Rabbit Ventricular Myocytes Research Articles

Wenxin Keli diminishes Ca2+ overload induced by hypoxia/reoxygenation in cardiomyocytes through inhibiting INaL and ICaL.

An increase in the late sodium current (INaL ) causes intracellular Na+ overload and subsequently intracellular Ca2+ ([Ca2+ ]i ) overload via the stimulated reverse Na+ -Ca2+ exchange (NCX). Wenxin Keli (WXKL) is an effective antiarrhythmic Chinese herb extract, but the underlying mechanisms are unclear. The INaL , NCX current (INCX ), L-type Ca2+ current (ICaL ), and action potentials were recorded using the whole-cell patch-clamp technique in rabbit ventricular myocytes. Myocyte [Ca2+ ]i transients were measured using a dual excitation fluorescence photomultiplier system. WXKL decreased the enhanced INaL , reverse INCX , diastolic [Ca2+ ]i , and the amplitude of Ca2+ transients induced by sea anemone toxin II (ATX II, a specific INaL channel opener) in a concentration-dependent manner. Hypoxia increased INaL , INCX , and diastolic [Ca2+ ]i , and decreased amplitude of [Ca2+ ]i transients. Hypoxia-reoxygenation aggravated these changes and induced spontaneous [Ca2+ ]i transients and hypercontraction in 86% cells (6/7). The application of WXKL during hypoxia or reoxygenation periods decreased the increased INaL , INCX , and diastolic [Ca2+ ]i , and prevented those events in 82% cells (9/11) under hypoxia-reoxygenation conditions. WXKL also inhibited the ICaL in a dose-dependent manner. Furthermore, WXKL shortened the action potential duration and completely abolished ATX II-induced early afterdepolarizations from 9/9 to /9. In isolated heart electrocardiogram recordings, WXKL inhibited ischemia-reperfusion induced ventricular premature beats and tachycardia. WXKL attenuated [Ca2+ ]i overload induced by hypoxia-reoxygenation in ventricular myocytes through inhibiting INaL and ICaL and prevents arrhythmias. This could, at least partly, contribute to the antiarrhythmic effects of WXKL.

Barbaloin inhibits ventricular arrhythmias in rabbits by modulating voltage-gated ion channels.

Barbaloin (10-β-D-glucopyranosyl-1,8-dihydroxy-3-(hydroxymethyl)-9(10H)-anthracenone) is extracted from the aloe plant and has been reported to have anti-inflammatory, antitumor, antibacterial, and other biological activities. Here, we investigated the effects of barbaloin on cardiac electrophysiology, which has not been reported thus far. Cardiac action potentials (APs) and ionic currents were recorded in isolated rabbit ventricular myocytes using whole-cell patch-clamp technique. Additionally, the antiarrhythmic effect of barbaloin was examined in Langendorff-perfused rabbit hearts. In current-clamp recording, application of barbaloin (100 and 200 μmol/L) dose-dependently reduced the action potential duration (APD) and the maximum depolarization velocity (Vmax), and attenuated APD reverse-rate dependence (RRD) in ventricular myocytes. Furthermore, barbaloin (100 and 200 μmol/L) effectively eliminated ATX II-induced early afterdepolarizations (EADs) and Ca2+-induced delayed afterdepolarizations (DADs) in ventricular myocytes. In voltage-clamp recording, barbaloin (10-200 μmol/L) dose-dependently inhibited L-type calcium current (ICa.L) and peak sodium current (INa.P) with IC50 values of 137.06 and 559.80 μmol/L, respectively. Application of barbaloin (100, 200 μmol/L) decreased ATX II-enhanced late sodium current (INa.L) by 36.6%±3.3% and 71.8%±6.5%, respectively. However, barbaloin up to 800 μmol/L did not affect the inward rectifier potassium current (IK1) or the rapidly activated delayed rectifier potassium current (IKr) in ventricular myocytes. In Langendorff-perfused rabbit hearts, barbaloin (200 μmol/L) significantly inhibited aconitine-induced ventricular arrhythmias. These results demonstrate that barbaloin has potential as an antiarrhythmic drug.

