Rabbit Myocytes Research Articles

Conductance Changes of Na+ Channels during the Late Na+ Current Flowing under Action Potential Voltage Clamp Conditions in Canine, Rabbit, and Guinea Pig Ventricular Myocytes.

Late sodium current (INa,late) is an important inward current contributing to the plateau phase of the action potential (AP) in the mammalian heart. Although INa,late is considered as a possible target for antiarrhythmic agents, several aspects of this current remained hidden. In this work, the profile of INa,late, together with the respective conductance changes (GNa,late), were studied and compared in rabbit, canine, and guinea pig ventricular myocytes using the action potential voltage clamp (APVC) technique. In canine and rabbit myocytes, the density of INa,late was relatively stable during the plateau and decreased only along terminal repolarization of the AP, while GNa,late decreased monotonically. In contrast, INa,late increased monotonically, while GNa,late remained largely unchanged during the AP in guinea pig. The estimated slow inactivation of Na+ channels was much slower in guinea pig than in canine or rabbit myocytes. The characteristics of canine INa,late and GNa,late were not altered by using command APs recorded from rabbit or guinea pig myocytes, indicating that the different shapes of the current profiles are related to genuine interspecies differences in the gating of INa,late. Both INa,late and GNa,late decreased in canine myocytes when the intracellular Ca2+ concentration was reduced either by the extracellular application of 1 µM nisoldipine or by the intracellular application of BAPTA. Finally, a comparison of the INa,late and GNa,late profiles induced by the toxin of Anemonia sulcata (ATX-II) in canine and guinea pig myocytes revealed profound differences between the two species: in dog, the ATX-II induced INa,late and GNa,late showed kinetics similar to those observed with the native current, while in guinea pig, the ATX-II induced GNa,late increased during the AP. Our results show that there are notable interspecies differences in the gating kinetics of INa,late that cannot be explained by differences in AP morphology. These differences must be considered when interpreting the INa,late results obtained in guinea pig.

Improved Ca2+ release synchrony following selective modification of Itof and phase 1 repolarization in normal and failing ventricular myocytes

Loss of ventricular action potential (AP) early phase 1 repolarization may contribute to the impaired Ca2+ release and increased risk of sudden cardiac death in heart failure. Therefore, restoring AP phase 1 by augmenting the fast transient outward K+ current (Itof) might be beneficial, but direct experimental evidence to support this proposition in failing cardiomyocytes is limited. Dynamic clamp was used to selectively modulate the contribution of Itof to the AP and Ca2+ transient in both normal (guinea pig and rabbit) and in failing rabbit cardiac myocytes. Opposing native Itof in non-failing rabbit myocytes increased Ca2+ release heterogeneity, late Ca2+ sparks (LCS) frequency and AP duration. (APD). In contrast, increasing Itof in failing myocytes and guinea pig myocytes (the latter normally lacking Itof) increased Ca2+ transient amplitude, Ca2+ release synchrony, and shortened APD. Computer simulations also showed faster Ca2+ transient decay (mainly due to fewer LCS), decreased inward Na+/Ca2+ exchange current and APD. When the Itof conductance was increased to ~0.2 nS/pF in failing cells (a value slightly greater than seen in typical human epicardial myocytes), Ca2+ release synchrony improved and AP duration decreased slightly. Further increases in Itof can cause Ca2+ release to decrease as the peak of the bell-shaped ICa-voltage relationship is passed and premature AP repolarization develops. These results suggest that there is an optimal range for Itof enhancement that may support Ca2+ release synchrony and improve electrical stability in heart failure with the caveat that uncontrolled Itof enhancement should be avoided.

Hyperglycemia regulates cardiac K+ channels via O-GlcNAc-CaMKII and NOX2-ROS-PKC pathways.

