Optical Action Potentials Research Articles

Beneficial Effect of Calcium Treatment for Hyperkalemia Is Not Due to "Membrane Stabilization".

Hyperkalemia is a common life-threatening condition causing severe electrophysiologic derangements and arrhythmias. The beneficial effects of calcium (Ca 2+ ) treatment for hyperkalemia have been attributed to "membrane stabilization," by restoration of resting membrane potential (RMP). However, the underlying mechanisms remain poorly understood. Our objective was to investigate the mechanisms underlying adverse electrophysiologic effects of hyperkalemia and the therapeutic effects of Ca 2+ treatment. Controlled experimental trial. Laboratory investigation. Canine myocytes and tissue preparations. Optical action potentials and volume averaged electrocardiograms were recorded from the transmural wall of ventricular wedge preparations ( n = 7) at baseline (4 mM potassium), hyperkalemia (8-12 mM), and hyperkalemia + Ca 2+ (3.6 mM). Isolated myocytes were studied during hyperkalemia (8 mM) and after Ca 2+ treatment (6 mM) to determine cellular RMP. Hyperkalemia markedly slowed conduction velocity (CV, by 67% ± 7%; p < 0.001) and homogeneously shortened action potential duration (APD, by 20% ± 10%; p < 0.002). In all preparations, this resulted in QRS widening and the "sine wave" pattern observed in severe hyperkalemia. Ca 2+ treatment restored CV (increase by 44% ± 18%; p < 0.02), resulting in narrowing of the QRS and normalization of the electrocardiogram, but did not restore APD. RMP was significantly elevated by hyperkalemia; however, it was not restored with Ca 2+ treatment suggesting a mechanism unrelated to "membrane stabilization." In addition, the effect of Ca 2+ was attenuated during L-type Ca 2+ channel blockade, suggesting a mechanism related to Ca 2+ -dependent (rather than normally sodium-dependent) conduction. These data suggest that Ca 2+ treatment for hyperkalemia restores conduction through Ca 2+ -dependent propagation, rather than restoration of membrane potential or "membrane stabilization." Our findings provide a mechanistic rationale for Ca 2+ treatment when hyperkalemia produces abnormalities of conduction (i.e., QRS prolongation).

Quantitative Assessment of Mitochondrial Morphology and Electrophysiological Function in the Diabetic Heart.

Mitochondria within a cardiomyocyte form a highly dynamic network that undergoes fusion and fission events in response to acute and chronic stressors, such as hyperglycemia and diabetes mellitus. Changes in mitochondrial architecture and morphology not only reflect their capacity for oxidative phosphorylation and ATP synthesis but also impact their subcellular localization and interaction with other organelles. The role of these ultrastructural abnormalities in modulating electrophysiological properties and excitation-contraction coupling remains largely unknown and warrants direct investigation considering the growing appreciation of the functional and structural coupling between the mitochondrial network, the calcium cycling machinery, and sarcolemmal ion channels in the cardiac myocyte. In this Methods in Molecular Biology chapter, we provide a protocol that allows for a quantitative assessment of mitochondrial shape and morphology in control and diabetic hearts that had undergone detailed electrophysiological measurements using high resolution optical action potential (AP) mapping.

NFATC1 modulation of atrial excitability in zebrafish.

Atrial fibrillation (AF) is a common cardiac arrhythmia with a strong component of heritability. We recently identified the cardiac transcription factor NFATC1 as a novel AF susceptibility gene in a family with autosomal dominant young-onset AF. To further explore the role for NFATC1 in cardiac excitability, we generated a nfatc1 null (KO) zebrafish using CRISPR/Cas9. The absence of nfatc1 resulted in increased event of ∼80% of sudden death (log rank test p=0.0001) in nfatc1 null fish in the juvenile stage (5‑7 weeks) with a grossly indistinguishable morphology in the fish or explanted heart. Furthermore, nfatc1 juvenile null fish showed significantly faster heart rates (95±3 bpm in KO (n=18) vs 70±4 bpm in WT (n=15), p=0.0001) and an increased incidence of arrhythmogenesis and spontaneous delayed afterdepolarizations (DADs) in explanted juvenile hearts (29 /40 atrial records in KO fish showed some form of irregular rhythm, versus 11/28 atrial records in WT (Chi-Squared, p=0.0013)). Optical action potentials (AP) with a voltage‑sensitive dye revealed an abbreviated AP in KO atrium (80±1 ms in KO (n=607) vs 91±1 ms in WT (n=441), p=0.0001), but not ventricle (186±3 ms in KO (n=109) vs 191±2 ms in WT (n=160), p=0.0001). Our data suggest NFATC1 modulates atrial excitability but not ventricular, which reinforces our initial assessment as an AF susceptibility gene. Experiments are ongoing to further define the mechanism behind the DADs we observed in the nfact1 null fish.

Role of AMPK pathway inactivation in high fat diet related cardiac electrophysiological remodeling.