Caveolae in Rabbit Ventricular Myocytes: Distribution and Dynamic Diminution after Cell Isolation

Caveolae are signal transduction centers, yet their subcellular distribution and preservation in cardiac myocytes after cell isolation are not well documented. Here, we quantify caveolae located within 100 nm of the outer cell surface membrane in rabbit single-ventricular cardiomyocytes over 8 h post-isolation and relate this to the presence of caveolae in intact tissue. Hearts from New Zealand white rabbits were either chemically fixed by coronary perfusion or enzymatically digested to isolate ventricular myocytes, which were subsequently fixed at 0, 3, and 8 h post-isolation. In live cells, the patch-clamp technique was used to measure whole-cell plasma membrane capacitance, and in fixed cells, caveolae were quantified by transmission electron microscopy. Changes in cell-surface topology were assessed using scanning electron microscopy. In fixed ventricular myocardium, dual-axis electron tomography was used for three-dimensional reconstruction and analysis of caveolae in situ. The presence and distribution of surface-sarcolemmal caveolae in freshly isolated cells matches that of intact myocardium. With time, the number of surface-sarcolemmal caveolae decreases in isolated cardiomyocytes. This is associated with a gradual increase in whole-cell membrane capacitance. Concurrently, there is a significant increase in area, diameter, and circularity of sub-sarcolemmal mitochondria, indicative of swelling. In addition, electron tomography data from intact heart illustrate the regular presence of caveolae not only at the surface sarcolemma, but also on transverse-tubular membranes in ventricular myocardium. Thus, caveolae are dynamic structures, present both at surface-sarcolemmal and transverse-tubular membranes. After cell isolation, the number of surface-sarcolemmal caveolae decreases significantly within a time frame relevant for single-cell research. The concurrent increase in cell capacitance suggests that membrane incorporation of surface-sarcolemmal caveolae underlies this, but internalization and/or micro-vesicle loss to the extracellular space may also contribute. Given that much of the research into cardiac caveolae-dependent signaling utilizes isolated cells, and since caveolae-dependent pathways matter for a wide range of other study targets, analysis of isolated cell data should take the time post-isolation into account.

A model of cardiac ryanodine receptor gating predicts experimental Ca2+-dynamics and Ca2+-triggered arrhythmia in the long QT syndrome

Abnormal Ca2+ handling is well-established as the trigger of cardiac arrhythmia in catecholaminergic polymorphic ventricular tachycardia and digoxin toxicity, but its role remains controversial in Torsade de Pointes (TdP), the arrhythmia associated with the long QT syndrome (LQTS). Recent experimental results show that early afterdepolarizations (EADs) that initiate TdP are caused by spontaneous (non-voltage-triggered) Ca2+ release from Ca2+-overloaded sarcoplasmic reticulum (SR) rather than the activation of the L-type Ca2+-channel window current. In bradycardia and long QT type 2 (LQT2), a second, non-voltage triggered cytosolic Ca2+ elevation increases gradually in amplitude, occurs before overt voltage instability, and then precedes the rise of EADs. Here, we used a modified Shannon-Puglisi-Bers model of rabbit ventricular myocytes to reproduce experimental Ca2+ dynamics in bradycardia and LQT2. Abnormal systolic Ca2+-oscillations and EADs caused by SR Ca2+-release are reproduced in a modified 0-dimensional model, where 3 gates in series control the ryanodine receptor (RyR2) conductance. Two gates control RyR2 activation and inactivation and sense cytosolic Ca2+ while a third gate senses luminal junctional SR Ca2+. The model predicts EADs in bradycardia and low extracellular [K+] and cessation of SR Ca2+-release terminate salvos of EADs. Ca2+-waves, systolic cell-synchronous Ca2+-release, and multifocal diastolic Ca2+ release seen in subcellular Ca2+-mapping experiments are observed in the 2-dimensional version of the model. These results support the role of SR Ca2+-overload, abnormal SR Ca2+-release, and the subsequent activation of the electrogenic Na+/Ca2+-exchanger as the mechanism of TdP. The model offers new insights into the genesis of cardiac arrhythmia and new therapeutic strategies.