Chronic hyperglycemia and diabetes lead to impaired cardiac repolarization, K+ channel remodeling and increased arrhythmia risk. However, the exact signaling mechanism by which diabetic hyperglycemia regulates cardiac K+ channels remains elusive. Here, we show that acute hyperglycemia increases inward rectifier K+ current (IK1), but reduces the amplitude and inactivation recovery time of the transient outward K+ current (Ito) in mouse, rat, and rabbit myocytes. These changes were all critically dependent on intracellular O-GlcNAcylation. Additionally, IK1 amplitude and Ito recovery effects (but not Ito amplitude) were prevented by the Ca2+/calmodulin-dependent kinase II (CaMKII) inhibitor autocamtide-2-related inhibitory peptide, CaMKIIδ-knockout, and O-GlcNAc-resistant CaMKIIδ-S280A knock-in. Ito reduction was prevented by inhibition of protein kinase C (PKC) and NADPH oxidase 2 (NOX2)-derived reactive oxygen species (ROS). In mouse models of chronic diabetes (streptozotocin, db/db, and high-fat diet), heart failure, and CaMKIIδ overexpression, both Ito and IK1 were reduced in line with the downregulated K+ channel expression. However, IK1 downregulation in diabetes was markedly attenuated in CaMKIIδ-S280A. We conclude that acute hyperglycemia enhances IK1 and Ito recovery via CaMKIIδ-S280 O-GlcNAcylation, but reduces Ito amplitude via a NOX2-ROS-PKC pathway. Moreover, chronic hyperglycemia during diabetes and CaMKII activation downregulate K+ channel expression and function, which may further increase arrhythmia susceptibility.

A potent antiarrhythmic drug N-methyl berbamine extends the action potential through inhibiting both calcium and potassium currents

N-methyl berbamine (N-MB) is a berberine derivative. Its analogue berbamine has been reported to have remarkable antiarrhythmic and ischemic protective effects. However, the pharmacological effects of N-MB are ill-defined. In this study, molecular docking was used to evaluate the binding of N-MB to CaV1.2 Ca2+ and KV11.1 K+ channels, and the effects of N-MB on action potential and ionic currents were observed in the ventricular myocytes of rabbits, HEK293 cells stably transfected with the hCaV1.2 gene and CHO cells stably transfected with hERG (human ether-a-go-go related gene). The results showed that N-MB was able to bind to both CaV1.2 and KV11.1 channels. Following a perfusion with N-MB, the durations of action potentials (APD20, APD50 and APD90) were extended, and the outward tail current, Itail, as well as the hERG current, IhERG, were inhibited, while the amplitude of action potential (APA) was only slightly reduced. N-MB also decreased the peak amplitude of the L-type Ca2+ channel current, ICaL, as well as the CaV1.2 current, ICaV1.2; this may limit the prolongation of APD. In conclusion, N-MB is a potent and natural antiarrhythmic multitarget drug that may elicit its antiarrhythmic effect through blocking both Ca2+ and K+ channel currents.

Effects and mechanism of digoxin on atrium electrical remodeling and susceptibility of atrial fibrillation in aged rabbits

