Guinea-pig Ventricle Research Articles

A Comparative Study of the Rapid (IKr) and Slow (IKs) Delayed Rectifier Potassium Currents in Undiseased Human, Dog, Rabbit, and Guinea Pig Cardiac Ventricular Preparations.

To understand the large inter-species variations in drug effects on repolarization, the properties of the rapid (IKr) and the slow (IKs) components of the delayed rectifier potassium currents were compared in myocytes isolated from undiseased human donor (HM), dog (DM), rabbit (RM) and guinea pig (GM) ventricles by applying the patch clamp and conventional microelectrode techniques at 37 °C. The amplitude of the E-4031-sensitive IKr tail current measured at -40 mV after a 1 s long test pulse of 20 mV, which was very similar in HM and DM but significant larger in RM and GM. The L-735,821-sensitive IKs tail current was considerably larger in GM than in RM. In HM, the IKs tail was even smaller than in DM. At 30 mV, the IKr component was activated extremely rapidly and monoexponentially in each studied species. The deactivation of the IKr component in HM, DM, and RM measured at -40 mV. After a 30 mV pulse, it was slow and biexponential, while in GM, the IKr tail current was best fitted triexponentially. At 30 mV, the IKs component activated slowly and had an apparent monoxponential time course in HM, DM, and RM. In contrast, in GM, the activation was clearly biexponential. In HM, DM, and RM, IKs component deactivation measured at -40 mV was fast and monoexponential, while in GM, in addition to the fast component, another slower component was also revealed. These results suggest that the IK in HM resembles that measured in DM and RM and considerably differs from that observed in GM. These findings suggest that the dog and rabbit are more appropriate species than the guinea pig for preclinical evaluation of new potential drugs expected to affect cardiac repolarization.

Vitamin C Deficiency Reduces Neurogenesis and Proliferation in the SVZ and Lateral Ventricle Extensions of the Young Guinea Pig Brain.

Although scurvy, the severe form of vitamin C deficiency, has been almost eradicated, the prevalence of subclinical vitamin C deficiency is much higher than previously estimated and its impact on human health might not be fully understood. Vitamin C is an essential molecule, especially in the central nervous system where it performs numerous, varied and critical functions, including modulation of neurogenesis and neuronal differentiation. Although it was originally considered to occur only in the embryonic brain, it is now widely accepted that neurogenesis also takes place in the adult brain. The subventricular zone (SVZ) is the neurogenic niche where the largest number of new neurons are born; however, the effect of vitamin C deficiency on neurogenesis in this key region of the adult brain is unknown. Therefore, through BrdU labeling, immunohistochemistry, confocal microscopy and transmission electron microscopy, we analyzed the proliferation and cellular composition of the SVZ and the lateral ventricle (LVE) of adult guinea pigs exposed to a vitamin-C-deficient diet for 14 and 21 days. We found that neuroblasts in the SVZ and LVE were progressively and significantly decreased as the days under vitamin C deficiency elapsed. The neuroblasts in the SVZ and LVE decreased by about 50% in animals with 21 days of deficiency; this was correlated with a reduction in BrdU positive cells in the SVZ and LVE. In addition, the reduction in neuroblasts was not restricted to a particular rostro–caudal area, but was observed throughout the LVE. We also found that vitamin C deficiency altered cellular morphology at the ultrastructural level, especially the cellular and nuclear morphology of ependymal cells of the LVE. Therefore, vitamin C is essential for the maintenance of the SVZ cell populations required for normal activity of the SVZ neurogenic niche in the adult guinea pig brain. Based on our results from the guinea pig brain, we postulate that vitamin C deficiency could also affect neurogenesis in the human brain.

Modulating cardiac conduction during metabolic ischemia with perfusate sodium and calcium in guinea pig hearts.

We previously demonstrated that altering extracellular sodium (Nao) and calcium (Cao) can modulate a form of electrical communication between cardiomyocytes termed "ephaptic coupling" (EpC), especially during loss of gap junction coupling. We hypothesized that altering Nao and Cao modulates conduction velocity (CV) and arrhythmic burden during ischemia. Electrophysiology was quantified by optically mapping Langendorff-perfused guinea pig ventricles with modified Nao (147 or 155 mM) and Cao (1.25 or 2.0 mM) during 30 min of simulated metabolic ischemia (pH 6.5, anoxia, aglycemia). Gap junction-adjacent perinexal width ( WP), a candidate cardiac ephapse, and connexin (Cx)43 protein expression and Cx43 phosphorylation at S368 were quantified by transmission electron microscopy and Western immunoblot analysis, respectively. Metabolic ischemia slowed CV in hearts perfused with 147 mM Nao and 2.0 mM Cao; however, theoretically increasing EpC with 155 mM Nao was arrhythmogenic, and CV could not be measured. Reducing Cao to 1.25 mM expanded WP, as expected during ischemia, consistent with reduced EpC, but attenuated CV slowing while delaying arrhythmia onset. These results were further supported by osmotically reducing WP with albumin, which exacerbated CV slowing and increased early arrhythmias during ischemia, whereas mannitol expanded WP, permitted conduction, and delayed the onset of arrhythmias. Cx43 expression patterns during the various interventions insufficiently correlated with observed CV changes and arrhythmic burden. In conclusion, decreasing perfusate calcium during metabolic ischemia enhances perinexal expansion, attenuates conduction slowing, and delays arrhythmias. Thus, perinexal expansion may be cardioprotective during metabolic ischemia. NEW & NOTEWORTHY This study demonstrates, for the first time, that modulating perfusate ion composition can alter cardiac electrophysiology during simulated metabolic ischemia.