Atrial-ventricular differences in rabbit cardiac voltage-gated Na+ currents: Basis for atrial-selective block by ranolazine

BackgroundClass 1 antiarrhythmic drugs are highly effective in restoring and maintaining sinus rhythm in atrial fibrillation patients but carry a risk of ventricular tachyarrhythmia. The antianginal agent ranolazine is a prototypic atrial-selective voltage-gated Na+ channel blocker but the mechanisms underlying its atrial-selective action remain unclear.ObjectiveThe present study examined the mechanisms underlying the atrial-selective action of ranolazine.MethodsWhole-cell voltage-gated Na+ currents (INa) were recorded at room temperature (∼22°C) from rabbit isolated left atrial and right ventricular myocytes.ResultsINa conductance density was ∼1.8-fold greater in atrial than in ventricular cells. Atrial INa was activated at command potentials ∼7 mV more negative and inactivated at conditioning potentials ∼11 mV more negative than ventricular INa. The onset of inactivation of INa was faster in atrial cells than in ventricular myocytes. Ranolazine (30 μM) inhibited INa in atrial and ventricular myocytes in a use-dependent manner consistent with preferential activated/inactivated state block. Ranolazine caused a significantly greater negative shift in voltage of half-maximal inactivation in atrial cells than in ventricular cells, the recovery from inactivation of INa was slowed by ranolazine to a greater extent in atrial myocytes than in ventricular cells, and ranolazine produced an instantaneous block that showed marked voltage dependence in atrial cells.ConclusionDifferences exist between rabbit atrial and ventricular myocytes in the biophysical properties of INa. The more negative voltage dependence of INa activation and inactivation, together with trapping of the drug in the inactivated channel, underlies an atrial-selective action of ranolazine.

Effects of diacetyl-liensinine on electrophysiology in rabbit ventricular myocytes

BackgroundDiacetyl-liensinine is a chemosynthetic derivative of liensinine, extracted from the seed embryo of Nelumbo nucifera Gaertn, in China. It has been found to have extensive anti- arrhythmic actions. The present study was designed to investigate the effects of diacetyl-liensinine on electro- physiology of myocytes.MethodsWe exposed rabbit ventricular myocytes to diacetyl-liensinine using standard whole-cell patch-clamp technique and measured the action potential, L-type calcium current (ICa-L), delayed rectifier potassium current (IK), transient outward potassium current (Ito) and inward rectifier potassium current (IK1).ResultsOur results showed that diacetyl-liensinine significantly prolonged action potential duration at 50 and 90% repolarization (APD50, APD90), at 10 and 30 μM, while shortened APD50 and APD90 at 100 μM. In addition, diacetyl-liensinine inhibited the ICa-L, IK, Ito and IK1 in a concentration-dependent manner.ConclusionsThe results suggest that diacetyl-liensinine might be a potential anti-arrhythmic agent.

β-Adrenergic induced SR Ca2 + leak is mediated by an Epac-NOS pathway

Cardiac β-adrenergic receptors (β-AR) and Ca2+-Calmodulin dependent protein kinase (CaMKII) regulate both physiological and pathophysiological Ca2+ signaling. Elevated diastolic Ca2+ leak from the sarcoplasmic reticulum (SR) contributes to contractile dysfunction in heart failure and to arrhythmogenesis. β-AR activation is known to increase SR Ca2+ leak via CaMKII-dependent phosphorylation of the ryanodine receptor. Two independent and reportedly parallel pathways have been implicated in this β-AR-CaMKII cascade, one involving exchange protein directly activated by cAMP (Epac2) and another involving nitric oxide synthase 1 (NOS1). Here we tested whether Epac and NOS function in a single series pathway to increase β-AR induced and CaMKII-dependent SR Ca2+ leak. Leak was measured as both Ca2+ spark frequency and tetracaine-induced shifts in SR Ca2+, in mouse and rabbit ventricular myocytes. Direct Epac activation by 8-CPT (8-(4-chlorophenylthio)-2′-O-methyl-cAMP) mimicked β-AR-induced SR Ca2+ leak, and both were blocked by NOS inhibition. The same was true for myocyte CaMKII activation (assessed via a FRET-based reporter) and ryanodine receptor phosphorylation. Inhibitor and phosphorylation studies also implicated phosphoinositide 3-kinase (PI3K) and protein kinase B (Akt) downstream of Epac and above NOS activation in this pathway. We conclude that these two independently characterized parallel pathways function mainly via a single series arrangement (β-AR-cAMP-Epac-PI3K-Akt-NOS1-CaMKII) to mediate increased SR Ca2+ leak. Thus, for β-AR activation the cAMP-PKA branch effects inotropy and lusitropy (by effects on Ca2+ current and SR Ca2+-ATPase), this cAMP-Epac-NOS pathway increases pathological diastolic SR Ca2+leak. This pathway distinction may allow novel SR Ca2+ leak therapeutic targeting in treatment of arrhythmias in heart failure that spare the inotropic and lusitropic effects of the PKA branch.