Although impaired AMPK signaling is a hallmark of metabolic diseases (MetD), which heighten arrhythmia risk, whether and how loss of AMPK activity per se promotes adverse electrophysiological (EP) remodeling independently of confounding factors in MetD remains unknown. We investigated the EP substrate of wildtype (WT) mice on high-fat diet (HFD) relative to those on normal diet (ND) and to mice expressing the muscle-specific AMPK kinase dead isoform (AMPK-KD). After 8-weeks of HFD, mice underwent hemodynamic analysis by echocardiography and ex vivo optical action potential (AP) mapping. While HFD-fed mice did not exhibit structural or hemodynamic changes by 8-weeks, they were more prone to pacing-induced VT than their ND counterparts (57% HFD vs 11% ND). Investigation of the EP substrate revealed a marked decrease in conduction velocity (CV) in HFD vs ND-fed mice (54.9±6.7 cm/s vs 71.6±10.2 cm/s, p<0.05) with no change in AP duration (APD). Importantly, we found similar CV slowing in AMPK-KD on ND as WT on HFD. Challenging AMPK-KD mice with HFD did not reduce CV further (45.4±10.6 cm/s HFD vs 49.0±11.1 cm/s ND). In contrast, APD was decreased exclusively in AMPK-KD mice on HFD, reflecting the importance of both factors (AMPK depletion and HFD) in the modulation of myocardial repolarization. Functionally, the subset of hearts that were prone to HFD-related VT exhibited decreased CV compared to those that were protected against VT. We examined the molecular correlates of myocardial conduction, and found a paradoxical increase in Nav1.5, total and Ser368-phosphorylated Cx43 in AMPK-KD, discounting overall protein expression as a factor in CV slowing. In conclusion, impaired AMPK signaling accounts for early CV defects in HFD prior to onset of structural or mechanical remodeling. Our findings highlight AMPK as a potentially powerful anti-arrhythmic target at early stages of MetD.

Abstract 15506: Optical Mapping in Atrial Engineered Heart Tissues Reveals Proarrhythmic Potential of the Anti-AF Agent Vernakalant

Pharmacotherapy for atrial fibrillation (AF) is confounded by ventricular proarrhythmia. Vernakalant (VER) belongs to a class of atrial selective channel blockers developed in attempts to convert AF safely. Yet, robust analysis of the effects of VER in human based tissues has not been performed. We hypothesized that engineered heart tissues (EHT) composed of atrial (A) or ventricular (V) human iPSCs would provide a robust platform for quantitative analysis of chamber specific electrophysiological (EP) effects of VER. Methods: iPSCs were differentiated into cardiomyocytes using biphasic Wnt signaling, insulin, and lactate purification. Atrial-like myocytes were enriched by adding 0.75 μM retinoic acid on days 4-6 to promote atrial gene expression and specification. Myocytes were seeded onto cryosectioned, laser cut and decellularized myocardial tissues and cultured under isometric conditions. EHTs were stained with high dose, water soluble di-2-ANEPEQ to enhance signal-to-noise while avoiding toxicity. Results: Staining with 25 μM di-2-ANEPEQ provided high fidelity optical AP signals that allowed comprehensive characterization of the EP substrate. A-EHT exhibited significantly shorter AP durations than V-EHT reflecting greater repolarizing drive (APD80, 180 ms vs 367 ms; p &lt; 0.0001, n = 4-5). Consistent with its role as an atrial-selective blocker, VER increased APD80 only in A-EHTs (180 ms vs 258 ms, p = 0.0027, n = 5). This beneficial property for combatting AF was countered by marked slowing of A-EHT conduction and AP upstroke velocity (20.6 cm/s vs 11.7 cm/s, p = 0.027, n = 5). Conclusions: Optical mapping of chamber specific EHTs confirms atrial specific nature of VER as an atrial repolarization prolonging agent but raises unexpected concerns regarding potential proarrhythmic toxicity related to atrial conduction slowing.

High-throughput optical action potential recordings in hiPSC-derived cardiomyocytes with a genetically encoded voltage indicator in the AAVS1 locus.

Cardiomyocytes (CMs) derived from human induced pluripotent stem cells (hiPSCs) represent an excellent in vitro model in cardiovascular research. Changes in their action potential (AP) dynamics convey information that is essential for disease modeling, drug screening and toxicity evaluation. High-throughput optical AP recordings utilizing intramolecular Förster resonance energy transfer (FRET) of the voltage-sensitive fluorescent protein (VSFP) have emerged as a substitute or complement to the resource-intensive patch clamp technique. Here, we functionally validated our recently generated voltage indicator hiPSC lines stably expressing CAG-promoter-driven VSFP in the AAVS1 safe harbor locus. By combining subtype-specific cardiomyocyte differentiation protocols, we established optical AP recordings in ventricular, atrial, and nodal CMs in 2D monolayers using fluorescence microscopy. Moreover, we achieved high-throughput optical AP measurements in single hiPSC-derived CMs in a 3D context. Overall, this system greatly expands the spectrum of possibilities for high-throughput, non-invasive and long-term AP analyses in cardiovascular research and drug discovery.