Objective: To investigate the effects and mechanism of digoxin on atrium electrical remodeling and susceptibility of atrial fibrillation (AF) in aged rabbits. Methods: Twenty aged male New Zealand rabbits were divided into aged group and aged plus digoxin group (n=10 each). Electrical parameters including heart rate (HR), RR and QT interval, ST segment and P wave dispersion from normal Ⅱ electrocardiogram, and the maximum upstroke velocity (Max(dv/dt)), plateau potential (plateau P), action potential duration of 10%, 20% and 90% (APD(10), APD(20), APD(90)) from recording of monophasic action potential (MAP), as well as atrial effective refractory period (AERP(200)) and dispersion (dERP(200)) with 200 ms of basic cycle length (BCL), and frequency self adaptation of AERP with 300 ms and 150 ms of BCLs (fERP) were recorded and compared between the 2 groups. BCLs and inducibility of AF post programmed electrical stimulation and Burst-pacing in left atrium tissue of rabbits in vivo were also analyzed. The L-type calcium current (I(Ca-L)) in 2 groups were recorded via whole-cell patch clamp technique, and the fluorescence intensity of intracellular free Ca(2+) was detected with Flup-3/AM loading by the laser scanning confocal microscope in enzymatically dissociated single rabbit atrial myocytes. Results: Compared with aged group, the heart rate was faster, RR and QT interval were obvious shorter, ST segment was raised and P wave dispersion was significantly increased in aged plus digoxin group (all P<0.05). Moreover, compared with aged group, the Max(dv/dt) and plateau P were obviously increased, APD(10) and APD(20) were significantly prolongated, and APD(9)0 was significantly shorter in aged plus digoxin group (all P<0.01). Otherwise, the fERP was markedly increased (0.81±0.15 vs. 0.67±0.05), and the induced rate of AF was obviously higher in aged plus digoxin group than in aged group (6/8 vs. 4/9) (all P<0.01). With voltage clamp model, digoxin significantly increased I(Ca-L) of atrial myocytes of aged rabbits, When command potential was 10 mV, the current densities of I(Ca-L) were significantly higher in digoxin group than that in aged group ((15.45±2.38) pA/pF vs. (7.03±1.69) pA/pF, P<0.01). Otherwise, the I-V curve of I(Ca-L) was downward shifted of all I-V curves in digoxin perfused aged atrial cells of rabbits. Moreover, the fluorescence intensities of intracellular free Ca(2+) was significantly higher in aged plus digoxin group than in aged group ((1 748±173) μmol/L vs. (478.13±87.63) μmol/L, P<0.01). Conclusion: Digoxin could aggravate the atrial electrical remodeling in atrium of aged rabbits, facilitate susceptibility of atrial fibrillation in aged rabbit, increased current density of I(Ca-L) and concentration of intracellular free Ca(2+), followed Ca(2+) overload and oscillations might be part of the underlying mechanisms.

Effect of KN93 on delayed afterdepolarization and calcium ion in ventricular myocytes of rabbits with heart failure

Objective To observe the effect of KN93, a CaMKⅡ inhibitor, on delayed afterdepolarization (DAD) and calcium ion in ventricular myocytes of rabbits with heart failure, and to investigate the effect of CaMKⅡ signaling pathway on trigged arrhythmia after heart failure. Methods Thirty male New Zealand White rabbits were randomized(random number) into the sham operated group (sham group), heart failure group (HF group) and heart failure with KN93 group (HF+KN93 group) (n=10 each group). The rabbit heart failure model was established by abdominal aortic constriction combined with aortic valve regurgitation. The ventricular myocytes were isolated by double enzyme digestion. The action potential and the transient inward current (Iti) were recorded by the whole-cell patch-clamp. The intracellular calcium transient was measured by the ion concentration measurement system. The main calcium transporter protein was detected by Western blotting. Data were analyzed by pCLAMP10.2. Statistical analysis was performed using SPSS 17.0. Comparisons among groups were conducted using ANOVA, and SNK-q multiple comparison procedure was utilized for post-hoc analysis. Results (1) After induction of heart failure, DAD and increment of trigger activity (TA) were observed in rabbit ventricular myocytes. Treatment of KN93 with 1.0 μmol/L reduced the events of DAD and TA. (2) After induction of heart failure, Iti densities were increased from -0.12±0.02 pA/pF to -0.95±0.06 pA/pF at the polarization potential of -50 mV (n=10, P<0.01). The current densities were reduced to -0.44±0.04 pA/pF after application of 1.0 μmol/L of KN93 (n=10, P<0.01). (3) KN93 led to decrement of intracellular calcium ion concentration and calcium transient amplitude, and acceleration of the decay process of calcium transient. (4) KN93 upregulated the expression of pPLN and SERCA2a, increased the uptake of intracellular calcium ion, downregulated the expression of NCX, decreased the Iti, and reduced the occurrence of DAD and TA. Conclusions KN93 can reduce the intracellular calcium ion concentration of the heart failure animal model, and the occurrence of the DAD and TA. CaMKⅡ may be a new therapeutic target for arrhythmias in the heart failure. Key words: KN93; Heart failure; Delayed afterdepolarization; Transient inward current; Calcium ion

