Action Potential Upstroke Velocity Research Articles

HiPSC-derived cardiomyocytes from Brugada Syndrome patients without identified mutations do not exhibit clear cellular electrophysiological abnormalities.

Brugada syndrome (BrS) is a rare cardiac rhythm disorder associated with sudden cardiac death. Mutations in the sodium channel gene SCN5A are found in ~20% of cases while mutations in other genes collectively account for <5%. In the remaining patients the genetic defect and the underlying pathogenic mechanism remain obscure. To provide insight into the mechanism of BrS in individuals without identified mutations, we here studied electrophysiological properties of cardiomyocytes (CMs) generated from human induced pluripotent stem cells (hiPSCs) from 3 BrS patients who tested negative for mutations in the known BrS-associated genes. Patch clamp studies revealed no differences in sodium current (INa) in hiPSC-CMs from the 3 BrS patients compared to 2 unrelated controls. Moreover, action potential upstroke velocity (Vmax), reflecting INa, was not different between hiPSC-CMs from the BrS patients and the controls. hiPSC-CMs harboring the BrS-associated SCN5A-1795insD mutation exhibited a reduction in both INa and Vmax, demonstrating our ability to detect reduced sodium channel function. hiPSC-CMs from one of the BrS lines demonstrated a mildly reduced action potential duration, however, the transient outward potassium current (Ito) and the L-type calcium current (ICa,L), both implicated in BrS, were not different compared to the controls. Our findings indicate that ion channel dysfunction, in particular in the cardiac sodium channel, may not be a prerequisite for BrS.

Multiple ion channel block by the cation channel inhibitor SKF-96365 in myocytes from the rabbit atrioventricular node

The atrioventricular node (AVN) of the cardiac conduction system coordinates atrial–ventricular excitation and can act as a subsidiary pacemaker. Recent evidence suggests that an inward background sodium current, I B,Na, carried by nonselective cation channels (NSCCs), contributes to AVN cell pacemaking. The study of the physiological contribution of I B,Na has been hampered, however, by a lack of selective pharmacological antagonists. This study investigated effects of the NSCC inhibitor SKF‐96365 on spontaneous activity, I B,Na, and other ionic currents in AVN cells isolated from the rabbit. Whole‐cell patch‐clamp recordings of action potentials (APs) and ionic currents were made at 35–37°C. A concentration of 10 μmol/L SKF‐96365 slowed spontaneous action potential rate by 13.9 ± 5.3% (n = 8) and slope of the diastolic depolarization from 158.1 ± 30.5 to 86.8 ± 30.5 mV sec−1 (P < 0.01; n = 8). Action potential upstroke velocity and maximum diastolic potential were also reduced. Under I B,Na‐selective conditions, 10 μmol/L SKF‐96365 inhibited I B,Na at −50 mV by 36.1 ± 6.8% (n = 8); however, effects on additional channel currents were also observed. Thus, the peak l‐type calcium current (I Ca,L) at +10 mV was inhibited by 38.6 ± 8.1% (n = 8), while the rapid delayed rectifier current, I Kr, tails at −40 mV following depolarization to +20 mV were inhibited by 55.6 ± 4.6% (n = 8). The hyperpolarization‐activated current, I f, was unaffected by SKF‐96365. Collectively, these results indicate that SKF‐96365 exerts a moderate inhibitory effect on I B,Na and slows AVN cell pacemaking. However, additional effects of the compound on I Ca,L and I Kr confound the use of SKF‐96365 to dissect out selectively the physiological role of I B,Na in the AVN.

Extracellular Matrix-Mediated Maturation of Human Pluripotent Stem Cell-Derived Cardiac Monolayer Structure and Electrophysiological Function.