Abstract P1098: Atrial Deletion Of AMPK Promotes Atrial Oxidative Stress And The Formation Of Delayed Afterdepolarizations Within The Inter-atrial Pulmonary Vein Junction

Atrial specific AMPK deletion leads to spontaneous onset of AF. Electrophysiological mechanisms underlying this heightened propensity remain unknown. We hypothesized that AMPK inactivation forms atrial triggered activity leading to atrial ectopy and AF initiation, in large part, by promoting atrial oxidative stress. Methods: We performed detailed ex vivo atrial optical action potential (AP) mapping on the posterior surface of hearts from mice with atrial deletion of AMPK a1/a2 (AMPK-dKO) (via Cre recombinase driven by sarcolipin) vs littermate controls (Ctrl), both during steady state pacing and following challenge with right atrial stimulus trains designed to promote calcium overload. Mitochondrial (mito) ultrastructure and markers of atrial oxidative stress were measured. To assess the functional impact of atrial oxidative stress, AMPK-dKO mice underwent chronic in vivo treatment with EUK-134 (EUK, 25mg/Kg SQ, 3X/wk) for 16 wks. Results: AMPK-dKO (7/11) but not Ctrl (0/5) hearts exhibited subthreshold delayed afterdepolarizations (DAD) that were consistently localized within a discrete inter-atrial region corresponding with the pulmonary veins (PV) junction. These triggers coincided with atrial oxidative stress indexed by a significant (p&lt;0.05) decrease in GSH/GSSG, PRDX3, SOD2, and increase in nitrotyrosine expression along with atrial mito swelling, decreased count and loss of cristae density. Chronic EUK treatment of AMPK-dKO suppressed the incidence (83% to 40%) and frequency (1.81±0.98/s to 0.083±0.08/s) of PV triggers abrogating the genesis of post-pacing atrial AP ectopy originating from the PV (4/5 vs 0/5). Serial in vivo ECG recordings in a subset of AMPK-dKO mice showed reorganization of the RR vs RR+1 relationship following 16 wks of EUK treatment consistent with decreased incidence of premature atrial complexes. Expression of total and S368 phosphorylated Cx43 were both reduced in the atria of AMPK-dKO mice vs Ctrl and restored by EUK reflecting reversal of pathological molecular remodeling by in vivo ROS scavenging. Conclusions: AMPK deletion promotes formation of left atrial ectopy from DAD sources within the PV region. AMPK plays a central role in the prevention of atrial oxidative stress and associated arrhythmias.

Increased cytosolic calcium buffering contributes to a cellular arrhythmogenic substrate in iPSC-cardiomyocytes from patients with dilated cardiomyopathy

Dilated cardiomyopathy (DCM) is a major risk factor for heart failure and is associated with the development of life-threatening cardiac arrhythmias. Using a patient-specific induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM) model harbouring a mutation in cardiac troponin T (R173W), we aim to examine the cellular basis of arrhythmogenesis in DCM patients with this mutation. iPSC from control (Ctrl) and DCM-TnT-R173W donors from the same family were differentiated into iPSC-CM and analysed through optical action potential (AP) recordings, simultaneous measurement of cytosolic calcium concentration ([Ca2+]i) and membrane currents and separately assayed using field stimulation to detect the threshold for AP- and [Ca2+]i-alternans development. AP duration was unaltered in TnT-R173W iPSC-CM. Nevertheless, TnT-R173W iPSC-CM showed a strikingly low stimulation threshold for AP- and [Ca2+]i-alternans. Myofilaments are known to play a role as intracellular Ca2+ buffers and here we show increased Ca2+ affinity of intracellular buffers in TnT-R173W cells, indicating increased myofilament sensitivity to Ca2+. Similarly, EMD57033, a myofilament Ca2+ sensitiser, replicated the abnormal [Ca2+]i dynamics observed in TnT-R173W samples and lowered the threshold for alternans development. In contrast, application of a Ca2+ desensitiser (blebbistatin) to TnT-R173W iPSC-CM was able to phenotypically rescue Ca2+ dynamics, normalising Ca2+ transient profile and minimising the occurrence of Ca2+ alternans at physiological frequencies. This finding suggests that increased Ca2+ buffering likely plays a major arrhythmogenic role in patients with DCM, specifically in those with mutations in cardiac troponin T. In addition, we propose that modulation of myofilament Ca2+ sensitivity could be an effective anti-arrhythmic target for pharmacological management of this disease.

Right predominant electrical remodeling in a pure model of pulmonary hypertension promotes reentrant arrhythmias.