The adhesion function of the sodium channel beta subunit (β1) contributes to cardiac action potential propagation.

Computational modeling indicates that cardiac conduction may involve ephaptic coupling - intercellular communication involving electrochemical signaling across narrow extracellular clefts between cardiomyocytes. We hypothesized that β1(SCN1B) -mediated adhesion scaffolds trans-activating NaV1.5 (SCN5A) channels within narrow (<30 nm) perinexal clefts adjacent to gap junctions (GJs), facilitating ephaptic coupling. Super-resolution imaging indicated preferential β1 localization at the perinexus, where it co-locates with NaV1.5. Smart patch clamp (SPC) indicated greater sodium current density (INa) at perinexi, relative to non-junctional sites. A novel, rationally designed peptide, βadp1, potently and selectively inhibited β1-mediated adhesion, in electric cell-substrate impedance sensing studies. βadp1 significantly widened perinexi in guinea pig ventricles, and selectively reduced perinexal INa, but not whole cell INa, in myocyte monolayers. In optical mapping studies, βadp1 precipitated arrhythmogenic conduction slowing. In summary, β1-mediated adhesion at the perinexus facilitates action potential propagation between cardiomyocytes, and may represent a novel target for anti-arrhythmic therapies.

Abstract 376: Acute Modulation of Sodium Channel Auxiliary Subunit β1-mediated Adhesion: a Novel Antiarrhythmic Strategy

Cardiac impulse propagation is thought to occur by direct cell to cell flow of current via connexin43 (Cx43) gap junctions (GJs). We recently demonstrated that cardiac sodium channels (Na v 1.5) localized within the perinexus, an intercalated disk (ID) nanodomain adjacent to Cx43 GJ, may enable ephaptic coupling between cardiac myocytes. We hypothesized that β1-mediated adhesion may closely approximate membranes within ID nanodomains, facilitating ephaptic coupling. Super-resolution STochastic Optical Reconstruction Microscopy-based Relative Localization Analysis (STORM-RLA) and immuno-electron microscopy identified two Na V 1.5 populations within the ID in guinea pig ventricles (GPVs): A perinexal population, accounting for 47% of ID-localized Na V 1.5 and a plicate population, co-distributing with N-Cadherin, accounting for 29%. β1 was preferentially localized to the perinexus (48% of ID-localized β1) over N-Cadherin-rich plicate regions (8%). βadp1, a novel peptide inhibitor of β1 adhesion, selectively and dose-dependently inhibited barrier function in β1-overexpressing 1610 cells in electric cell-substrate impedance spectroscopy studies. Neither βadp1 nor a scrambled control peptide (βadp1-scr) affected I Na or action potentials in isolated GPV myocytes. However, βadp1 reduced peak current recorded from Na V 1.5 clusters adjacent Cx43-EGFP at cell-cell contacts using scanning ion conductance microscopy-guided patch clamp. In GPVs, βadp1 (100 μM) compromised the diffusion-resistance of the ID as assessed by perfusion of fixable fluorescent dyes. βadp1 (48±4 μm) but not βadp1-scr (22±1 μm) increased perinexal intermembrane spacing compared to control GPVs (17±1μm). Optical mapping revealed that βadp1 but not βadp1-scr slowed conduction in GPVs and iPSC-derived cardiomyocyte monolayers. Importantly, in GPVs, βadp1 increased conduction anisotropy and precipitated spontaneous tachyarrhythmias in a dose-dependent manner. Thus, β1-mediated adhesion generates close apposition between Na V 1.5-rich perinexal membranes, facilitating ephaptic conduction in the heart. Importantly, β1-mediated adhesion may be a target for novel, mechanistically-driven antiarrhythmic therapy.

Argon Exposure Induces Postconditioning in Myocardial Ischemia-Reperfusion.