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) monolayers generated to date display an immature embryonic-like functional and structural phenotype that limits their utility for research and cardiac regeneration. In particular, the electrophysiological function of hPSC-CM monolayers and bioengineered constructs used to date are characterized by slow electric impulse propagation velocity and immature action potential profiles. Here, we have identified an optimal extracellular matrix for significant electrophysiological and structural maturation of hPSC-CM monolayers. hPSC-CM plated in the optimal extracellular matrix combination have impulse propagation velocities ≈2× faster than previously reported (43.6±7.0 cm/s; n=9) and have mature cardiomyocyte action potential profiles, including hyperpolarized diastolic potential and rapid action potential upstroke velocity (146.5±17.7 V/s; n=5 monolayers). In addition, the optimal extracellular matrix promoted hypertrophic growth of cardiomyocytes and the expression of key mature sarcolemmal (SCN5A, Kir2.1, and connexin43) and myofilament markers (cardiac troponin I). The maturation process reported here relies on activation of integrin signaling pathways: neutralization of β1 integrin receptors via blocking antibodies and pharmacological blockade of focal adhesion kinase activation prevented structural maturation. Maturation of human stem cell-derived cardiomyocyte monolayers is achieved in a 1-week period by plating cardiomyocytes on PDMS (polydimethylsiloxane) coverslips rather than on conventional 2-dimensional cell culture formats, such as glass coverslips or plastic dishes. Activation of integrin signaling and focal adhesion kinase is essential for significant maturation of human cardiac monolayers.

Effects of Late Sodium Current (INAL) Blockade during Acute Myocardial Ischemia

Background: Ischemia ischaracterized by an overproduction of toxic metabolites well-known enhancers of the late sodium current (INaL). Ranolazine (RAN), a blocker of INaL, improves recovery during reperfusion. However, during ischemia diastolic potential depolarization might limit INa recruiting; thus, whether INaL is actually enhanced during acute ischemia is still unknown.Aim: To test the functional contribution of INaL in a cellular model of acute ischemia.Methods: Rat ventricular myocytes were exposed for 7 min to a (normoxic) ischemia-mimic solution (ISC); to evaluate the role of INaL, RAN (10μM) or TTX (1μM) were added throughout the protocol. INaL was isolated as TTX (1μM)-sensitive current during action potentials (APs) elicited at 1Hz. Cell shortening, cytosolic Na+ (Nai) and Ca2+ (Cai) were monitored in field-stimulated myocytes (1Hz). The sarcolemma (s) and mitochondrial (m) Na+/Ca2+exchanger (NCX) were blocked by SEA-0400 (1μM) and CGP37157 (1μM) respectively.Results: During ISC, loss of contraction was followed by partial recovery. While APs persisted throughout ISC, diastolic potential markedly depolarized and AP upstroke velocity decreased. In spite of this, ISC induced INaL enhancement, which was completely prevented by RAN. ISC increased Nai and Cai, which were partially prevented by RAN; TTX affected ISC-induced Nai enhancement only. SEA-0400 boosted cytosolic Cai and sarcoplasmic reticulum (SR) Ca2+content during ISC. SEA-0400 effects were sharply curtailed by RAN and partially prevented by TTX. CGP37157 prevented RAN effects during sNCX blockade.Conclusion: During ISC, INaL increased in spite of AP changes and contributed to Nai increment. Unexpectedly, sNCX blockade unrevealed RAN and TTX effects on Cai. These results suggest Na+-dependent, but sNCX-independent Ca2+ accumulation mechanisms during acute ischemia. Indeed, mNCX appears to be involved in the INaL induced cytosolic Ca2+ accumulation.

Activation of Reverse Na/Ca Exchanger by Skeletal Na Channel Isoform Increases Excitation-Contraction Coupling Efficiency in Rabbit Cardiomyocytes

Our prior work has shown that TTX-sensitive sodium current (INa) affects sarcoplasmic reticular (SR) Ca release. This current activates early reverse NCX. The resulting Ca entry primes the dyadic cleft, which appears to increase Ca channel coupling fidelity (we refer to this as the priming mechanism). The skeletal isoform Nav1.4 is the main TTX-sensitive Na channel expressed in adult rabbit ventricular cardiomyocytes. Here we tested the hypothesis that this is the principal isoform involved in the priming mechanism. We evoked action potentials (AP) in isolated rabbit ventricular cells loaded with fluo-4, and simultaneously recorded Ca transients before and after the application of either relatively low doses of TTX (100 nM) or the specific Nav1.4 inhibitor μ-Conotoxin GIIIB (1 μM). 100 nM of TTX will effectively block all skeletal and the neuronal Navs (Nav1.1 is also present with a low level of expression). However this concentration will have a negligible (5%) effect on the cardiac isoform. There was a more prominent AP upstroke velocity reduction in TTX (73±1%, n=4) than in GIIB (81±1%, n=6, P<0.05). This can be expected given the relative contribution of each Nav isoform to the phase 0 of the AP. However, the effects of either drug on SR Ca release (measured as the transient maximum upstroke velocity) were identical (69±0.03% in TTX (n=5) vs. 69±3% in GIIIB (n=6), P<0.05). Furthermore, this reduction in SR Ca release is comparable to the value that we obtained previously when total INa was inactivated with a ramp. This result suggests that the Nav skeletal isoform is the main Nav isoform involved in regulating the efficiency of excitation-contraction coupling in rabbit cardiomyocytes.