Electrophysiological (EP) properties have been studied mainly in the monocrotaline model of pulmonary arterial hypertension (PAH). Findings are confounded by major extrapulmonary toxicities, which preclude the ability to draw definitive conclusions regarding the role of PAH per se in EP remodeling. The purpose of this study was to investigate the EP substrate and arrhythmic vulnerability of a new model of PAH that avoids extracardiopulmonary toxicities. Sprague-Dawley rats underwent left pneumonectomy (Pn) followed by injection of the vascular endothelial growth factor inhibitor Sugen-5416 (Su/Pn). Five weeks later, cardiac magnetic resonance imaging was performed invivo, optical action potential (AP) mapping exvivo, and molecular analyses invitro. Su/Pn rats exhibited right ventricular (RV) hypertrophy and were highly prone to pacing-induced ventricular tachycardia/fibrillation (VT/VF). Underlying this susceptibility was disproportionate RV-sided prolongation of AP duration, which promoted formation of right-sided AP alternans at physiological rates. While propagation was impaired at all rates in Su/Pn rats, the extent of conduction slowing was most severe immediately before the emergence of interventricular lines of block and onset of VT/VF. Measurement of the cardiac wavelength revealed a decrease in Su/Pn relative to control. Nav1.5 and total connexin 43 expression was not altered, while connexin 43 phosphorylation was decreased in PAH. Col1a1 and Col3a1 transcripts were upregulated coinciding with myocardial fibrosis. Once generated, VT/VF was sustained by multiple reentrant circuits with a lower frequency of RV activation due to wavebreak formation. In this pure model of PAH, we document RV-predominant remodeling that promotes multiwavelet reentry underlying VT. The Su/Pn model represents a severe form of PAH that allows the study of EP properties without the confounding influence of extrapulmonary toxicity.

B-AB01-02 EPICARDIAL ADIPOSITY MEDIATED LOCAL INFLAMMATION PROMOTES ARRHYTHMOGENESIS IN OBESE DONOR HEARTS

Epicardial adiposity has emerged as an independent risk factor for VT/VF. However, the molecular mechanisms by which the accumulation of epicardial fat contributes to VT/VF development remains unelucidated. We hypothesized that epicardial adipose tissue (EAT) paracrine signaling promotes local inflammation mediated arrhythmogenesis in obese human hearts. To test our hypothesis, we characterized transcriptome profile of the EAT and the underlying myocardial tissue in normal, overweight, and obese hearts as well as their susceptibility to arrhythmias. Tissue samples from the right ventricle (RV) EAT as well as the underlying myocardium were collected from donor hearts (N=9) and processed for RNA sequencing as well as western blotting studies. Arrhythmia susceptibility was investigated in the same hearts using optical mapping studies on RV wedge preparations. Briefly, the tissue was paced using a dynamic restitution protocol (S1S1) after intervention with isoproterenol (100nM). Optical action potentials (OAPs) and electrograms (EGMs) were recorded to track EADs, DADs, and the premature ventricular complexes (PVCs). The pro-inflammatory markers (IL-6, IL-1β, TLR-2, NLRP-3) were upregulated in EAT of obese hearts when compared with EAT in normal hearts. Additionally, the fibrotic markers (TGFβ, MMP2) were also upregulated in myocardial tissue from obese hearts in comparison with normal hearts. Tissue wedge preparations from obese hearts (2.45±1.4) registered higher incidence of PVCs per min when compared with normal (0.6±0.3) and overweight (0.96±0.2) hearts in presence of β-adrenergic stimulation. Moreover, CaMKII protein expression was higher in obese hearts (1.57±0.46) compared to normal hearts (0.89±0.29) (p<0.05). Our results suggest that EAT paracrine signaling promotes arrhythmias in obese hearts via local inflammation mediated functional and structural remodeling of the underlying cardiac tissue.

High heart rate associated early repolarization causes J-waves in both zebra finch and mouse.

High heart rates are a feature of small endothermic—or warm‐blooded—mammals and birds. In small mammals, the QT interval is short, and local ventricular recordings reveal early repolarization that coincides with the J‐wave on the ECG, a positive deflection following the QRS complex. Early repolarization contributes to short QT‐intervals thereby enabling brief cardiac cycles and high heart rates. We therefore hypothesized high hearts rates associate with early repolarization and J‐waves on the ECG of endothermic birds. We tested this hypothesis by comparing isolated hearts of zebra finches and mice and recorded pseudo‐ECGs and optical action potentials (zebra finch, n = 8; mouse, n = 8). In both species, heart rate exceeded 300 beats per min, and total ventricular activation was fast (QRS < 10 ms). Ventricular activation progressed from the left to the right ventricle in zebra finch, whereas it progressed from apex‐to‐base in mouse. In both species, the early repolarization front followed the activation front, causing a positive J‐wave in the pseudo‐ECG. Inhibition of early repolarization by 4‐aminopyridine reduced J‐wave amplitude in both species. Action potential duration was similar between ventricles in zebra finch, whereas in mouse the left ventricular action potential was longer. Accordingly, late repolarization had opposite directions in zebra finch (left‐right) and mouse (right‐left). This caused a similar direction for the zebra finch J‐wave and T‐wave, whereas in the mouse they were discordant. Our findings demonstrate that early repolarization and the associated J‐wave may have evolved by convergence in association with high heart rates.