Cardioprotection against ischemia-reperfusion (I/R) damages remains a major concern during prehospital management of acute myocardial infarction. Noble gases have shown beneficial effects in preconditioning studies. Because emergency proceedings in the context of myocardial infarction require postconditioning strategies, we evaluated the effects of argon in such protocols on mammalian cardiac tissue. In rat, cardiac I/R was induced in vivo by transient coronary artery ligature and cardiac functions were evaluated by magnetic resonance imaging. Hypoxia-reoxygenation (H/R)-induced arrhythmias were evaluated in vitro using intracellular microelectrodes on both rat-isolated ventricle and a model of border zone in guinea pig ventricle. Hypoxia-reoxygenation loss of contractile force was assessed in human atrial appendages. In those models, postconditioning was induced by 5 minutes application of argon at the time of reperfusion. In the in vivo model, I/R produced left ventricular ejection fraction decrease (24%) and wall motion score increase (36%) which was prevented when argon was applied in postconditioning. In vitro, argon postconditioning abolished H/R-induced arrhythmias such as early after depolarizations, conduction blocks, and reentries. Recovery of contractile force in human atrial appendages after H/R was enhanced in the argon group, increasing from 51% ± 2% in the nonconditioned group to 83% ± 7% in the argon-treated group ( P < .001). This effect of argon was abolished in the presence of wortmannin and PD98059 which inhibit prosurvival phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt) and MEK/extracellular receptor kinase 1/2 (ERK 1/2), respectively, or in the presence of the mitochondrial permeability transition pore opener atractyloside, suggesting the involvement of the reperfusion injury salvage kinase pathway. Argon has strong cardioprotective properties when applied in conditions of postconditioning and thus appears as a potential therapeutic tool in I/R situations.

The Sodium Channel Auxiliary Subunit β1 is Structurally Critical for Cardiac Conduction: Evidence from the Single Molecule Scale to the Whole Organ

Electrical impulse propagation in the heart's ventricles is thought to occur by means of direct flow of electric current from cell to cell via gap junctions (GJs) composed of connexin43 (Cx43). We recently demonstrated that cardiac sodium channels (Nav1.5) localized within the perinexus, an intercalated disk (ID) nanodomain adjacent to Cx43 GJ, may enable ephaptic coupling between cardiac myocytes. We hypothesized that the cell adhesion function of β1 may contribute to close intermembrane apposition within these nanodomains and thereby facilitate ephaptic coupling. Super-resolution STochastic Optical Reconstruction Microscopy-based Relative Localization Analysis (STORM-RLA) identified two populations of NaV1.5 within the ID in guinea pig ventricles: One, localized to the perinexus, within 200nm of Cx43 GJs, accounted for 47% of ID-localized NaV1.5 and the other, which co-distributed with N-Cadherin to plicate ID regions, accounted for 29% of ID-localized NaV1.5. These findings were confirmed by immuno-electron microscopy. Confocal microscopy revealed β1 enrichment at the ID, and suggested preferential localization to GJ-enriched interplicate ID regions. STORM-RLA confirmed the preferential localization of β1 to interplicate ID regions: Perinexal nanodomains accounted for 48% of ID-localized β1, whereas N-Cadherin-rich plicate regions accounted for only 8%. In order to test the role of β1 adhesion in the heart, we developed a novel peptide inhibitor of β1 adhesion - βadp1. In electric cell-substrate impedance spectroscopy studies, it inhibited the barrier function in β1-overexpressing 1610 cells but not in native 1610 cells. In guinea pig ventricles, βadp1 (100 µM) compromised the diffusion-resistance of the ID as assessed by perfusion of fixable fluorescent dyes: A 0.5kD molecule preferentially accumulated within IDs in control hearts but not in βadp1-treated hearts. Perinexal intermembrane spacing measured by transmission electron microscopy was increased in βadp1-treated hearts (48 ± 4 µm) compared to untreated hearts (17± 1µm) and those treated with a scrambled control peptide, βadp1-scr (22 ± 1 µm). Notably, neither peptide affected membrane spacing at other ID locations. In optical mapping studies, βadp1 but not βadp1-scr preferentially decreased transverse conduction velocity, increased anisotropy and precipitated spontaneous tachyarrhythmias in a dose-dependent manner. In conclusion, these results suggest a critical role of β1-mediated adhesion in generating close apposition between NaV1.5-rich perinexal membranes and thereby, facilitating ephaptic conduction in the heart. Further, these data highlight β1-mediated adhesion as a target for novel, mechanistically-driven antiarrhythmic therapy.

Abstract 13129: Acute Inhibition of Sodium Channel Beta Subunit (β1) -mediated Adhesion is Highly Proarrhythmic