Electrophysiological Properties of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes Cultured on a Flexible Matrigel Substrate

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) provide a unique tool to evaluate cellular properties in disease models, investigate underlying mechanisms and optimize cell-based therapies. We recently reported that after plating 30 day old single hiPSC-CMs on undiluted Matrigel “Mattress” (thickness ∼0.5 mm) for 5 days, mattress hiPSC-CMs developed a rod-like cell shape, aligned myofilament structure, increased sarcomere length and exhibited robust cell shortening. Here, we compared the electrophysiological properties of mattress hiPSC-CMs to hiPSC-CMs cultured on standard substrates (control) and acutely-isolated adult rabbit CMs. The action potentials of mattress hiPSC-CMs were comparable to rabbit CMs. Compared to control, mattress hiPSC-CMs had significantly increased the action potential upstroke velocity due to two-fold increase in sodium current (INa) (−68.61±11.63 pA·pF-1 in mattress vs. −29.66±7.50 pA·pF-1 in control at −30mV). The inward rectifier current (IK1) is responsible for the maintenance of resting membrane potential and contributes to phase 3 of the action potential. Although resting potential and action potential duration was not different from rabbit CMs, IK1 density in mattress hiPSC-CMs was nine-time smaller than in rabbit CMs, but there was no difference between mattress and control hiPSC-CMs (−4.86±0.66 pA·pF-1 in mattress vs. −43.75±4.13 pA·pF-1 in rabbit vs. −3.98±0.83 pA·pF-1 in control at −120 mV). The funny current (If) is an inward current activating at hyperpolarized membrane potential, which is detectable in the developing heart but absent in the adult ventricle and contributes to the spontaneous beating of hiPSC-CMs. Mattress hiPSC-CMs exhibit robust If that was not significantly different from control (−6.22±1.31 pA·pF-1 in mattress vs.-5.60±1.43 pA·pF-1 in control at −120 mV). In summary, the matrigel mattress method enables the rapid generation of robustly contracting single hiPSC-CMs and increases INa, with negligible effect on IK1 or If.

Matrigel Mattress: A Method for the Generation of Single Contracting Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

The lack of measurable single-cell contractility of human-induced pluripotent stem cell-derived cardiac myocytes (hiPSC-CMs) currently limits the utility of hiPSC-CMs for evaluating contractile performance for both basic research and drug discovery. To develop a culture method that rapidly generates contracting single hiPSC-CMs and allows quantification of cell shortening with standard equipment used for studying adult CMs. Single hiPSC-CMs were cultured for 5 to 7 days on a 0.4- to 0.8-mm thick mattress of undiluted Matrigel (mattress hiPSC-CMs) and compared with hiPSC-CMs maintained on a control substrate (<0.1-mm thick 1:60 diluted Matrigel, control hiPSC-CMs). Compared with control hiPSC-CMs, mattress hiPSC-CMs had more rod-shape morphology and significantly increased sarcomere length. Contractile parameters of mattress hiPSC-CMs measured with video-based edge detection were comparable with those of freshly isolated adult rabbit ventricular CMs. Morphological and contractile properties of mattress hiPSC-CMs were consistent across cryopreserved hiPSC-CMs generated independently at another institution. Unlike control hiPSC-CMs, mattress hiPSC-CMs display robust contractile responses to positive inotropic agents, such as myofilament calcium sensitizers. Mattress hiPSC-CMs exhibit molecular changes that include increased expression of the maturation marker cardiac troponin I and significantly increased action potential upstroke velocity because of a 2-fold increase in sodium current (INa). The Matrigel mattress method enables the rapid generation of robustly contracting hiPSC-CMs and enhances maturation. This new method allows quantification of contractile performance at the single-cell level, which should be valuable to disease modeling, drug discovery, and preclinical cardiotoxicity testing.