Abstract 14897: Activation Dyssynchrony Between Contact Multielectrode Mapping and Subsurface Near-Infrared Optical Mapping During Human Atrial Fibrillation

Due to complex 3D human atrial structure, atrial fibrillation (AF) mapping with multielectrode arrays (MEA) mostly represents surface activation. Therefore, MEA may not properly visualize patient specific AF mechanisms, which impairs ablation outcomes. Conversely, near-infrared optical mapping (NIOM) visualizes subsurface intramural activation and can efficiently reveal reentrant drivers responsible for AF maintenance. Delay between surface activation seen by MEA electrograms (EGMs) vs subsurface activation seen by NIOM optical action potentials (OAPs) occurs due to dyssynchrony between myocardium layers, especially during AF. Coronary perfused explanted human atria (n=7) were mapped with NIOM (0.3-0.9mm 2 resolution) and 64-electrode MEA (3mm 2 resolution). Unipolar EGMs were analyzed for the steepest negative deflection. The delay between [-dV/dtmax] of unipolar EGMs and corresponding optical action potentials (OAPs) was compared in 500ms and 300ms pacing and AF. Subsequent structural analysis was done by 9.4T MRI (154-180μm 3 resolution) with gadolinium enhancement and histology. Delay between EGM and OAP local activation times rate-dependently and heterogeneously increased from 6±3 ms and 10±4 ms during 500ms and 300ms pacing to 15±11 ms (with maximum delay 47±18 ms) during pacing induced AF (average cycle length 124±65ms). Large local OAP-EGM delay, seen during AF, correlates with higher fibrosis percentage and fiber twist (p&lt;0.05). NIOM identified reentrant drivers maintaining AF, which were incorrectly visualized as multiple breakthroughs in 68% of MEA maps (n=22). Higher frequency leads to an increased activation discrepancy between EGM and NIOM caused by increased dyssynchrony in regions of higher fibrosis percentage and fiber twist, which may prevent MEA from proper identification of AF drivers in diseased fibrotic human atria. Reannotation of EGM activation based on NIOM may be required for correct AF mechanisms visualization.

Membrin/GOSR2 is a novel NaV1.5-interacting protein modulating cardiac conduction

Abstract Background Genome-wide association studies have associated a locus spanning GOSR2 with QRS- and QT-interval. GOSR2 encodes Membrin, a protein located at the cis-Golgi, which plays a role in protein trafficking. Altered trafficking of the cardiac sodium channel (NaV1.5), encoded by SCN5A, has been shown to reduce cardiac conduction. Purpose To explore the modulatory role of Membrin on cardiac conduction and sodium channel availability. Methods and results Tandem Affinity Purification in H10 cells (derived from neonatal rat cardiomyocytes) overexpressing the NaV1.5 C-terminus identified Membrin as a putative interactor of NaV1.5. We subsequently confirmed the interaction between NaV1.5 and Membrin by means of a co-immunoprecipitation assay in HEK293A cells that overexpress NaV1.5 and Membrin. To investigate whether Membrin affects cardiac conduction we recorded optical action potentials from the left ventricle (LV) of Langendorff-perfused hearts from Gosr2+/− mice and wild type (WT) littermate controls. Conduction velocity was measured at steady state pacing (cycle length 120ms) and at the minimal possible cycle length (S2min), during S1S2 pacing. Longitudinal conduction velocity was increased in Gosr2+/− mice compared to WT at steady state- (76.44 vs. 67.00 cm/s) as well as at S2min (62.00 vs. 51.86 cm/s, p=0.039, n=10 and 9, resp.). Single cell patch-clamp studies revealed a shortened action potential duration at 90% repolarization at all pacing frequencies (390 vs 342 V/s at 2Hz, p=0.036) in isolated mid-LV cardiomyocytes of Gosr2+/− mice compared to WT. In addition, the maximal upstroke velocity was increased in Gosr2+/− mid-LV cardiomyocytes at frequencies of 6Hz and higher (390 vs 342 V/s at 6Hz, p=0.044). Conclusion Our findings identify Membrin as a novel interacting protein of NaV1.5 and a modulator of cardiac conduction. We propose that Membrin acts through ion channel trafficking or by modulating the posttranslational maturation of ion channels. Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): Leducq foundation

Optical mapping of human embryonic stem cell-derived cardiomyocyte graft electrical activity in injured hearts