Multiscale Determinants of Delayed Afterdepolarization Amplitude in Cardiac Tissue

Spontaneous calcium (Ca) waves in cardiac myocytes underlie delayed afterdepolarizations (DADs) that trigger cardiac arrhythmias. How these subcellular/cellular events overcome source-sink factors in cardiac tissue to generate DADs of sufficient amplitude to trigger action potentials is not fully understood. Here, we evaluate quantitatively how factors at the subcellular scale (number of Ca wave initiation sites), cellular scale (sarcoplasmic reticulum (SR) Ca load), and tissue scale (synchrony of Ca release in populations of myocytes) determine DAD features in cardiac tissue using a combined experimental and computational modeling approach. Isolated patch-clamped rabbit ventricular myocytes loaded with Fluo-4 to image intracellular Ca were rapidly paced during exposure to elevated extracellular Ca (2.7 mmol/L) and isoproterenol (0.25 μmol/L) to induce diastolic Ca waves and subthreshold DADs. As the number of paced beats increased from 1 to 5, SR Ca content (assessed with caffeine pulses) increased, the number of Ca wave initiation sites increased, integrated Ca transients and DADs became larger and shorter in duration, and the latency period to the onset of Ca waves shortened with reduced variance. In silico analysis using a computer model of ventricular tissue incorporating these experimental measurements revealed that whereas all of these factors promoted larger DADs with higher probability of generating triggered activity, the latency period variance and SR Ca load had the greatest influences. Therefore, incorporating quantitative experimental data into tissue level simulations reveals that increased intracellular Ca promotes DAD-mediated triggered activity in tissue predominantly by increasing both the synchrony (decreasing latency variance) of Ca waves in nearby myocytes and SR Ca load, whereas the number of Ca wave initiation sites per myocyte is less important.

Inhibition of late sodium current suppresses calcium-related ventricular arrhythmias by reducing the phosphorylation of CaMK-II and sodium channel expressions

Cardiac arrhythmias associated with intracellular calcium inhomeostasis are refractory to antiarrhythmic therapy. We hypothesized that late sodium current (INa) contributed to the calcium-related arrhythmias. Monophasic action potential duration at 90% completion of repolarization (MAPD90) was significantly increased and ventricular arrhythmias were observed in hearts with increased intracellular calcium concentration ([Ca2+]i) by using Bay K 8644, and the increase became greater in hearts treated with a combination of ATX-II and Bay K 8644 compared to Bay K 8644 alone. The prolongations caused by Bay K 8644 and frequent episodes of ventricular tachycardias, both in absence and presence of ATX-II, were significantly attenuated or abolished by late INa inhibitors TTX and eleclazine. In rabbit ventricular myocytes, Bay K 8644 increased ICaL density, calcium transient and myocyte contraction. TTX and eleclazine decreased the amplitude of late INa, the reverse use dependence of MAPD90 at slower heart rate, and attenuated the increase of intracellular calcium transient and myocyte contraction. TTX diminished the phosphorylation of CaMKII-δ and Nav 1.5 in hearts treated with Bay K 8644 and ATX-II. In conclusion, late INa contributes to ventricular arrhythmias and its inhibition is plausible to treat arrhythmias in hearts with increased [Ca2+]i

Quantitative analysis of the Ca2+ -dependent regulation of delayed rectifier K+ current IKs in rabbit ventricular myocytes.