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

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

Local Gradients in Electrotonic Loading Modulate the Local Effective Refractory Period: Implications for Arrhythmogenesis in the Infarct Border Zone.

Ectopic electrical activity that originates in the peri-infarct region can give rise to potentially lethal re-entrant arrhythmias. The spatial variation in electrotonic loading that results from structural remodelling in the infarct border zone may increase the probability that focal activity will trigger electrical capture, but this has not previously been investigated systematically. This study uses in-silico experiments to examine the structural modulation of effective refractory period on ectopic beat capture. Informed by 3-D reconstructions of myocyte organization in the infarct border zone, a region of rapid tissue expansion is abstracted to an idealized representation. A novel metric is introduced that defines the local electrotonic loading as a function of passive tissue properties and boundary conditions. The effective refractory period correlates closely with local electrotonic loading, while the action potential duration, conduction, and upstroke velocity reduce in regions of increasing electrotonic load. In the presence of focal ectopic stimuli, spatial variation in effective refractory period can cause unidirectional conduction block providing a substrate for reentrant arrhythmias. Consequently, based on the observed results, a possible novel mechanism for arrhythmogenesis in the infarct border zone is proposed.

Late Sodium Current in Human Atrial Cardiomyocytes from Patients in Sinus Rhythm and Atrial Fibrillation.

Slowly inactivating Na+ channels conducting “late” Na+ current (INa,late) contribute to ventricular arrhythmogenesis under pathological conditions. INa,late was also reported to play a role in chronic atrial fibrillation (AF). The objective of this study was to investigate INa,late in human right atrial cardiomyocytes as a putative drug target for treatment of AF. To activate Na+ channels, cardiomyocytes from transgenic mice which exhibit INa,late (ΔKPQ), and right atrial cardiomyocytes from patients in sinus rhythm (SR) and AF were voltage clamped at room temperature by 250-ms long test pulses to -30 mV from a holding potential of -80 mV with a 100-ms pre-pulse to -110 mV (protocol I). INa,late at -30 mV was not discernible as deviation from the extrapolated straight line IV-curve between -110 mV and -80 mV in human atrial cells. Therefore, tetrodotoxin (TTX, 10 μM) was used to define persistent inward current after 250 ms at -30 mV as INa,late. TTX-sensitive current was 0.27±0.06 pA/pF in ventricular cardiomyocytes from ΔKPQ mice, and amounted to 0.04±0.01 pA/pF and 0.09±0.02 pA/pF in SR and AF human atrial cardiomyocytes, respectively. With protocol II (holding potential -120 mV, pre-pulse to -80 mV) TTX-sensitive INa,late was always larger than with protocol I. Ranolazine (30 μM) reduced INa,late by 0.02±0.02 pA/pF in SR and 0.09±0.02 pA/pF in AF cells. At physiological temperature (37°C), however, INa,late became insignificant. Plateau phase and upstroke velocity of action potentials (APs) recorded with sharp microelectrodes in intact human trabeculae were more sensitive to ranolazine in AF than in SR preparations. Sodium channel subunits expression measured with qPCR was high for SCN5A with no difference between SR and AF. Expression of SCN8A and SCN10A was low in general, and lower in AF than in SR. In conclusion, We confirm for the first time a TTX-sensitive current (INa,late) in right atrial cardiomyocytes from SR and AF patients at room temperature, but not at physiological temperature. While our study provides evidence for the presence of INa,late in human atria, the potential of such current as a target for the treatment of AF remains to be demonstrated.

Changes in electrical activity of working myocardium under condition of if current inhibition.

The study examined the effect of ZD7288, a blocker of hyperpolarization-activated "funny" current If, on electrical activity in working atrial and ventricular myocardium in rats. In concentrations range from 3×10(-6) to 3×10(-5) M, the agent significantly increased the duration of action potentials at 50 and 90% repolarization levels in both atrial and ventricular myocardium at the fixed stimulation rate of 5 Hz. The blocker affected neither resting potential nor the upstroke velocity of action potential. In patch-clamp experiments, ZD7288 selectively inhibited If current, but produced no effect on delayed rectifier potassium currents that determine the rate of repolarization. The described effects of ZD7288 are not related to its non-specific effects on the ionic currents except If.