BackgroundHuman embryonic stem cell-derived cardiomyocytes (hESC-CMs) show tremendous promise for cardiac regeneration, but the successful development of hESC-CM-based therapies requires improved tools to investigate their electrical behavior in recipient hearts. While optical voltage mapping is a powerful technique for studying myocardial electrical activity ex vivo, we have previously shown that intra-cardiac hESC-CM grafts are not labeled by conventional voltage-sensitive fluorescent dyes. We hypothesized that the water-soluble voltage-sensitive dye di-2-ANEPEQ would label engrafted hESC-CMs and thereby facilitate characterization of graft electrical function and integration.MethodsWe developed and validated a novel optical voltage mapping strategy based on the simultaneous imaging of the calcium-sensitive fluorescent protein GCaMP3, a graft-autonomous reporter of graft activation, and optical action potentials (oAPs) derived from di-2-ANEPEQ, which labels both graft and host myocardium. Cardiomyocytes from three different GCaMP3+ hESC lines (H7, RUES2, or ESI-17) were transplanted into guinea pig models of subacute and chronic infarction, followed by optical mapping at 2 weeks post-transplantation.ResultsUse of a water-soluble voltage-sensitive dye revealed pro-arrhythmic properties of GCaMP3+ hESC-CM grafts from all three lines including slow conduction velocity, incomplete host-graft coupling, and spatially heterogeneous patterns of activation that varied beat-to-beat. GCaMP3+ hESC-CMs from the RUES2 and ESI-17 lines both showed prolonged oAP durations both in vitro and in vivo. Although hESC-CMs partially remuscularize the injured hearts, histological evaluation revealed immature graft structure and impaired gap junction expression at this early timepoint.ConclusionSimultaneous imaging of GCaMP3 and di-2-ANEPEQ allowed us to acquire the first unambiguously graft-derived oAPs from hESC-CM-engrafted hearts and yielded critical insights into their arrhythmogenic potential and line-to-line variation.

Low-Noise Fluorescent Infrared Detection of Single-Cell Cardiac Action Potentials

Optical recordings with voltage-sensitive fluorescent dyes enable non-invasive action potential (AP) measurements, which are useful for high-throughput drug screening. However, optical measurements are hampered by dye rundown, photobleaching, and phototoxicity. Long-lasting infrared dyes overcome these limitations, but optical recordings from single-cells are difficult due to low signal-to-noise ratios (SNR). Using the voltage-sensitive infrared dye, Di-4-ANBDQBS (Cytovolt1), we developed an optical system consisting of matched hardware, biological, and dye components to produce a low-noise fluorescent infrared detection system for single-cell AP recording. We combined a high-sensitivity photodiode-based detector, a high-stability LED excitation source, and an optimized optical filter set for enhanced single-cell optical AP recordings. We validated our system by correlating membrane voltage to fluorescence under single-cell patch clamp, and showed the fluorescence signal is highly-linear with membrane voltage. We also optimized dye concentration and staining method along with emission/excitation wavelengths to yield maximum ΔF/F and thus maximum SNR. To correlate membrane voltages to optical voltage signals, we performed simultaneous patch clamp and optical recordings on HEK293 cells and human induced pluripotent stem cell derived cardiomyocytes (hIPSC-CMs). Low-noise recordings, with minimal dye rundown, were obtained from single-cells. Optical signals were recorded in response to a voltage ramp, and averaged R2 values from linear fits of fluorescence versus voltage were 0.88+0.04, 0.97+0.007, 0.97+0.004 and 0.98+0.004 for 50, 75, 100 and 150 μM of Di‑4‑ANBDQBS, respectively, in HEK293 cells. ΔF/F measurements were 10.5+0.3%, 14.9+1.8%, 13.8+2.5% and 13.6+3.5% for 50, 75, 100 and 150 μM of dye, respectively. For hiPSC-CMs, ΔF/F measurements from simultaneous voltage and fluorescence of APs were up to 23.1% per 100 mV. Taken together, these data demonstrate the feasibility of high fidelity single cell voltage measurements in a simple system not requiring photomultiplier tubes or laser excitation.

NS5806 Induces Electromechanically Discordant Alternans and Arrhythmogenic Voltage-Calcium Dynamics in the Isolated Intact Rabbit Heart.

Background: NS5806 activates the transient outward potassium current Ito, and has been claimed to reproduce Brugada Syndrome (BrS) in ventricular wedge preparations. Ito modulates excitation-contraction coupling, which is critical in alternans dynamics. We explored NS5806-arrhythmogenic effects in the intact whole heart and its impact on alternans.Methods: Langendorff-perfused rabbit hearts (n = 20) underwent optical AP and Ca mapping during pacing at decremental cycle lengths (CL). Spontaneous arrhythmias and pacing-induced alternans was characterized at baseline (BL), after perfusing with NS5806, before and after adding verapamil (VP), and SEA0400 (SEA, n = 5 each), to modulate Ca-current and Na-Ca exchange, the main AP-Ca coupling mechanisms.Results: NS5806 induced BrS-like ECG features in 6 out of 20 hearts. NS5806 prolonged steady-state (3 Hz) action potential duration (APD) by 16.8%, Ca decay constant by 34%, and decreased conduction velocity (CV) by 52.6%. After NS5806 infusion, spontaneous ventricular ectopy (VE) and AP/Ca alternans occurred. Pacing-induced alternans during NS5806 infusion occurred at longer CL and were AP/Ca discordant from its onset. Spatially discordant alternans after NS5806 infusion had non-propagation-driven nodal line distribution. No spontaneous phase-2 reentry occurred. Under NS5806 + VP, alternans became AP/Ca concordant and only induced in two out of five; NS5806 + SEA did not affect alternans but suppressed spontaneous ectopy.Conclusions: NS5806 disrupts AP-Ca coupling and leads to Ca-driven, AP/Ca-discordant alternans and VE. Despite BrS-like ECG features, no spontaneous sustained arrhythmias or phase-2 reentry occurred. NS5806 does not fully reproduce BrS in the intact rabbit heart.

