the same time to assess myocardial strains. Patients with ≥15% reduction in end-systolic volume at 6 months were classified as responders. ARI decreased in responders (263 ± 46 ms vs 246 ± 47 ms, P < .01) and increased in nonresponders (235 ± 23 ms vs 261 ± 20 ms; P < .01). Time-to-peak radial, circumferential, and longitudinal strains increased in responders (41 ± 27, 35 ± 25, 56 ± 37 ms; P < .01) and decreased in nonresponders (-58 ± 26, -47 ± 26, -64 ± 27 ms; P < .01). There was a nonlinear correlation between changes in time-to-peak strain and ARIs (Spearman correlation coefficient r ≥ 0.70; P < .01). Baseline QRS duration >145 ms and QRS duration shortening with biventricular pacing were associated with ARI shortening following CRT. Changes in ventricular wall mechanics predict local APD lengthening or shortening during CRT. Nonresponders have a worsening of myocardial strain and local APD. Baseline QRS duration >145 ms and QRS duration shortening with biventricular pacing identified patients who exhibited improvement in APD.

Abstract 20168: Enhanced Endothelial Nitric Oxide Production and Cardiac Dysfunction in an Inducible Endothelial-Specific miR-130a Mouse Model

Introduction: MicroRNA-130a (miR130a) has been shown to be important in cardiovascular gene regulation in both normal and diseased states. We recently reported on the regulation of connexin43 by miR130a. Methods: We created an inducible mouse model to express miR-130a in endothelial cells under the control of the VE-cadherein promoter (VE-cad-miR130a). Overexpression of miR-130a was initiated in adult mice at weaning, approximately 4 weeks post-birth. Results: Survival analysis revealed &gt;50% mortality by 9 weeks of age with overexpression of miR-130a. By 9 weeks of age, LV function was reduced (26.4% ± 4 vs 33.6% ± 6 in controls, p&lt;0.001, n=10). Prior to the onset of LV dysfunction; we found numerous abnormalities in cardiac function. First, we noted significant bradycardia on ambulatory telemetric recordings (389bpm ± 90 vs 534bpm ± 72 in controls, p&lt;0.0001, n=3). Moreover, the cardiac response to β-AR stimulation was blunted in VE-Cad-miR130a mice relative to control when challenged with isoproterenol. Interestingly, sinus node recovery time was significantly prolonged, supportive of sinus node dysfunction. While predominately in sinus rhythm, we found episodes of spontaneous atrial tachyarrhythmias, which were confirmed on invasive electrophysiologic testing. To investigate further, we performed patch clamp measurements of action potential duration (APD) in atrial cariomyocytes. APD was significantly prolonged in VE-Cad-miR130a myocytes (APD90 325ms vs 60ms, p&lt;0.00001, n=3). Next, we performed invasive blood measure measurements and found a significant decrease in mean arterial blood pressure (76.9mmHg ± 3 vs 94.3mmHg ± 5 in controls, p&lt;0.01, n=4). Based on previous reports of increased nitric oxide in the setting of loss of endothelial connexin43, we found significant elevation of NO concentration in VE-Cad-miR130a. Inhibition of NO synthase via IP administration of L-NAME increased MAP in VE-Cad-miR130a to a maximum steady-state mimicking that of control mice. Conclusions: Overexpression of miR-130a in endothelial cells promotes impaired cardiovascular function and abnormal atrial conduction. These results suggest a newly identified role for endothelial miR-130a in the development of hypotension and cardiac dysrhythmias.

Oscillatory behavior of ventricular action potential duration in heart failure patients at respiratory rate and low frequency.