Abstract 14019: Electrophysiological Effects of the Late Sodium Current Inhibitor GS967 in Scn5a -1798insD Mouse and Human SCN5A -1795insD iPSC-derived Cardiomyocytes

Background and Aim: Selective inhibition of cardiac late sodium current (I Na,L ) is an emerging target in the treatment of ventricular arrhythmias. The electrophysiological effects of GS967, a potent I Na,L inhibitor, were investigated in an overlap syndrome model of both gain and loss of sodium channel function, comprising cardiomyocytes derived from human SCN5A -1795insD induced pluripotent stem cells (iPSC-CMs) and mice carrying the homologous mutation Scn5a -1798insD. Methods and Results: On patch-clamp analysis, isolated mouse Scn5a -1798insD cardiomyocytes and human SCN5A -1795insD iPSC-CMs showed decreased peak I Na and action potential (AP) upstroke velocity (Vmax) and increased I Na,L and AP duration at 90% repolarization (APD 90 ) as compared to wild-type. GS967 (50-300 nM) significantly decreased APD 90 in mouse Scn5a -1798insD cardiomyocytes by 8±2% (mean±SEM) at 50 nM (n=7), 13±3% at 100 nM (n=11) and 20±5% at 300 nM (n=6) (all p &lt;0.01 vs. control), without affecting Vmax. GS967 (300 nM) selectively inhibited I NaL in mouse Scn5a -1798insD cardiomyocytes (GS967-sensitive current of 0.7±0.1 pA/pF, n=6), but had no effect on peak I Na . Furthermore, GS967 (100 nM) suppressed fast (5 Hz) pacing-induced afterpotentials and triggered activity. In human SCN5A -1795insD iPSC-CMs (n=6), GS967 (300 nM) significantly reduced APD 90 without affecting the resting membrane potential or Vmax. In Langendorff-perfused, isolated mouse Scn5a -1798insD hearts (n=5), GS967 (300 nM) had no effect on ventricular activation time or conduction velocity (as assessed by epicardial mapping). Conclusion: Selective inhibition of I NaL by GS967 attenuated AP prolongation and prevented pro-arrhythmic activity in mouse Scn5a -1798insD cardiomyocytes and human SCN5A-1795insD iPSC-CMs, thus suppressing the gain-of-function features of this overlap syndrome mutation. Importantly, these beneficial actions of GS967 occurred in the absence of deleterious effects on sodium channel availability or cardiac conduction, despite a pre-existing decrease in peak I Na . Thus, selective inhibition of I Na,L constitutes a promising pharmacological treatment of cardiac channelopathies associated with enhanced I NaL , even in overlap syndromes whereby peak I Na is decreased.

Dynamic excitation states and firing patterns are controlled by sodium channel kinetics in myenteric neurons: a simulation study

Enteric neurons located in the gastro-intestinal tract are of particular importance to control digestive functions such as motility and secretion. In our recent publication, we showed that mouse myenteric neurons exhibit 2 types of tetrodotoxin-resistant Na+ currents: a fast inactivating Na+ current produced by Nav1.5 channels, present in nearly all myenteric neurons, and a persistent Na+ current attributed to Nav1.9 channels, restricted to the intrinsic primary afferent neurons (IPANs). By combination of experimental recording and computer simulation we found that Nav1.5 contributed to the upstroke velocity of action potentials (APs), whereas Nav1.9 opposed AP repolarization. Here, we detailed the Na+, Ca2+ and K+ currents used in our computational model of IPAN. We refined the prototype cell to reproduce the sustained firing pattern recorded in situ. As shown in experimental conditions we demonstrated that Nav1.9 channels critically determine the up-state life-time and thus, are essential to sustain tonic firing.