The effect of allicin on the ventricular myocytes of rabbit with heart failure

Objective To observe the effect of allicin on the action potential duration (APD) and L-type calcium current (ICa, L) in the ventricular myocytes of rabbits with heart failure in order to explore the mechanisms of therapeutic effect of allicin on cardiac arrhythmias complicated with heart failure. Methods Forty-five New Zealand White male rabbits were randomly (random number) assigned to 3 groups (n=15 in each group): sham operated group (sham group), heart failure group (HF group), and heart failure treated with allicin group (HF+All group). The rabbit heart failure model was established by abdominal aortic constriction coupled with aortic regurgitation, the ventricular myocytes were obtained by enzyme double digestion, and the whole cell clamp was used to record action potential and calcium current.The action potential duration (APD), ICa, L and gating mechanism were observed during heart failure and allicin administered. Data were processed with pCLAMP version 10.2. Statistical analysis was performed using SPSS 17.0. Comparisons among groups were carried out using ANOVA, and SNK-q was used for multiple comparison as post-hoc. Results (1) Prolonged APD was found during heart failure, APD50 was prolonged from (93.4±4.7) ms in sham group to (115.5±6.2) ms in HF group(P<0.01). After administration of allicin 30 μmol/L, APD50 was shortened to (105.2±5.5) ms (P<0.05). (2) The density of ICa,L increased during heart failure, peak current density increased increased from (-8.4±0.6) pA/pF in sham group to (-15.1±1.1) pA/pF while 0 mV attained at depolarizations (P<0.01). After administration of allicin 30 μmol/L, the current density reduced to (-10.1±0.8) pA/pF ( P<0.01). The effect of allicin presented in both voltage dependent and consentration dependent manner. (3) According to the gating mechanism study, the main mechanism of lowering the density of ICa, L by allicin after heart failure was the acceleration of the steady inactivation of the channel, and the de-escalation of the recovery kinetic after the inactivation of the channel. Conclusions Allcin can be used to reduce the calcium current of ventricular myocytes in animal heart failure model, it has the potential of clinical use in treating cardiac arrhythmias during heart failure. Key words: Allicin; Heart failure; Rabbit; Ventricular myocytes; Action potential; L- calcium current

N-Methylberbamine extends the action potential of the cardiac ventricular myocytes of rabbits through inhibiting an outward rectifying current

N-Methylberbamine extends the action potential of the cardiac ventricular myocytes of rabbits through inhibiting an outward rectifying current

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.

A Parsimonious Model of the Rabbit Action Potential Elucidates the Minimal Physiological Requirements for Alternans and Spiral Wave Breakup.

Elucidating the underlying mechanisms of fatal cardiac arrhythmias requires a tight integration of electrophysiological experiments, models, and theory. Existing models of transmembrane action potential (AP) are complex (resulting in over parameterization) and varied (leading to dissimilar predictions). Thus, simpler models are needed to elucidate the “minimal physiological requirements” to reproduce significant observable phenomena using as few parameters as possible. Moreover, models have been derived from experimental studies from a variety of species under a range of environmental conditions (for example, all existing rabbit AP models incorporate a formulation of the rapid sodium current, INa, based on 30 year old data from chick embryo cell aggregates). Here we develop a simple “parsimonious” rabbit AP model that is mathematically identifiable (i.e., not over parameterized) by combining a novel Hodgkin-Huxley formulation of INa with a phenomenological model of repolarization similar to the voltage dependent, time-independent rectifying outward potassium current (IK). The model was calibrated using the following experimental data sets measured from the same species (rabbit) under physiological conditions: dynamic current-voltage (I-V) relationships during the AP upstroke; rapid recovery of AP excitability during the relative refractory period; and steady-state INa inactivation via voltage clamp. Simulations reproduced several important “emergent” phenomena including cellular alternans at rates > 250 bpm as observed in rabbit myocytes, reentrant spiral waves as observed on the surface of the rabbit heart, and spiral wave breakup. Model variants were studied which elucidated the minimal requirements for alternans and spiral wave break up, namely the kinetics of INa inactivation and the non-linear rectification of IK.The simplicity of the model, and the fact that its parameters have physiological meaning, make it ideal for engendering generalizable mechanistic insight and should provide a solid “building-block” to generate more detailed ionic models to represent complex rabbit electrophysiology.