Introduction: The sodium channel auxiliary subunit and cell adhesion molecule β1 (SCN1b) is enriched at cardiomyocyte intercalated disks (ID) along with the pore-forming subunit Na V 1.5. We recently demonstrated that the perinexus, an Na V 1.5-rich ID nanodomain located near connexin43 (Cx43) gap junctions (GJ), is important for cell-to-cell electrical coupling. Disrupting close membrane apposition within the perinexus was highly proarrhythmic. Hypothesis: β1-mediated adhesion is key to close membrane apposition within the perinexus; therefore, inhibiting β1-mediated adhesion may disrupt perinexal structure, impair conduction and precipitate arrhythmias. Methods and Results: Na V 1.5 and β1 localization within ID nanodomains was quantitatively assessed from super-resolution STochastic Optical Reconstruction Microscopy (STORM) images of en face IDs from adult guinea pig ventricles. Na V 1.5 and β1 were located 63±15 and 75±25 nm from Cx43 GJ; 31% of both Na V 1.5 and β1 localized to GJ perinexi. β1-mediated adhesion was acutely inhibited by βadp1, a novel peptide mimetic of β1’s putative extracellular adhesion domain. βadp1 decreased cellular adhesion as assessed by Electric Cell-Substrate Impedance Spectroscopy (ECIS) in β1-overexpressing 1610 cells in a dose-dependent manner but not in native 1610 cells. Perinexal intermembrane spacing as measured by Transmission Electron Microscopy (TEM) increased 3-fold from 17±1 nm in vehicle controls (0.25% DMSO) to 49±5 nm (p&lt;0.05) in 100 μM βadp1-treated hearts; however, intermembrane spacing at other ID locations was unaltered (22±1 vs. 24±1 nm). Optical mapping revealed preferential slowing of transverse conduction (8.0±0.7 vs. 16.5±1.2 cm/s, p&lt;0.05) and increased anisotropy (3.9±0.2 vs. 2.6±0.2, p&lt;0.05) in βadp1-treated hearts relative to vehicle controls. Further, 3 of 4 βadp1-treated hearts exhibited spontaneous ventricular tachycardias. Conclusions: These data support a role for β1-mediated adhesion in maintaining close apposition between Na V 1.5-rich perinexal membranes. Importantly, acute inhibition of β1-mediated adhesion resulted in disruption of close membrane apposition within the GJ perinexus and precipitated severe conduction slowing and spontaneous arrhythmias.

Abstract 13422: Novel Calcium Therapy for Preserving Cardiac Conduction During Myocardial Inflammation

Introduction: Diseases like ischemia, myocarditis and heart failure are associated with myocardial inflammation and elevated cytokines such as Tumor Necrosis Factor α (TNFα). TNFα modulates gap junctional coupling (GJC) which may then alter cardiac conduction velocity (CV) . Yet, the relationship between TNFα and GJC is controversial. TNFα also modulates extracellular edema, and edema slows CV via an alternative mode of intercellular communication called ephaptic coupling (EpC). However, the relationship between TNFα, CV, GJC, and EpC remains poorly understood. Hypothesis: TNFα modulates cardiac conduction velocity (CV) by altering EpC. Methods: CV was quantified from optically mapped guinea pig ventricles. The width of the candidate cardiac ephapse called the perinexus (WP) was measured by transmission electron microscopy. Connexin43 (Cx43) expression, phosphorylation and distribution around the myocyte was determined by western blotting and confocal immunofluorescence. Results: TNFα anisotropically slowed conduction at 90 minutes relative to time controls (0.21±0.01 vs 0.24±0.01m/s). While TNFα increased WP and reduced EpC (24.2±1.12 vs 18.6±0.86 nm), TNFα did not significantly modify total Cx43 expression or phosphorylation, although Cx43 localization at the intercalated disc was reduced relative to time controls (81% vs 86%). Physiologically elevating calcium concentration in the presence of TNFα (TNFα + 2.5mM calcium) restored CV to time control values within 15 mins and further improved conduction above time control values at 90 mins. This CV increase correlated with restoration of WP to control values at both 0 and 90 mins. Interestingly, at 90 mins, TNFα + 2.5mM calcium also increased total Cx43 expression as well as intercalated disc localization relative to 90mins of TNFα alone(86%). Conclusions: TNFα reduces CV by increasing WP and redistributing Cx43. With TNFα, 2.5mM calcium acutely (15 mins) improves CV presumably by increasing EpC, and over 90 mins, improves CV above control values possibly by the increase in both EpC and GJC . Taken together, the data suggests that physiologic ionic modulation could be an effective therapy to prevent cardiac arrhythmias.

Blocking effect of carvedilol, a beta‐blocker on Na+/Ca2+ exchange current in isolated cardiac ventricular cells of guinea pig

BackgroundCarvedilol has a1, b1, and b2‐adrenergic blocking effects together with the effects such as vasodilating, anti‐ischemic, anti‐oxidant, anti‐apoptotic, anti‐proliferate and anti‐inflammatory activities. Carvedilol inhibited IKr, IKs, ICa‐L and Ito in rabbit cardiac ventricular myocytes. On the other hand, metoprolol, a nonselective b‐blocker also has blocking several channels, including IK1, ICa and Ito in hearts. Murayama et al. (2013) reported that carvedilol and its analogues inhibit delayed after depolarizations (DADs) in Langendorff‐perfused rabbit hearts. It is well known that DADs are caused by the Na+/Ca2+ exchange current (INCX).MethodsWe examined the effects of carvedilol and metoprolol, two b‐blockers, on INCX by using the whole‐cell voltage‐clamp method in isolated guinea pig ventricles and CCL39 fibroblast cells expressing dog cardiac Na+/Ca2+ exchanger (NCX1).ResultsCarvedilol suppressed INCX in a concentration‐dependent manner but metoprolol did not in isolated guinea pig cardiac ventricular myocytes. IC50 values for the Ca2+ influx (outward) and efflux (inward) components of INCX were 69.7 μM and 61.5 μM, respectively. Carvedilol at 100 μM inhibited INCX in CCL39 cells expressing wild type NCX1 similarly to mutant NCX1 without the intracellular regulatory loop. Carvedilol at 30 μM abolished ouabain‐induced DADs in isolated cardiac ventricular myocytes.ConclusionsCarvedilol suppressed bi‐directional INCX in a concentration‐dependent manner with the IC50 value of approximately 65 μM. We conclude that carvedilol inhibits NCX1 at supratherapeutic concentrations.