Electrical Excitation of the Heart in a Basal Vertebrate, the European River Lamprey (Lampetra fluviatilis)

Hagfishes and lampreys (order Cyclostomata) are living representatives of an ancient group of jawless vertebrates (class Agnatha). Studies on cyclostome hearts may provide insights into the evolution of the vertebrate heart and thereby increase our understanding of cardiac function in higher vertebrates, including mammals. To this end, electrical excitability of the heart in a basal vertebrate, the European river lamprey (Lampetra fluviatilis), was examined. Ion currents of cardiac myocytes, action potentials (APs) of atrial and ventricular muscle, and electrocardiogram (in vivo) were measured using the patch-clamp method, intracellular microelectrodes, and trailing wires, respectively. The characteristic features of fairly high heart rate (28.4 ± 3 beats min(-1)) and short AP duration (550 ± 44 and 122.1 ± 28.5 for ventricle and atrium, respectively) at low ambient temperature (5°C) are shared with cold-active teleost fishes. However, the ion current basis of the ventricular AP differs from that of other fishes. For inward currents, sodium current density (INa) is lower and calcium current density (ICa) higher than in teleost ventricles, while the kinetics of INa is slow and that of ICa is fast in comparison. Among the ventricular repolarizing currents, the delayed rectifier K(+) current is smaller than in myocytes of several teleost species. Unlike mammalian hearts, ATP-sensitive K(+) channels are constitutively open under normoxic conditions, thus contributing to negative resting membrane potential and repolarization of APs. Upstroke velocity of AP (5.4 ± 0.9 and 6.3 ± 0.6 V s(-1) for ventricular and atrial myocytes, respectively) is slower than in teleost hearts. Excitability of the lamprey heart seems to possess both primitive and advanced characteristics. Short APs are appropriate to support brief and vigorous contractions (in common with higher vertebrates), while relatively low AP upstroke velocities enable only relatively slow propagation of contraction over the heart.

NORMAL DEVELOPMENT OF CONDUCTION VELOCITY IN RATS IS PREVENTED BY FRUCTOSE-FAT FEEDING

Background: Diabetes and metabolic syndrome causes cardiac electrical abnormalities that increase the risk for arrhythmias and sudden death. We have established fructose-fat fed rats (FFFRs) as a prediabetic model, which after 6 weeks of feeding exhibit intramyocardial lipid accumulation, decreased cardiac conduction velocity (CV), and increased risk of ventricular fibrillation following ischemia–reperfusion. CV slowing was not explained by changes in Naþ, Kþ, or gap junction channel function; however, CV may be altered by a depolarized resting membrane potential (RMP). Therefore, the aim of this study was to investigate the time dependence of diet-induced cardiac lipid accumulation, CV disturbances, and changes in RMP and action potential (AP) morphology. Methods: Four-week-old male Sprague-Dawley rats were fed ad libitum with either normal chow or a diet consisting of 60% fat in the chow and 10% fructose in the drinking water. After 1, 3, and 6 weeks of feeding, we measured cardiac CV in right ventricular tissue strips with extracellular electrodes, AP morphology, and RMP using intracellular microelectrodes, and triglyceride content by thin layer chromatography. Results: CV increased from 68.5 0.24 cm/s to 77.7 0.23 cm/s from 1 to 6 weeks in control rats, whereas FFFRs failed to increase CV during the same period (68.9 0.23 cm/s after 1 week vs 67.9 0.23 cm/s after 6 weeks). Intramyocardial triglyceride levels were already significantly higher in FFFRs compared to controls, after 3 weeks of feeding, a time point when CV was not significantly different between controls and FFFRs (70.5 0.15 cm/s vs 70.3 0.20 cm/s). RMP and AP morphology were not different between the 2 groups at any point, except for time to 20% repolarization (APD20), which was significantly longer for FFFRs after 6 weeks (5.69 0.33 ms vs 4.62 0.43 ms). Conclusions: Rats on normal chow increase CV between 1 and 6 weeks of feeding (approximate age 5–6 and 10–11 weeks, respectively), whereas fructose-fat feeding prevents CV development. Lipid accumulation is already present after 3 weeks of fructose-fat feeding, preceding CV slowing. CV slowing could not be explained by changes in RMP or AP upstroke velocity.

Structural heterogeneity modulates effective refractory period: a mechanism of focal arrhythmia initiation.