Genetically Encoded Biosensors Reveal PKA Hyperphosphorylation on the Myofilaments in Rabbit Heart Failure.

In heart failure, myofilament proteins display abnormal phosphorylation, which contributes to contractile dysfunction. The mechanisms underlying the dysregulation of protein phosphorylation on myofilaments is not clear. This study aims to understand the mechanisms underlying altered phosphorylation of myofilament proteins in heart failure. We generate a novel genetically encoded protein kinase A (PKA) biosensor anchored onto the myofilaments in rabbit cardiac myocytes to examine PKA activity at the myofilaments in responses to adrenergic stimulation. We show that PKA activity is shifted from the sarcolemma to the myofilaments in hypertrophic failing rabbit myocytes. In particular, the increased PKA activity on the myofilaments is because of an enhanced β2 adrenergic receptor signal selectively directed to the myofilaments together with a reduced phosphodiesterase activity associated with the myofibrils. Mechanistically, the enhanced PKA activity on the myofilaments is associated with downregulation of caveolin-3 in the hypertrophic failing rabbit myocytes. Reintroduction of caveolin-3 in the failing myocytes is able to normalize the distribution of β2 adrenergic receptor signal by preventing PKA signal access to the myofilaments and to restore contractile response to adrenergic stimulation. In hypertrophic rabbit myocytes, selectively enhanced β2 adrenergic receptor signaling toward the myofilaments contributes to elevated PKA activity and PKA phosphorylation of myofilament proteins. Reintroduction of caveolin-3 is able to confine β2 adrenergic receptor signaling and restore myocyte contractility in response to β adrenergic stimulation.

A novel heterozygous mutation in cardiac calsequestrin causes autosomal dominant catecholaminergic polymorphic ventricular tachycardia

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a lethal inherited arrhythmia syndrome characterized by adrenergically stimulated ventricular tachycardia. Mutations in the cardiac ryanodine receptor gene (RYR2) cause an autosomal dominant form of CPVT, while mutations in the cardiac calsequestrin 2 gene (CASQ2) cause an autosomal recessive form. The aim of this study was to clinically and genetically evaluate a large family with severe autosomal dominant CPVT. Clinical evaluation of family members was performed, including detailed history, physical examination, electrocardiogram, exercise stress test, and autopsy review of decedents. We performed genome-wide linkage analysis in 12 family members and exome sequencing in 2 affected family members. In silico models of mouse and rabbit myocyte electrophysiology were used to predict potential disease mechanisms. Severe CPVT with dominant inheritance in 6 members was diagnosed in a large family with 2 sudden deaths, 2 resuscitated cardiac arrests, and multiple appropriate implantable cardioverter-defibrillator shocks. A comprehensive analysis of cardiac arrhythmia genes did not reveal a pathogenic variant. Exome sequencing identified a novel heterozygous missense variant in CASQ2 (Lys180Arg) affecting a highly conserved residue, which cosegregated with disease and was absent in unaffected family members. Genome-wide linkage analysis confirmed a single linkage peak at the CASQ2 locus (logarithm of odds ratio score 3.01; θ = 0). Computer simulations predicted that haploinsufficiency was unlikely to cause the severe CPVT phenotype and suggested a dominant negative mechanism. We show for the first time that a variant in CASQ2 causes autosomal dominant CPVT. Genetic testing in dominant CPVT should include screening for heterozygous CASQ2 variants.