Heart Rate and Extracellular Sodium and Potassium Modulation of Gap Junction Mediated Conduction in Guinea Pigs.

Background: Recent studies suggested that cardiac conduction in murine hearts with narrow perinexi and 50% reduced connexin43 (Cx43) expression is more sensitive to relatively physiological changes of extracellular potassium ([K+]o) and sodium ([Na+]o).Purpose: Determine whether similar [K+]o and [Na+]o changes alter conduction velocity (CV) sensitivity to pharmacologic gap junction (GJ) uncoupling in guinea pigs.Methods: [K+]o and [Na+]o were varied in Langendorff perfused guinea pig ventricles (Solution A: [K+]o = 4.56 and [Na+]o = 153.3 mM. Solution B: [K+]o = 6.95 and [Na+]o = 145.5 mM). Gap junctions were inhibited with carbenoxolone (CBX) (15 and 30 μM). Epicardial CV was quantified by optical mapping. Perinexal width was measured with transmission electron microscopy. Total and phosphorylated Cx43 were evaluated by western blotting.Results: Solution composition did not alter CV under control conditions or with 15μM CBX. Decreasing the basic cycle length (BCL) of pacing from 300 to 160 ms decreased CV uniformly with both solutions. At 30 μM CBX, a change in solution did not alter CV either longitudinally or transversely at BCL = 300 ms. However, reducing BCL to 160 ms caused CV to decrease more in hearts perfused with Solution B than A. Solution composition did not alter perinexal width, nor did it change total or phosphorylated serine 368 Cx43 expression. These data suggest that the solution dependent CV changes were independent of altered perinexal width or GJ coupling. Action potential duration was always shorter in hearts perfused with Solution B than A, independent of pacing rate and/or CBX concentration.Conclusions: Increased heart rate and GJ uncoupling can unmask small CV differences caused by changing [K+]o and [Na+]o. These data suggest that modulating extracellular ionic composition may be a novel anti-arrhythmic target in diseases with abnormal GJ coupling, particularly when heart rate cannot be controlled.

Abstract 11138: Superresolution Studies of Sodium Channels Within Intercalated Disk Microdomains Suggest Novel Arrhythmia Mechanism

Pore-forming (Nav1.5) and auxiliary (β1; SCN1b) subunits of cardiac sodium channels are enriched at the cardiomyocyte intercalated disk (ID). Mathematical models suggest that this may facilitate conduction via ephaptic mechanisms. We recently demonstrated Nav1.5 enrichment (gSTED superresolution microscopy) and close membrane apposition (&lt;10 nm; electron microscopy) within the perinexus, a microdomain surrounding connexin43 (Cx43) gap junctions (GJ). These data identified the perinexus as a candidate structure for the cardiac ephapse. Further studies using gSTED and STORM superresolution microscopy revealed Nav1.5 and β1 enrichment within ID regions not containing dense clusters of Cx43 and N-Cadherin. Notably, both were identified within the perinexus: Overall, 22% of Nav1.5 &amp; β1 were located within perinexal regions while only 2 and 5% respectively overlapped with Cx43 clusters. Importantly, acute interstitial edema (AIE) increased intermembrane distance at perinexal, but not at non-perinexal sites in adult guinea pig myocardium. Functionally, this correlated with decreased transverse conduction velocity (CV-T; 15.2±0.3 vs. 19.6±0.1cm/s) and increased anisotropic ratio (AR; 3.0±0.2 vs. 2.8±0.1) relative to control, in perfused guinea pig ventricles. Nav1.5 blockade (0.5 μM flecainide) by itself decreased CV (18%) without changing AR. However, Nav1.5 inhibition during AIE preferentially decreased CV-T (13.0±0.6cm/s), increased AR (3.3±0.2) and increased spontaneous arrhythmias (7/9 vs. 4/11) compared to AIE alone. Notably, only a computer model including ephaptic coupling and the ID localization of Nav1.5 could recapitulate these results. Next we investigated the role of β1 in ephaptic coupling: Electrical cell-substrate impedance spectroscopy of 1610 cells heterologously overexpressing β1 revealed 3-fold higher paracellular resistance relative to native 1610 cells. These data along with the known cell adhesion function of β1 in neural tissue suggest that β1-mediated adhesion may facilitate close membrane apposition within the perinexus. Taken together, our results identify β1-mediated adhesion as a novel determinant of anisotropic conduction and potential antiarrhythmic target.