Reductions in electrotonic loading around regions of structural and electrophysiological heterogeneity may facilitate capture of focal triggered activity, initiating reentrant arrhythmias. How electrotonic loading, refractoriness and capture of focal ectopics depend upon the intricate nature of physiological structural anatomy, as well as pathological tissue remodelling, however, is not well understood. In this study, we performed computational bidomain simulations with anatomically-detailed models representing the rabbit left ventricle. We used these models to quantify the relationship between local structural anatomy and spatial heterogeneity in action potential (AP) characteristics, electrotonic currents and effective refractory periods (ERPs) under pacing and restitution protocols. Regions surrounding vessel cavities, in addition to tissue surfaces, had significantly lower peak downstream electrotonic currents than well coupled myocardium ( vs A/cm2), with faster maximum AP upstroke velocities ( vs mV/ms), although noticeably very similar APDs ( vs ms) and AP restitution properties. Despite similarities in APDs, ERPs in regions of low electrotonic load in the vicinity of surfaces, intramural vessel cavities and endocardial structures were up to ms shorter compared to neighbouring well-coupled tissue, leading to regions of sharp ERP gradients. Consequently, focal extra-stimuli timed within this window of ERP heterogeneity between neighbouring regions readily induced uni-directional block, inducing reentry. Most effective induction sites were within channels of low ERPs between large vessels and epicardium. Significant differences in ERP driven by reductions in electrotonic loading due to fine-scale physiological structural heterogeneity provides an important mechanism of capture of focal activity and reentry induction. Application to pathological ventricles, particularly myocardial infarction, will have important implications in anti-arrhythmia therapy.

Specialized Functions of Nav1.5 and Nav1.9 Channels in Electrogenesis of Myenteric Neurons in Intact Mouse Ganglia

Voltage-gated sodium (Nav) channels play a central role in gastrointestinal physiology because they transmit depolarizing impulses in enteric neurons, thereby enabling the coordination of intestinal motility. However, little is known about the ion channel machinery that specifies firing pattern of enteric neurons. Here, we used in situ patch-clamp recording of myenteric neurons from mice to define functionally the Nav channel subtypes responsible for the electrical signature of myenteric neurons. We found that mouse myenteric neurons exhibit two types of tetrodotoxin-resistant Na(+) currents: an early inactivating Na(+) current (INaT) and a persistent Na(+) current (INaP). INaT was encountered in all myenteric neurons, whereas INaP was preferentially found in Dogiel type II sensory neurons. Knock-out mouse studies, in combination with pharmacological assays, indicate that INaT is carried by the Scn5a-encoded "cardiac" Nav1.5, whereas INaP is attributed to the Scn11a-encoded Nav1.9. Current-clamp experiments show that Nav1.9 flows at subthreshold voltages, generating tonic firing. In addition, action potential (AP) clamp reveals that Nav1.5 contributes to the upstroke velocity of APs, whereas Nav1.9, which remains active during the falling phase, opposes AP repolarization. We developed a computational model of a Dogiel type II myenteric neuron that successfully reproduces all experimentally observed phenomena and highlights the differential roles of Nav1.5 and Nav1.9 in the control of excitability. Our data illustrate how excitability can be finely tuned to provide specific firing templates by the selective deployment of Nav1.5 and Nav1.9 isoforms. We propose that Nav-dependent ENS disorders of excitability may play important roles in the pathogenesis of digestive diseases.

0150: PDZ domain proteins interacting with Nav1.5 differentially regulate Nav1.5 channel pools in mouse cardiomyocytes