Oscillations of arterial pressure occur spontaneously at a frequency of approximately 0.1 Hz coupled with synchronous oscillations of sympathetic nerve activity (“Mayer waves”). This study investigated the extent to which corresponding oscillations may occur in ventricular action potential duration (APD). Fourteen ambulatory (outpatient) heart failure patients with biventricular pacing devices were studied while seated upright watching movie clips to maintain arousal. Activation recovery intervals (ARI) as a measure of ventricular APD were obtained from unipolar electrograms recorded from the LV epicardial pacing lead during steady state RV pacing from the device. Arterial blood pressure was measured non-invasively (Finapress) and respiration monitored. Oscillations were quantified using time frequency and coherence analysis. Oscillatory behavior of ARI at the respiratory frequency was observed in all subjects. The magnitude of the ARI variation ranged from 2.2 to 6.9 ms (mean 5.0 ms). Coherence analysis showed a correlation with respiratory oscillation for an average of 43% of the recording time at a significance level of p < 0.05. Oscillations in systolic blood pressure in the Mayer wave frequency range were observed in all subjects for whom blood pressure was recorded (n = 13). ARI oscillation in the Mayer wave frequency range was observed in 6/13 subjects (46%) over a range of 2.9 to 9.2 ms. Coherence with Mayer waves at the p < 0.05 significance level was present for an average of 29% of the recording time. In ambulatory patients with heart failure during enhanced mental arousal, left ventricular epicardial APD (ARI) oscillated at the respiratory frequency (approximately 0.25 Hz). In 6 patients (46%) APD oscillated at the slower Mayer wave frequency (approximately 0.1 Hz). These findings may be important in understanding sympathetic activity-related arrhythmogenesis.

P134Increased beta-adrenergic inotropy in ventricular myocardium from Trpm4 knockout mice

TRPM4 have an extensive role in regulating intracellular calcium dependent cell functions in many cells including mast cells, chromaffin cells and recently in neurons. TRPM4 proteins constitute calcium activated, but calcium impermeable, non-selective cation channels and are expressed both in atrial and ventricular cardiomyocytes. The Trpm4 gene has recently been associated with several disorders, including cardiac conduction diseases such as Progressive Familial Heart Block Type 1, Right Bundle Branch Block and Brugada Syndrome. The physiological function of TRPM4 in the heart remains however incompletely understood. The objective of this study was to establish the role of TRPM4 in left ventricular muscle function. We used TRPM4 knockout mice and performed patch-clamp experiments, membrane potential measurements, microfluorometry, contractility measurements and in vivo pressure-volume loop analysis. We demonstrate that TRPM4 proteins are functionally present in mouse ventricular myocytes and are activated upon calcium induced calcium release. Measurements of action potential duration show that TRPM4 is active during the repolarization of the ventricular action potential. We show a significantly decreased time for 50 % and 90 % repolarization in Trpm4 knockout ventricular myocytes. We also provide evidence that this change in action potential shape leads to an increased driving force for the L-type calcium current during the action potential. Moreover, in vivo pressure-volume analysis and in vitro contraction strength measurement on isolated papillary muscles displays an increased β-adrenergic inotropic response in TRPM4 knockout mice. Taking together these data, our results show that functional TRPM4 proteins are novel determinants of the inotropic effect of β-adrenergic stimulation on the ventricular heart muscle.

Increased β-Adrenergic Inotropy in Ventricular Myocardium From Trpm4 −/− Mice

The Trpm4 gene has recently been associated with several disorders, including cardiac conduction diseases and Brugada syndrome. Transient receptor potential member 4 (TRPM4) proteins constitute Ca2+ -activated, but Ca2+ -impermeable, nonselective cation channels and are expressed both in atrial and in ventricular cardiomyocytes. The physiological function of TRPM4 in the heart remains, however, incompletely understood. To establish the role of TRPM4 in cardiac muscle function. We used TRPM4 knockout mice and performed patch-clamp experiments, membrane potential measurements, microfluorometry, contractility measurements, and in vivo pressure-volume loop analysis. We demonstrate that TRPM4 proteins are functionally present in mouse ventricular myocytes and are activated on Ca2+ -induced Ca2+ release. In Trpm4(-/-) mice, cardiac muscle displays an increased β-adrenergic inotropic response both in vitro and in vivo. Measurements of action potential duration show a significantly decreased time for 50% and 90% repolarization in Trpm4(-/-) ventricular myocytes. We provide evidence that this change in action potential shape leads to an increased driving force for the L-type Ca2+ current during the action potential, which explains the altered contractility of the heart muscle. Our results show that functional TRPM4 proteins are novel determinants of the inotropic effect of β-adrenergic stimulation on the ventricular heart muscle.