Superresolution Microscopy Reveals Sodium Channel Localization within Intercalated Disk Microdomains: Implications for Ephaptic Coupling

Pore-forming (Nav1.5) and auxiliary (β1; SCN1b) subunits of cardiac sodium channels are enriched at the cardiomyocyte intercalated disk (ID). Mathematical models suggest that this may facilitate conduction via ephaptic mechanisms. We previously demonstrated anisotropic conduction slowing during acute interstitial edema (AIE), possibly due to weakened ephaptic coupling. Here we assessed Nav1.5 and β1 localization to ID microdomains using electron microscopy (EM) and super-resolution microscopy (gSTED, STORM) and used optical mapping and computer modeling to investigate the implications for ephaptic conduction in the heart. gSTED and STORM revealed Nav1.5 and β1 enrichment within ID regions not containing dense clusters of Cx43 and N-Cadherin. Notably, both were identified within the perinexus, a microdomain surrounding Cx43 gap junctions. Overall, 22% of Nav1.5 was located within perinexal regions while only 2% was within Cx43 clusters. EM revealed closer membrane apposition at perinexal ( 10nm) under control conditions. AIE increased intermembrane distance at perinexal, but not at non-perinexal sites. Functionally, this correlated with decreased transverse conduction velocity (CV-T; 15.2±0.3 vs. 19.6±0.1cm/s) and increased anisotropic ratio (AR; 3.0±0.2 vs. 2.8±0.1) relative to control, in perfused guinea pig ventricles. Next, we investigated AIE effects on Nav1.5 function in conduction. Nav1.5 blockade (0.5 µM flecainide) by itself decreased CV (18%) without changing AR. However, Nav1.5 inhibition during AIE preferentially decreased CV-T (13.0±0.6cm/s), increased AR (3.3±0.2) and increased spontaneous arrhythmias (7/9 vs. 4/11) compared to AIE alone. Notably, only a computer model including ephaptic coupling and the ID localization of Nav1.5 could recapitulate these results. In summary, sodium channel complexes localized to ID microdomains such as the perinexus may enable ephaptic conduction in the heart. Further, Nav1.5 functional availability and perinexal membrane spacing emerge as novel determinants of anisotropic conduction.

Mechanoelectric feedback does not contribute to the Frank-Starling relation in the rat and guinea pig heart

Mechanoelectric feedback (MEF) is the process by which mechanical forces on the myocardium can alter its electrical properties. The effect can be large enough to induce ectopic beats or fibrillation. However, the role of MEF at physiological levels of mechanical stress is not clear. We have investigated alteration in action potential morphology in rat and guinea pig ventricle and in rat atrial tissue at levels of stretch near the plateau of the Frank-Starling curve. Stretch of &gt;100 mm.Hg End Diastolic Left Ventricular Pressure (EDLVP) or rapidly applied stretch (EDLVP increased by 25 mm.Hg within 100 ms) often triggered ectopic beats in isolated rat and guinea-pig hearts. However, ventricular epicardial monophasic action potentials (MAPs) recorded during stretch to EDLVP up to 30 mm. Hg showed no consistent changes in action potential duration (at APD20, APD50 or APD80) in either species. MAP recording detected APD prolongation with very small concentrations of 4-AP (10 μM), confirming the discrimination of the recording technique. In isolated rat atrial strips, no changes in intracellular action potential morphology or membrane potential were seen when stretched to levels producing an optimum increase in contractility. We conclude that alteration in action potential morphology with stretch does not contribute to the Frank-Starling relation in ventricle of rat or guinea-pig isolated heart, or in rat atrial tissue.

Direct conversion of quiescent cardiomyocytes to pacemaker cells by expression of Tbx18

The heartbeat originates within the sinoatrial node (SAN), a small structure containing <10,000 genuine pacemaker cells. If the SAN fails, the ∼5 billion working cardiomyocytes downstream of it become quiescent, leading to circulatory collapse in the absence of electronic pacemaker therapy. Here we demonstrate conversion of rodent cardiomyocytes to SAN cells in vitro and in vivo by expression of Tbx18, a gene critical for early SAN specification. Within days of in vivo Tbx18 transduction, 9.2% of transduced, ventricular cardiomyocytes develop spontaneous electrical firing physiologically indistinguishable from that of SAN cells, along with morphological and epigenetic features characteristic of SAN cells. In vivo, focal Tbx18 gene transfer in the guinea-pig ventricle yields ectopic pacemaker activity, correcting a bradycardic disease phenotype. Myocytes transduced in vivo acquire the cardinal tapering morphology and physiological automaticity of native SAN pacemaker cells. The creation of induced SAN pacemaker (iSAN) cells opens new prospects for bioengineered pacemakers.