Cardiomyocyte-Specific STIM1 (Stromal Interaction Molecule 1) Depletion in the Adult Heart Promotes the Development of Arrhythmogenic Discordant Alternans.

STIM1 (stromal interaction molecule 1) is a calcium (Ca2+) sensor that regulates cardiac hypertrophy by triggering store-operated Ca2+ entry. Because STIM1 binding to phospholamban increases sarcoplasmic reticulum Ca2+ load independent of store-operated Ca2+ entry, we hypothesized that it controls electrophysiological function and arrhythmias in the adult heart. Inducible myocyte-restricted STIM1-KD (STIM1 knockdown) was achieved in adult mice using an αMHC (α-myosin heavy chain)-MerCreMer system. Mechanical and electrophysiological properties were examined using echocardiography in vivo and optical action potential (AP) mapping ex vivo in tamoxifen-induced STIM1flox/flox-Cretg/- (STIM1-KD) and littermate controls for STIM1flox/flox (referred to as STIM1-Ctl) and for Cretg/- without STIM deletion (referred to as Cre-Ctl). STIM1-KD mice (N=23) exhibited poor survival compared with STIM1-Ctl (N=22) and Cre-Ctl (N=11) with >50% mortality after only 8-days of cardiomyocyte-restricted STIM1-KD. STIM1-KD but not STIM1-Ctl or Cre-Ctl hearts exhibited a proclivity for arrhythmic behavior, ranging from frequent ectopy to pacing-induced ventricular tachycardia/ventricular fibrillation (VT/VF). Examination of the electrophysiological substrate revealed decreased conduction velocity and increased AP duration (APD) heterogeneity in STIM1-KD. These features, however, were comparable in VT/VF(+) and VT/VF(-) hearts. We also uncovered a marked increase in the magnitude of APD alternans during rapid pacing, and the emergence of a spatially discordant alternans profile in STIM1-KD hearts. Unlike conduction velocity slowing and APD heterogeneity, the magnitude of APD alternans was greater (by 80%, P<0.05) in VT/VF(+) versus VT/VF(-) STIM1-KD hearts. Detailed phase mapping during the initial beats of VT/VF identified one or more rotors that were localized along the nodal line separating out-of-phase alternans regions. In an adult murine model with inducible and myocyte-specific STIM1 depletion, we demonstrate for the first time the regulation of spatially discordant alternans by STIM1. Early mortality in STIM1-KD mice is likely related to enhanced susceptibility to VT/VF secondary to discordant APD alternans.

Examination of the Effects of Conduction Slowing on the Upstroke of Optically Recorded Action Potentials.

IntroductionThe upstroke of optical action potentials (APs) recorded from intact hearts are generally recognized to be slower than those recorded with microelectrodes. This is thought to reflect spatial signal averaging within the volume of tissue that makes up the optical signal. However, to date, there has been no direct experimental study on the relationship between conduction velocity (CV) and optical AP upstroke morphology in the intact heart. Notably, it is known that sodium channel block and gap junction inhibition, which both slow CV, exert differential effects on the upstroke velocity of microelectrode-recorded APs. Whether such differences are evident in optical APs is not known. The present study sought to determine the relationship between tissue CV and optical AP upstroke velocity in intact mouse hearts.Materials and MethodsIsolated, perfused mouse hearts were stained with the potentiometric dye Rh-237. Fluorescent signals were recorded from across the anterior surface of the left and right ventricles during constant pacing. Maximum rate of change in fluorescence (dF/dtmax) and tissue CV were assessed in control conditions, during an acute period of low-flow ischemia, and following perfusion of flecainide (1–3 μmol/L), a sodium channel blocker, or carbenoxolone (10–50 μmol/L), a gap junction inhibitor.ResultsDuring epicardial pacing, an anisotropic pattern was observed in both activation and dF/dtmax maps, with more rapid optical AP upstroke velocities orientated along the fastest conduction paths (and vice versa). Low-flow ischemia resulted in a time-dependent slowing of ventricular CV, which was accompanied by a concomitant reduction in optical AP upstroke velocity. All values returned to baseline on tissue reperfusion. Both flecainide and carbenoxolone were associated with a concentration-dependent reduction in CV and decrease in optical AP upstroke velocity, despite distinct mechanisms of action. Similar responses to carbenoxolone were observed for low- (156 μm pixel with) and high- (20 μm pixel width) magnification recordings. Comparison of data from all interventions revealed a linear relationship between CV and upstroke dF/dt.ConclusionIn intact mouse hearts, slowing of optical AP upstroke velocity is directly proportional to the change in CV associated with low-flow ischemia, sodium channel block, and gap junction inhibition.