[Ca2+ ]i enhanced rabbit ventricular slowly activating delayed rectifier K+ current (IKs ) by negatively shifting the voltage dependence of activation and slowing deactivation, similar to perfusion of isoproterenol. Rabbit ventricular rapidly activating delayed rectifier K+ current (IKr ) amplitude and voltage dependence were unaffected by high [Ca2+ ]i . When measuring or simulating IKs during an action potential, IKs was not different during a physiological Ca2+ transient or when [Ca2+ ]i was buffered to 500nm. The slowly activating delayed rectifier K+ current (IKs ) contributes to repolarization of the cardiac action potential (AP). Intracellular Ca2+ ([Ca2+ ]i ) and β-adrenergic receptor (β-AR) stimulation modulate IKs amplitude and kinetics, but details of these important IKs regulators and their interaction are limited. We assessed the [Ca2+ ]i dependence of IKs in steady-state conditions and with dynamically changing membrane potential and [Ca2+ ]i during an AP. IKs was recorded from freshly isolated rabbit ventricular myocytes using whole-cell patch clamp. With intracellular pipette solutions that controlled free [Ca2+ ]i , we found that raising [Ca2+ ]i from 100 to 600nm produced similar increases in IKs as did β-AR activation, and the effects appeared additive. Both β-AR activation and high [Ca2+ ]i increased maximally activated tail IKs , negatively shifted the voltage dependence of activation, and slowed deactivation kinetics. These data informed changes in our well-established mathematical model of the rabbit myocyte. In both AP-clamp experiments and simulations, IKs recorded during a normal physiological Ca2+ transient was similar to IKs measured with [Ca2+ ]i clamped at 500-600nm. Thus, our study provides novel quantitative data as to how physiological [Ca2+ ]i regulates IKs amplitude and kinetics during the normal rabbit AP. Our results suggest that micromolar [Ca2+ ]i , in the submembrane or junctional cleft space, is not required to maximize [Ca2+ ]i -dependent IKs activation during normal Ca2+ transients.

Differential responses of rabbit ventricular and atrial transient outward current (Ito) to the Ito modulator NS5806.

Transient outward potassium current (Ito) in the heart underlies phase 1 repolarization of cardiac action potentials and thereby affects excitation–contraction coupling. Small molecule activators of Ito may therefore offer novel treatments for cardiac dysfunction, including heart failure and atrial fibrillation. NS5806 has been identified as a prototypic activator of canine Ito. This study investigated, for the first time, actions of NS5806 on rabbit atrial and ventricular Ito. Whole cell patch‐clamp recordings of Ito and action potentials were made at physiological temperature from rabbit ventricular and atrial myocytes. 10 μmol/L NS5806 increased ventricular Ito with a leftward shift in Ito activation and accelerated restitution. At higher concentrations, stimulation of Ito was followed by inhibition. The EC 50 for stimulation was 1.6 μmol/L and inhibition had an IC 50 of 40.7 μmol/L. NS5806 only inhibited atrial Ito (IC 50 of 18 μmol/L) and produced a modest leftward shifts in Ito activation and inactivation, without an effect on restitution. 10 μmol/L NS5806 shortened ventricular action potential duration (APD) at APD 20‐APD 90 but prolonged atrial APD. NS5806 also reduced atrial AP upstroke and amplitude, consistent with an additional atrio‐selective effect on Na+ channels. In contrast to NS5806, flecainide, which discriminates between Kv1.4 and 4.x channels, produced similar levels of inhibition of ventricular and atrial Ito. NS5806 discriminates between rabbit ventricular and atrial Ito, with mixed activator and inhibitor actions on the former and inhibitor actions against the later. NS5806 may be of significant value for pharmacological interrogation of regional differences in native cardiac Ito.

Electrophysiology of Hypokalemia and Hyperkalemia.

Electrophysiology of Hypokalemia and Hyperkalemia.