R-Cepia1er as a New Tool to Directly Measure [Ca] in the Sarcoplasmic Reticulum of Ventricular Myocytes

In cardiomyocytes, [Ca] within the sarcoplasmic reticulum (SR) determines, in part, the amplitude of SR Ca release which, in turn, governs myocardial contraction. Therefore, it is critical to understand the molecular mechanisms that regulate [Ca]SR handling. Until recently, the best tool available to directly measure [Ca]SR was a low affinity Ca indicator (Fluo-5N). However this dye presents several difficulties, including species-limitation (it does not work in small rodents), dye extrusion, and non-specific cellular localization. Recently a new genetically encoded family of Ca indicators has been generated, named calcium-measuring organelle-entrapped protein indicators (CEPIA). Here we tested the red emitting fluorescence Ca indicator targeted to the SR, R-CEPIA1er, as a tool to directly measure [Ca]SR dynamics in ventricular myocytes. Infection of rabbit and rat myocytes with an adenovirus expressing the R-CEPIA1er gene, displayed prominent localization of the indicator in the SR and nuclear envelope. Calibration of R-CEPIA1er in rabbit myocytes resulted in a Kd of 617 microM, suggesting that this sensor is sensitive in the whole physiological range of [Ca]SR. [Ca]SR dynamics measured with R-CEPIA1er were tested in rabbit myocytes and compared to Fluo5-N signal. We found that the time course of the [Ca]SR depletion and fractional [Ca]SR release (FR) induced by an action potential was similar between R-CEPIA1er and Fluo-5N. R-CEPIA1er fluorescence did not decline during experiments, indicating lack of dye extrusion or photobleaching. Furthermore, this Ca sensor allowed us to reliably measure [Ca]SR dynamics in rat myocytes. Measurements of [Ca]SR with R-CEPIA1er can be combined with [Ca]cyt indicators (e.g. Fluo-4) to obtain detailed information regarding Ca homeostasis in cardiac myocytes. Moreover, R-CEPIA1er is an optimal tool that can be used to study [Ca]SR handling in commonly used transgenic animals (such as mice and rats).

Mitochondrial Permeability Transition Pore Opening Promotes Calcium Alternans and Waves in Ventricular Myocytes

The interactions between mitochondria and sarcoplasmic reticulum (SR) calcium (Ca) release form a positive feedback loop, which plays important roles in cardiac excitation-contraction coupling and arrhythmogenesis. We developed a spatially-distributed model for rabbit ventricular myocytes, which consists of a three-dimensional network of coupled mitochondria and Ca release units (CRUs). The L-type Ca channels, the ryanodine receptors, and the mitochondrial permeability transition pores (mPTPs) were simulated using random Markov transitions. We then carried out computer simulations using the detailed ventricular cell model to investigate the effects of mitochondrial depolarization on intracellular Ca cycling dynamics. We show that opening of the mPTPs that cause depolarization of a small portion of the mitochondria promotes Ca alternans and Ca waves due to Ca extrusion from mitochondria elevating SR Ca and enhancing CRU coupling. In experiments of isolated rabbit and mouse ventricular myocytes, we applied FCCP to cause mitochondrial depolarization (measured by TMRM) and mPTP opening (measured by calcein loss from the matrix). For rabbit myocytes at pacing cycle length (PCL) 400 ms, no Ca alternans was observed under control conditions, but Ca alternans appeared 2 minutes after FCCP treatment (50-100 nM) coincident with matrix calcein loss indicating mPTP opening. At the same PCL, spontaneous Ca release was observed 2 minutes after FCCP treatment. For mouse myocytes at PCL 200 ms, no Ca alternans was observed under control condition, but Ca alternans appeared 2 minutes after FCCP treatment. The FCCP-mediated Ca alternans was prevented by the mPTP inhibitor cyclosporine A (CsA). FCCP treatment also sped up Ca oscillations which slowed after CsA. These results show that mitochondrial depolarization and mPTP opening can result in Ca alternans and waves which are known to be arrhythmogenic, mechanistically linking metabolic stress to cardiac arrhythmogenesis.