Increased Nav 1.5 channel late current underlies prolongation of action potential duration of ventricular myocytes from aged guinea pigs

<h3>Background</h3> The action potential duration (APD) of aged hearts is prolonged. Because the late Na⁺ current (I_NaL) has been shown to play a significant role in modulating cardiac APD, we sought to determine whether the age-related prolongation of APD is due to an increase in endogenous I_NaL. <h3>Methods</h3> Experiments were performed on ventricular myocytes isolated from the hearts of young (one-month old) or aged (one-year old) guinea pigs. The APD and the amplitude of I_NaL were determined using the whole-cell patch-electrode technique at 36 ^oC. The APD was measured at 50% repolarization (APD₅₀). I_NaL was activated by 250-ms voltage-clamp pulses from -90 to -30 mV under conditions wherein K⁺ and Ca²⁺ currents were inhibited. The amplitude of I_NaL was calculated as the average current during the last 100-ms of the voltage clamp. <h3>Results</h3> The APD₅₀ of myocytes from aged hearts (323±9 ms, n=29) was significantly longer than those from young hearts (229±6 ms, n=43, p < 0.001). The selective I_NaL inhibitor GS967 (0.01 - 1 µmol/L, n=7) and the Na⁺ channel blocker tetrodotoxin (0.1 - 10 µmol/L, n=9) shortened the APD₅₀ of myocytes from aged hearts by 24±4 to 44±4% and by 32±4 to 45±4%, respectively (p < 0.001). Consistent with the results of APD measurements, the amplitude of I_NaL of aged myocytes (-0.51±0.05 pA/pF, n=17) was significantly greater than that of young myocytes (-0.22±0.02 pA/pF, n=27, p < 0.001). MTSEA (methanethiosulfonate ethylammonium, 2 mmol/L), a Na_v1.5 channel blocker, reduced the I_NaL of young and aged myocytes by 94±3% (n = 10) and 96±1% (n = 8), respectively, suggesting that the Na_v 1.5 channel is the major contributor to I_NaL of both young and aged guinea pig ventricular myocytes. <h3>Conclusion</h3> The present results suggest that 1) an increase in endogenous I_NaL is responsible for the prolongation of APD in aged guinea pig ventricular myocytes, and 2) Na_v 1.5 channels are the major contributor to the increased I_NaL.

Mechanisms of Steeper APD Restitution in Rat Failing Right Ventricular Myocytes

Pulmonary artery hypertension (PAH) causes the right ventricle (RV) to become hypertrophied and fail. During this process the RV undergoes electrophysiological remodeling with resultant changes in action potential duration (APD). This study evaluated changes in APD restitution in a rodent model of PAH.Male Wistar rats were given a single i.p. injection of monocrotaline (60 mg/kg) or an equivalent volume of saline. When clinical symptoms of heart failure became apparent (3-4 weeks later) animals were euthanized and the hearts excised. RV myocytes were enzymatically isolated and used for a number of electrophysiological measurements. These included: measurements of APD at pacing rates between 1 and 9 Hz; an action potential (AP) clamp to impose the AP recorded at 1 Hz at pacing rates of 1, 2, 5 and 7 Hz; and measurements of APD during application of increased negative current pulses between 1 and 500 pA in amplitude at 1 and 5 Hz.Myocytes from the failing right ventricle had a significantly longer APD and steeper APD steeper restitution curve compared to sham cells. Despite this greater APD shortening, under action potential clamp, compensation currents in failing myocytes were smaller in amplitude as pacing frequency increased. Consistent with this observation, injection of negative current caused a greater decrease in APD90 in failing cells (P < 0.0001-0.05, n = 6-7 myocytes, ANOVA, Tukeys post-test).These findings show that cells isolated from the monocrotaline treated RV required less current to cause a decrease in APD, which is consistent with an increased membrane resistance. Previous studies have reported a decreased expression of potassium channels in monocrotaline-treated myocytes, which may be a contributing factor responsible for the results observed.Supported by the MRC.