A New Class of Antiarrhythmics for Late ICA,L

Early Afterdepolarizations (EADs) of the cardiac action potential (AP) are membrane potential oscillations arising during phase 2-3 and well-recognized cellular triggers of lethal cardiac arrhythmias. The L-type Ca2+ current (ICa,L) is a major regenerative inward current during these phases of the AP, and is therefore highly relevant to EAD upstroke and propagation at the tissue level. We recently discovered that late ICa,L reduction produced by minimally altering specific ICa,L biophysical properties (smaller pedestal ICa,L or more depolarized steady-state activation), potently suppresses EAD occurrence in rabbit ventricular myocytes under dynamic clamp. Based on these findings, we hypothesize that drugs able to mimic these late ICa,L modifications could constitute a new Class of antiarrhythmics that do not compromise inotropy. We are studying the antiarrhythmic effects of two pilots drugs, roscovitine and pregabalin, that we found to reduce late ICa,L in rabbit ventricular myocytes via two distinct mechanisms. Specifically, roscovitine reduced the non-inactivating (pedestal) ICa,L while leaving peak ICa,L intact and pregabalin induced a depolarizing shift of the steady-state activation. In this study we assessed the antiarrhythmic potential of these pilot compounds that reduce late ICa,L. We tested the ability of these drugs to suppress EADs in ventricular myocytes induced by oxidative stress (H2O2). The perfusion of roscovitine (20µM) or pregabalin (1mM) dramatically suppressed EAD occurrence (roscovitine: from 81.37%±8.1% to 0% APs with EADs; pregabalin from 64.5±17.7% to 6.6±3.8%) and restored normal AP duration. Moreover, both drugs suppressed EAD-mediated VT/VF in isolated perfused aged rat hearts, restoring sinus rhythm (roscovitine: 5/6; pregabalin: 2/2 hearts). These studies set the basis for the development of a conceptually new Class of antiarrhythmics (L-type Ca2+ channel gating modifiers) that selectively reduce late ICa,L, avoiding the negative inotropy caused by traditional CaV channel blockers used as Class IV Antiarrhythmics.

The Role of RyR2 Intersubunit Cross-Linking in SR Ca Mishandling in Failing Heart

We previously reported that oxidation of RyR2 caused formation of disulfide bonds between two subunits within the channel - intersubunit cross-linking (XL). Because intersubunit dynamics within the RyR2 determines the channel gating, XL should play an important role in RyR2 regulation. We found a strong positive correlation between the degree of RyR2 XL and sarcoplasmic reticulum (SR) Ca leak in rabbit ventricular myocytes. When the XL reached the maximum level, further oxidation of RyR2 did not enhance SR Ca leak.

Diffusion in the Transverse-Axial Tubule System of Cardiac Myocytes

Excitation-contraction (E-C) coupling in cardiac ventricular myocytes is critically dependent on the structure of transverse-axial (t-) tubules, which are invaginations of the surface sarcolemma. Many currents have been shown to be preferentially located at the t-tubular membrane, including the L-Type calcium current, Na-Ca exchange, tetrodotoxin-sensitive Na and steady-state K currents (Orchard, Pasek, and Brette. 2009. Exp. Physiol. 94: 509-519). It has been suggested that membrane folding within the t-system may introduce a slow diffusion zone, which may have implications for ion balance for these currents during the E-C coupling cycle (Hong et al. 2014. Nat. Med. 20: 624-632.). To investigate diffusion in the t-tubule system, we examined t-tubule structure in rabbit and mouse ventricular myocytes as two common experimental models, using electron tomography. Cells were also superfused with solutions containing solutes of varying molecular weight to examine their penetration into the t-system. Fluorescence Recovery After Photobleaching (FRAP) was used to examine transport within the t-tubules of quiescent myocytes. We found that despite marked differences in t-tubule diameter and complexity in the two species, there were relatively small differences in solute penetration below ∼70kDa, and in the time-course of fluorescence recovery during FRAP. These data suggest that gross differences in t-tubule structure do not have marked effects on the rate of diffusion within t-tubules. This work was supported by the BHF and MRC

The effect of PKA-mediated phosphorylation of ryanodine receptor on SR Ca2 + leak in ventricular myocytes