Potassium channel activators differentially modulate the effect of sodium channel blockade on cardiac conduction

Diminished repolarization reserve contributes to the arrhythmogenic substrate in many disease states. Pharmacological activation of K(+) channels has been suggested as a potential antiarrhythmic therapy in such conditions. Having previously demonstrated that I(K1) and I(Kr) can modulate cardiac conduction, we tested here the effects of pharmacological I(KATP) and I(Ks) activation on cardiac conduction and its dependence on the sodium current (I(Na)). Bath electrocardiograms (ECGs) recorded from Langendorff-perfused guinea pig ventricles revealed QRS prolongation during I(KATP) activation by pinacidil but not during I(Ks) activation by R-L3 relative to control. In contrast, when I(Na) was partially blocked by flecainide, R-L3 but not pinacidil prolonged the QRS relative to flecainide alone. Conduction velocity (θ) was quantified by optical mapping during epicardial pacing. Both longitudinal (θ(L)) and transverse (θ(T)) θ were reduced by pinacidil (by 10 ± 1 and 9 ± 3%, respectively) and R-L3 (by 11 ± 2% and 15 ± 4%, respectively). Flecainide decreased θ(L) by 33 ± 4% and θ(T) by 36 ± 5%. Whereas pinacidil did not further slow θ relative to flecainide alone, R-L3 decreased both θ(L) and θ(T). Pharmacological activation of I(KATP) and I(Ks) slows cardiac conduction; however, they demonstrate diverse effects on θ dependence on I(Na) blockade. These findings may have significant implications for the use of K(+) channel activators as antiarrhythmic drugs and for patients with Na(+) channel abnormalities or being treated with Na(+) channel blockers.

A Novel Role for Ephaptic Coupling in Cardiac Conduction: An Experimental and Modeling Study

Introduction: We demonstrated that edema slows conduction (θ and increases anisotropy (ARθ). Existing mathematical models incorporating only gap junctional (Gj) coupling cannot explain these data. We hypothesized that inhibiting ion channels during edema would unmask ephaptic coupling effects. Methods: θ and ARθ were quantified by optical mapping in Langendorff-perfused guinea pig ventricles. Mannitol (26.1g/l) was perfused to increase VIS. INa, which partially colocalizes with Gj, was inhibited by flecainide (0.5μM). IK1, which exhibits low intercalated disk colocalization with Gj, was inhibited by BaCl2 (10 μM). Conduction was mathematically modeled in a 2D tissue slab with both Gj and ephaptic coupling and appropriate INa and IK1 cellular distributions. Results: During control, longitudinal (θL) and transverse (θT) θ were 51.9±1.0cm/s and 21.2±0.6cm/s respectively; ARθ was 2.5±0.1. Flecainide decreased both θL and θT by 17±2% (n=5, p<0.05 vs. control). BaCl2 increased both θL and θT by 23±2% (n=4, p<0.05 vs. control). Neither altered ARθ (p=ns vs. control). Mannitol decreased θL (16±3%, n=3, p<0.05) and θT (26±2%, n=3, p<0.05) and increased ARθ to 3.0±0.1 (n=3, p<0.05), relative to control. Importantly, flecainide + mannitol decreased θL and θT by 17±2 and 9±2% respectively (p<0.05), raising ARθ to 3.3±0.2 (p<0.05) relative to mannitol alone. BaCl2 + mannitol increased θT (by 15±3%, p<0.05) but not θL relative to mannitol alone, thereby lowering ARθ to 2.6±0.1 (p<0.05). Interestingly, the computer model of edema and ephaptic coupling recapitulated experimental findings where reducing INa decreased θ and increased ARθ while reducing IK1 increased θ and decreased ARθ. Conclusions: INa and IK1 blockade isotropically affected θ by themselves, but preferentially altered θT during edema. This is the first demonstration that altering ionic currents can alter ARθ, and the mechanism may involve ephaptic coupling.

Lineage Reprogramming from Cardiomyocytes to Pacemaker Cells via a Single Transcription Factor