Lights, camera, path splitter: a new approach for truly simultaneous dual optical mapping of the heart with a single camera

BackgroundOptical mapping of transmembrane voltage and intracellular calcium is a powerful tool for investigating cardiac physiology and pathophysiology. However, simultaneous dual mapping of two fluorescent probes remains technically challenging. We introduce a novel, easy-to-use approach that requires a path splitter, single camera and excitation light to simultaneously acquire voltage and calcium signals from whole heart preparations, which can be applied to other physiological models – including neurons and isolated cardiomyocytes.ResultsComplementary probes were selected that could be excited with a single wavelength light source. Langendorff-perfused hearts (rat, swine) were stained and imaged using a sCMOS camera outfitted with an optical path splitter to simultaneously acquire two emission fields at high spatial and temporal resolution. Voltage (RH237) and calcium (Rhod2) signals were acquired concurrently on a single sensor, resulting in two 384 × 256 images at 814 frames per second. At this frame rate, the signal-to-noise ratio was 47 (RH237) and 85 (Rhod2). Imaging experiments were performed on small rodent hearts, as well as larger pig hearts with sufficient optical signals. In separate experiments, each dye was used independently to assess crosstalk and demonstrate signal specificity. Additionally, the effect of ryanodine on myocardial calcium transients was validated – with no measurable effect on the amplitude of optical action potentials. To demonstrate spatial resolution, ventricular tachycardia was induced –resulting in the novel finding that spatially discordant calcium alternans can be present in different regions of the heart, even when electrical alternans remain concordant. The described system excels in providing a wide field of view and high spatiotemporal resolution for a variety of cardiac preparations.ConclusionsWe report the first multiparametric mapping system that simultaneously acquires calcium and voltage signals from cardiac preparations, using a path splitter, single camera and excitation light. This approach eliminates the need for multiple cameras, excitation light patterning or frame interleaving. These features can aid in the adoption of dual mapping technology by the broader cardiovascular research community, and decrease the barrier of entry into panoramic heart imaging, as it reduces the number of required cameras.

Abstract 239: Selective Right-sided Electrical Remodeling In A Pure Model Of Pulmonary Hypertension Promotes Micro-reentrant Arrhythmias

Background: Pulmonary arterial hypertension (PAH) is characterized by elevated mean PA pressures that increase right ventricular (RV) afterload, leading to RV failure. Whereas structural and mechanical decompensation in PAH are well established, the extent of electrophysiological (EP) remodeling and arrhythmia propensity are far less clear. This is, in large part, because PAH-related EP properties have been exclusively studied in the monocrotaline (MCT) model, which is confounded by major extra-pulmonary toxicities, including liver failure and direct myocardial damage. To address this knowledge gap, we performed a comprehensive evaluation of the EP substrate, including arrhythmia dynamics, in a new pure model of PAH. Methods: Sprague Dawley rats underwent surgical left pneumonectomy followed by injection of the VEGF inhibitor Sugen 5416 (S/P), or underwent no intervention (CTRL). 5 wks later, rats were euthanized for high-resolution optical action potential (AP) mapping studies in ex vivo perfused hearts. Results: 6/6 S/P vs 0/5 CTRL hearts were prone to pacing-induced VT (P&lt;.05). Underlying this differential susceptibility was disproportionate RV sided prolongation of AP duration in S/P. This, in turn, promoted the formation of right-sided AP alternans at physiological rates in S/P but not CTRL. Correspondingly, KCND2 mRNA transcripts were downregulated by 80X in the RV of S/P relative to CTRL. While propagation was impaired at all rates in S/P, the most profound decrease in conduction velocity (53%) was always observed immediately prior to VT initiation. Nav1.5 and Cx43 mRNA expression were not altered in S/P. In contrast Col1a1, Col3a1, and fibronectin transcripts were upregulated implicating fibrosis in impaired conduction. The average wavelength was significantly decreased in S/P relative to CTRL (P&lt;.05). Once generated, VT was sustained by multiple microreentrant circuits likely facilitated by the short wavelengths. Conclusion: In this pure model of PAH, we document highly selective RV-sided EP remodeling that facilitates the initiation of multi-wavelet micro-reentry underlying VT. The S/P model represents a severe form of PAH that allows the study of EP properties without the confounding influence of extra-pulmonary toxicity.