Functional impact of cardiac ryanodine receptor (type 2 RyR or RyR2) phosphorylation by protein kinase A (PKA) remains highly controversial. In this study, we characterized a functional link between PKA-mediated RyR2 phosphorylation level and sarcoplasmic reticulum (SR) Ca2+ release and leak in permeabilized rabbit ventricular myocytes. Changes in cytosolic [Ca2+] and intra-SR [Ca2+]SR were measured with Fluo-4 and Fluo-5N, respectively. Changes in RyR2 phosphorylation at two PKA sites, serine-2031 and -2809, were measured with phospho-specific antibodies. cAMP (10μM) increased Ca2+ spark frequency approximately two-fold. This effect was associated with an increase in SR Ca2+ load from 0.84 to 1.24mM. PKA inhibitory peptide (PKI; 10μM) abolished the cAMP-dependent increase of SR Ca2+ load and spark frequency. When SERCA was completely blocked by thapsigargin, cAMP did not affect RyR2-mediated Ca2+ leak. The lack of a cAMP effect on RyR2 function can be explained by almost maximal phosphorylation of RyR2 at serine-2809 after sarcolemma permeabilization. This high RyR2 phosphorylation level is likely the consequence of a balance shift between protein kinase and phosphatase activity after permeabilization. When RyR2 phosphorylation at serine-2809 was reduced to its “basal” level (i.e. RyR2 phosphorylation level in intact myocytes) using kinase inhibitor staurosporine, SR Ca2+ leak was significantly reduced. Surprisingly, further dephosphorylation of RyR2 with protein phosphatase 1 (PP1) markedly increased SR Ca2+ leak. At the same time, phosphorylation of RyR2 at serine 2031 did not significantly change under identical experimental conditions. These results suggest that RyR2 phosphorylation by PKA has a complex effect on SR Ca2+ leak in ventricular myocytes. At an intermediate level of RyR2 phosphorylation SR Ca2+ leak is minimal. However, complete dephosphorylation and maximal phosphorylation of RyR2 increases SR Ca2+ leak.

Adrenomedullin Stimulates Porcine Sinus Node Automaticity

Adrenomedullin (AM) is a vasodilatory and chronotropic peptide that has cardioprotective properties in settings of ischemia-reperfusion. Specifically, the 52-amino acid peptide hormone has been shown to reduce infarct size and potentially be protective against malignant arrhythmias. AM reduces L-type calcium currents in rabbit ventricular myocytes, and can possibly be utilized as an antiarrhythmic drug. However, at present it is unclear whether AM has cardiac electrophysiological effects in vivo. In the present study, we investigated whether AM affects sinus and atrioventricular nodal functions, cardiac pacing thresholds, refractory properties and intracardiac conduction intervals in an intact porcine model (Norwegian landrace and Yorkshire hybrids; n=12). Hemodynamic and electrophysiological parameters were recorded at baseline and following 60 min of AM-infusion (100 ng/kg/min). The hemodynamic effects of AM were characterized by a reduced mean arterial pressure (76 ± 9 vs. 57 ± 6; p<0.05; mmHg), tachycardia (91 ± 13 vs. 112 ± 15; p<0.05; beats/min) and an augmented cardiac index (131 ± 20 vs. 176 ± 28; p<0.05; ml/min/kg). The sinus cycle length was reduced (674 ± 89 vs. 546 ± 73; p<0.05; ms), sinoatrial conduction time was unaltered (79 ± 24 vs. 72 ± 19; ns; ms), and the sinus node recovery time (SNRT) was reduced at paced cycle lengths of 425 ms, 375 ms and 325 ms (average SNRT 1034 ± 449 vs.704 ± 141; p<0.05; ms). Diastolic pacing thresholds, effective refractory periods, Wenckebach cycle length and intracardiac conduction intervals were unaffected by AM. In conclusion, AM stimulates sinus node function with a reduced sinus cycle length and increased automaticity but has no further detectable intracardiac electrophysiological effects. Whether the increased automaticity is a direct effect on the sinus node or an indirect effect mediated through the autonomic nervous system remains to be determined.

A Dynamical Threshold for Cardiac Delayed Afterdepolarization-Mediated Triggered Activity

Ventricular myocytes are excitable cells whose voltage threshold for action potential (AP) excitation is ∼−60 mV at which INa is activated to give rise to a fast upstroke. Therefore, for a short stimulus pulse to elicit an AP, a stronger stimulus is needed if the resting potential lies further away from the INa threshold, such as in hypokalemia. However, for an AP elicited by a long duration stimulus or a diastolic spontaneous calcium release, we observed that the stimulus needed was lower in hypokalemia than in normokalemia in both computer simulations and experiments of rabbit ventricular myocytes. This observation provides insight into why hypokalemia promotes calcium-mediated triggered activity, despite the resting potential lying further away from the INa threshold. To understand the underlying mechanisms, we performed bifurcation analyses and demonstrated that there is a dynamical threshold, resulting from a saddle-node bifurcation mainly determined by IK1 and INCX. This threshold is close to the voltage at which IK1 is maximum, and lower than the INa threshold. After exceeding this dynamical threshold, the membrane voltage will automatically depolarize above the INa threshold due to the large negative slope of the IK1-V curve. This dynamical threshold becomes much lower in hypokalemia, especially with respect to calcium, as predicted by our theory. Because of the saddle-node bifurcation, the system can automatically depolarize even in the absence of INa to voltages higher than the ICa,L threshold, allowing for triggered APs in single myocytes with complete INa block. However, because INa is important for AP propagation in tissue, blocking INa can still suppress premature ventricular excitations in cardiac tissue caused by calcium-mediated triggered activity. This suppression is more effective in normokalemia than in hypokalemia due to the difference in dynamical thresholds.