The heartbeat originates in a small number of highly-specialized pacemaker cells found in the sinoatrial node (SAN). In an effort to create pacemaker cells from working cardiomyocytes, we expressed Tbx18, a transcription factor indispensible for embryonic development of the SAN. Within days, Tbx18-transduced neonatal rat cardiomyocytes (NRVMs) exhibited rhythmic spontaneous electrical firing. The mechanism of induced pacemaker activity resembled that of SAN cells, with enhanced voltage- and Ca2+-dependent “clock” pathways. The maximum diastolic potential was more positive in Tbx18-NRVMs in comparison to controls (−47±10 mV vs. −73±6 mV) with accompanying reduction in IK1. Line-scan confocal imaging of Tbx18-NRVMs loaded with Rhod-2 resolved localized Ca2+ release events (LCRs) that preceded each whole-cell Ca2+ transient resembling LCRs observed in native SAN cells. LCRs were not detected in control NRVMs. Spontaneous Ca2+-transients in Tbx18-NRVMs were suppressed on superfusion with ryanodine (10 μM) by 47±6% in comparison to 12±2% suppression in controls, indicative of increased relevance of [Ca2+]i stores. A 2.3-fold increase in [Ca2+]i stores was observed in Tbx18-NRVMs which can be attributed to increased sarcoplasmic reticulum Ca2+-ATPase 2a (SERCA2a) activity due to downregulation or phosphorylation of phospholamban (PLN), an inhibitor of SERCA. Tbx18-overexpression led to relative increase in the protein level of phosphorylated PLN (Ser16) similar to higher p-PLN level observed in the adult SAN relative to the left ventricle. In addition, Tbx18-NRVMs exhibited increased levels of cAMP and HCN4, shrank in size with disorganized myofibrils, and underwent epigenetic modifications consistent with durable reprogramming. Focal Tbx18 gene transfer in the guinea-pig ventricle consistently created ectopic pacemaker activity in vivo with a rhythm of 40±20 beats/min (n=5/7). Taken together, the data demonstrate faithful reprogramming of ventricular myocytes to pacemaker cells by a single transcription factor, Tbx18.

NCX is an important determinant for premature ventricular activity in a drug-induced model of Andersen–Tawil syndrome

Andersen-Tawil syndrome (ATS1)-associated ventricular arrhythmias are initiated by premature ventricular activity (PVA) resulting from diastolic Ca(2+) (Ca(D)) accumulation. We hypothesized that relatively high Na(+)-Ca(2+) exchanger (NCX) expression coupled with slower Ca(2+) uptake may constitute an arrhythmogenic substrate during drug-induced ATS1 (DI-ATS1). DI-ATS1 was induced with 10 µmol/L BaCl(2) and 2 mmol/L [K(+)](o). Ca(2+) transients and action potentials were optically mapped from Langendorff-perfused guinea pig ventricles. Intracellular Ca(2+) handling was modulated by either direct NCX inhibition with 5 µmol/L KB-R7943 or by sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2a) inhibition with cyclopiazonic acid (CPA). During DI-ATS1, PVA was more frequent in left ventricular (LV)-base (LVB) vs. LV-apex (LVA) (2.2 ± 0.8 vs. 0.6 ± 0.3 PVA/10 min), consistent with greater Ca(D) (1.65 ± 0.13 vs. 1.42 ± 0.09 normalized-Ca(D) units) and western blot-assessed NCX protein expression (81.2 ± 30.9%) in LVB relative to LVA. Further, regions of high NCX (LVB) evidenced a shorter PVA coupling interval relative to regions of low NCX expression (LVA, 67.7 ± 3.5 vs. 78.5 ± 3.6%). Inhibiting NCX during DI-ATS1 lowered the incidence of ventricular tachycardias (VTs, 0 vs. 25%) and PVA (1.5 ± 0.4 vs. 4.3 ± 1.4 PVA/10 min), but it did not affect PVA coupling intervals in LVB nor LVA (70.8 ± 4.3 vs. 73.8 ± 2.5%). Conversely, inhibition of SERCA2a with CPA, thereby increasing the role of NCX in Ca(2+) handling, significantly increased the incidence of VTs and PVA relative to DI-ATS1 alone, while decreasing the PVA coupling interval in all regions. PVA preferentially occurs in regions of enhanced NCX expression with relatively slower Ca(2+) uptake and during perfusion of CPA which further reduces sarcoplasmic reticular Ca(2+) uptake.

The alteration of asymmetric dimetilarginine (ADMA) levels in cardiac and gastrocnemius muscles following radioactive iodine application in guinea pigs and the effect of L-carnitine on this alteration

Purpose: Tissue levels of asymmetric dimetilarginine (ADMA) and symmetric dimetilarginine (SDMA) were investigated in cardiac ventricle and gastrocnemius muscles of guinea pigs treated with radioactive iodine (RAI) alone or in combination with L-carnitine (LC).Material and methods: Group 1 received no treatment (control group). Group 2 received a total dose of 30 mCi−1kg−1 body weight iodine-131 alone. Group 3 received 200 mg−1kg−1 of LC for 10 days alone. Group 4 received 200 mg−1kg−1 of LC plus RAI therapy. Free thyroid hormones, ADMA and SDMA concentrations were measured.Results: Serum free thyroid hormone concentrations were found decreased in the RAI and LC-RAI groups after RAI application. A significant decrease in ADMA and SDMA concentration was observed in ventricle muscle following RAI application. The LC-RAI group had significantly decreased ADMA levels in ventricle muscle compared with those of the control group. Similarly, SDMA concentrations in ventricle and gastrocnemius muscles of the LC-RAI groups were significantly lower than those of the control groups.Conclusions: Our results indicated that RAI appears to exert an inhibitory effect on ADMA and SDMA levels of ventricular muscle. LC administration when given adjuvant to RAI therapy may cause a marked decrease in ADMA concentrations of both ventricular and gastrocnemius muscles.