Sympathetic innervation of the anterior left ventricular wall by the right and left stellate ganglia

The sympathetic nervous system is thought to play a role in the genesis of ventricular tachyarrhythmias (VT). Left and added right cardiac sympathectomy have been shown to reduce the burden of arrhythmias in the setting of a VT storm. However, the contribution of the right stellate ganglion (RSG) and the left stellate ganglion (LSG) to the innervation of the anterior left ventricular (LV) wall is not well understood. To evaluate the innervation of the anterior LV wall by the LSG and the RSG. The heart and stellate ganglia were exposed via sternotomy in pigs with normal hearts (n = 8). A 20-electrode catheter was placed on the anterior LV wall to record activation recovery interval (ARI), a surrogate measure of action potential duration. A microdialysis catheter was inserted in a similar location to sample interstitial norepinephrine (NE) content. ARI and NE measurements were recorded at baseline and during LSG and RSG stimulation. LSG stimulation shortened ARI by 17.1% ± 10.5% (mean ± standard error), while RSG stimulation shortened ARI by 42.1% ± 15.7%, P = .04 (LSG vs RSG). LSG stimulation increased interstitial NE levels by 200% ± 65%, while RSG stimulation increased the NE content by 260% ± 40% (P = .012). LSG stimulation increased dispersion in ARI from 376.0 ± 83.7 ms(2) to 1242.5 ± 566 ms(2) (P = .03) and caused ventricular fibrillation in 2 pigs. During RSG stimulation, dispersion increased from 419 ± 65.8 to 474.8 ± 81 ms(2) (P = .4). Both the LSG and the RSG provide significant innervation to the anterior LV wall as demonstrated by both ARI shortening and NE concentrations. LSG stimulation significantly increases ARI dispersion. This study provides mechanistic insight into the beneficial effects of left sympathectomy and the additional role of right sympathectomy in reducing arrhythmias in patients with anterior myocardial scars and VT storm.

159 Pilot Study exploring the regional repolarisation instability index in relation to myocardial heterogeneity and prediction of ventricular arrhythmia and death

<h3>Introduction</h3> There is a need for better sudden cardiac death (SCD) risk markers. Mounting evidence suggests that the mechanism underlying risk of ventricular arrhythmia (VA) is increased heterogeneity of electrical restitution. We investigated a novel measure of action potential duration (APD) restitution heterogeneity: the Regional Repolarisation Instability Index (R2I2) and correlated it with peri-infarct zone (PIZ) a cardiac magnetic resonance (CMR) anatomic marker of VA risk. <h3>Methods</h3> Blinded retrospective study of 30 patients with ischaemic cardiomyopathy assessed for an implantable cardioverter defibrillator. The R2I2 was derived from high resolution 12 lead ECG recorded during programmed electrical stimulation (PES). ECG surrogates were used to plot APD as a function of diastolic interval; the R2I2 was the maximal value of the mean squared residuals of the mean points for anterior, inferior and lateral leads normalised to the mean value for the total population. PIZ was measured from late gadolinium enhanced CMR images using the full width half maximum technique. <h3>Results</h3> Seven patients reached the endpoint of VA/death (median follow-up 24 months). R2I2 &gt; median was found to be predictive of VA/death independent of PES result, left ventricular ejection fraction and QRS duration (6/14 vs 1/15 p=0.031). Modest correlation was seen between the R2I2 and PIZ (r=0.41 p=0.057) (Abstract 159 figure 1). <h3>Conclusions</h3> In this pilot study of ischaemic cardiomyopathy patients, the R2I2 was shown to be an electrical measure of VA/death risk with a moderately strong correlation with an anatomic measure of arrhythmic substrate, the extent of PIZ. The R2I2 may add value to existing markers of VA/death and merits further investigation.