The cardiac sodium channel Na V 1.5 initiates the action potential (AP) and is essential for conduction. The last three residues of Na V 1.5 (Ser-Ile-Val) constitute a PDZ domain-binding motif (SIV) that interacts with PDZ proteins such as syntrophin proteins and SAP97 defining distinct subsets of Na V 1.5 multi-protein complexes, respectively at the lateral membrane and the intercalated discs (ID) of cardiomyocytes. Previous results with dystrophin-deficient mice showed that disruption of the dystrophin-syntrophin macromolecular complex impairs excitability and impulse propagation by reducing the expression of Na V 1.5 and syntrophin at the lateral membrane of cardiomyocytes. Recently, we investigated the in vivo role of the Na V 1.5 SIV motif by characterizing mice bearing a truncation of this motif (ΔSIV). Western blots of ΔSIV hearts displayed reduced levels of Na V 1.5, which corresponded to a 35% decrease in cardiomyocyte wholecell I Na and AP upstroke velocity. Immunostainings revealed a specific loss of Na V 1.5 channels in ΔSIV cardiomyocytes at lateral membranes concomitant to a 60% decrease in I Na recorded at the lateral membrane. However, Na V 1.5 expression at ID and T-tubules was not altered. No I Na decrease was observed at the ID. Epicardial mapping of ΔSIV hearts showed decreased conduction velocity that manifested as prolongation of the QRS interval in ECGs. When SAP97 expression was constitutively suppressed in the heart of knock-out mice, I Na and AP maximal upstroke velocity were unchanged. However, AP duration was greatly increased, suggesting that the expression and function of other ion channels is modified. These data reflect the in vivo significance of the PDZ-domain-binding SIV motif in correct expression of Na V 1.5 channels at the lateral membrane and underline the functional role of lateral Na V 1.5 channels in cardiac conduction. In addition, the results confirm the existence of distinct pools of Na V 1.5 channels within the cardiomyocytes and suggest a complex regulation and organization of the Na V 1.5 pool at the ID, where compensatory mechanisms could be developped when SAP97 expression is constitutively suppressed.

Investigations of the Navβ1b sodium channel subunit in human ventricle; functional characterization of the H162P Brugada syndrome mutant

Brugada syndrome (BrS) is a rare inherited disease that can give rise to ventricular arrhythmia and ultimately sudden cardiac death. Numerous loss-of-function mutations in the cardiac sodium channel Nav1.5 have been associated with BrS. However, few mutations in the auxiliary Navβ1-4 subunits have been linked to this disease. Here we investigated differences in expression and function between Navβ1 and Navβ1b and whether the H162P/Navβ1b mutation found in a BrS patient is likely to be the underlying cause of disease. The impact of Navβ subunits was investigated by patch-clamp electrophysiology, and the obtained in vitro values were used for subsequent in silico modeling. We found that Navβ1b transcripts were expressed at higher levels than Navβ1 transcripts in the human heart. Navβ1 and Navβ1b coexpressed with Nav1.5 induced a negative shift on steady state of activation and inactivation compared with Nav1.5 alone. Furthermore, Navβ1b was found to increase the current level when coexpressed with Nav1.5, Navβ1b/H162P mutated subunit peak current density was reduced by 48% (-645 ± 151 vs. -334 ± 71 pA/pF), V1/2 steady-state inactivation shifted by -6.7 mV (-70.3 ± 1.5 vs. -77.0 ± 2.8 mV), and time-dependent recovery from inactivation slowed by >50% compared with coexpression with Navβ1b wild type. Computer simulations revealed that these electrophysiological changes resulted in a reduction in both action potential amplitude and maximum upstroke velocity. The experimental data thereby indicate that Navβ1b/H162P results in reduced sodium channel activity functionally affecting the ventricular action potential. This result is an important replication to support the notion that BrS can be linked to the function of Navβ1b and is associated with loss-of-function of the cardiac sodium channel.

Seasonal acclimatization of the cardiac action potential in the Arctic navaga cod (Eleginus navaga, Gadidae)

Freshwater fishes of north-temperate latitudes adjust electrical excitability of the heart to seasonal temperature changes by changing expression levels of ion channel isoforms. However, little is known about thermal responses of action potential (AP) in the hearts of marine polar fishes. To this end, we examined cardiac AP in the atrial myocardium of the Arctic navaga cod (Eleginus navaga) from the White Sea (Russia) acclimatized to winter (March) and summer (September) seasons. Acute increases in temperature from 4 to 10 °C were associated with increases in heart rate, maximum velocity of AP upstroke and negative resting membrane potential, while duration of AP was shortened in both winter-acclimatized and summer-acclimatized navaga hearts. In winter, there was a compensatory shortening (41.1%) of atrial AP duration and this was associated with a strong increase in transcript expression of Erg K(+) channels, known to produce the rapid component of the delayed rectifier K(+) current, I(Kr). Smaller increases were found in the expression of Kir2.1 channels that produce the inward rectifier K(+) current, I(K1). These findings indicate that the heart of navaga cod has a good acclimatory capacity in electrical excitation of cardiac myocytes, which enables cardiac function in the cold-eurythermal waters of the subarctic White Sea.