Electrophysiological Determination of Submembrane Na+ Concentration in Cardiac Myocytes

In the heart, Na+ is a key modulator of the action potential, Ca2+ homeostasis, energetics, and contractility. Because Na+ currents and cotransport fluxes depend on the Na+ concentration in the submembrane region, it is necessary to accurately estimate the submembrane Na+ concentration ([Na+]sm). Current methods using Na+-sensitive fluorescent indicators or Na+ -sensitive electrodes cannot measure [Na+]sm. However, electrophysiology methods are ideal for measuring [Na+]sm. In this article, we develop patch-clamp protocols and experimental conditions to determine the upper bound of [Na+]sm at the peak of action potential and its lower bound at the resting state. During the cardiac cycle, the value of [Na+]sm is constrained within these bounds. We conducted experiments in rabbit ventricular myocytes at body temperature and found that 1) at a low pacing frequency of 0.5 Hz, the upper and lower bounds converge at 9 mM, constraining the [Na+]sm value to ∼9 mM; 2) at 2 Hz pacing frequency, [Na+]sm is bounded between 9 mM at resting state and 11.5 mM; and 3) the cells can maintain [Na+]sm to the above values, despite changes in the pipette Na+ concentration, showing autoregulation of Na+ in beating cardiomyocytes.

Tolterodine reduces veratridine-augmented late INa, reverse-INCX and early afterdepolarizations in isolated rabbit ventricular myocytes.

The augmentation of late sodium current (INa.L) not only causes intracellular Na+ accumulation, which results in intracellular Ca2+ overload via the reverse mode of the Na+/Ca2+ exchange current (reverse-INCX), but also prolongs APD and induces early afterdepolarizations (EAD), which can lead to arrhythmia and cardiac dysfunction. Thus, the inhibition of INa.L is considered to be a potential way for therapeutic intervention in ischemia and heart failure. In this study we investigated the effects of tolterodine (Tol), a competitive muscarinic receptor antagonist, on normal and veratridine (Ver)-augmented INa.L, reverse-INCX and APD in isolated rabbit ventricular myocytes, which might contribute to its cardioprotective activity. Rabbit ventricular myocytes were prepared. The INa.L and reverse-INCX were recorded in voltage clamp mode, whereas action potentials and Ver-induced early afterdepolarizations (EADs) were recorded in current clamp mode. Drugs were applied via superfusion. Tol (3-120 nmol/L) concentration-dependently inhibited the normal and Ver-augmented INa.L with IC50 values of 32.08 nmol/L and 42.47 nmol/L, respectively. Atropine (100 μmol/L) did not affect the inhibitory effects of Tol (30 nmol/L) on Ver-augmented INa.L. In contrast, much high concentrations of Tol was needed to inhibit the transient sodium current (INa.T) with an IC50 value of 183.03 μmol/L. In addition, Tol (30 nmol/L) significantly shifted the inactivation curve of INa.T toward a more depolarizing membrane potential without affecting its activation characteristics. Moreover, Tol (30 nmol/L) significantly decreased Ver-augmented reverse-INCX. Tol (30 nmol/L) increased the action potential duration (APD) by 16% under the basal conditions. Ver (20 μmol/L) considerably extended the APD and evoked EADs in 18/24 cells (75%). In the presence of Ver, Tol (30 nmol/L) markedly decreased the APD and eliminated EADs (0/24 cells). Tol inhibits normal and Ver-augmented INaL and decreases Ver-augmented reverse-INCX. In addition, Tol reverses the prolongation of the APD and eliminates the EADs induced by Ver, thus prevents Ver-induced arrhythmia.