Abstract 17193: Inhibiting Late Sodium Current Attenuates Isoproterenol-Induced Transient Inward Current and Delayed Afterdepolarizations in Ventricular Myocytes

Introduction: The β-adrenergic agonist isoproterenol (ISO) is known to induce the arrhythmogenic transient inward current (I Ti ) and delayed afterdepolarization (DAD) via a stimulation of L-type Ca 2+ current. Recent studies found that ISO-induced DADs in cardiac tissues are inhibited by GS967, a selective blocker of the late Na + current (I NaL ). Thus, we hypothesize that I NaL contributes to the actions of ISO, and selective inhibition of this current will reduce ISO-induced I Ti and DADs. Methods: Transmembrane currents and action potentials of rabbit and guinea pig (GP) ventricular myocytes were recorded using the whole-cell patch-clamp technique. ISO (0.1 μM), GS967 (1 μM) and the Na + channel blocker tetrodotoxin (TTX, 3 μM) were used in the experiments. Results: In rabbit myocytes, application of ISO caused an increase in the amplitude of I NaL from -0.10±0.03 to -0.32±0.04 pA/pF (n = 17, p &lt; 0.05). The ISO-stimulated I NaL was inhibited by GS967 and TTX. In one series of experiments, ISO increased the I NaL from -0.14±0.04 to -0.35±0.06 pA/pF, and GS967 applied in the presence of ISO reduced the current to -0.14±0.03 pA/pF (n = 9, p &lt; 0.05). In another series of experiments, the amplitude of I NaL was increased by ISO from -0.17±0.08 to -0.41±0.09 pA/pF, and was decreased to -0.09±0.08 pA/pF when TTX was applied with ISO (n = 5, p &lt; 0.05). Application of ISO also induced I Ti and DADs. GS967 applied in the presence of ISO inhibited the amplitude of I Ti by 52±6%, from -1.79±0.30 to -0.87±0.16 pA/pF (n = 8, p &lt; 0.05). Consistent with the inhibition of I Ti , GS967 suppressed the amplitude of ISO-induced DADs by 56±12%, from 6.54±1.59 to 3.22±1.27 mV (n = 5, p &lt; 0.05). Similarly, in GP myocytes ISO-induced I Ti and DADs were decreased by GS967 from -1.14±0.21 to -0.73±0.16 pA/pF (n = 7, p &lt; 0.05) and from 7.16±0.59 to 4.67±0.24 mV (n = 5, p &lt; 0.05), respectively. Conclusions: An increased I NaL is likely to contribute to the proarrhythmic effects of ISO in cardiac myocytes. GS967 significantly attenuated ISO-induced I NaL , I Ti and DADs, suggesting that inhibiting this current could be an effective strategy to antagonize the arrhythmogenic actions of β-adrenergic stimulation.

Heterogeneous distribution of INa-L determines interregional differences in rate adaptation of repolarization.

The QT interval, an index of ventricular repolarization, is rate dependent. Exaggerated rate dependence of repolarization is arrhythmogenic. Whether there are differences in rate-dependent repolarization among different heart chambers is unknown. The purpose of this study was to test our hypothesis that heterogeneous distribution in late sodium current (INa-L) is responsible for interregional differences in rate adaptation of repolarization. Action potential duration (APD), QT intervals, and their rate adaptation were studied in isolated arterially perfused ventricular and atrial wedge preparations. APD and INa-L were recorded in isolated rabbit myocytes using the microelectrode and the whole-cell patch clamp technique, respectively. There were distinct interregional differences in repolarization and its rate adaptations among different cardiac chambers: the left ventricle exhibited the longest QT and APD with the greatest rate dependence, followed by the right ventricle, whereas the atria had the shortest APD without significant rate dependence. Interestingly, INa-L densities distributed heterogeneously and matched to interregional APDs and rate dependences as follows: left ventricle > right ventricle > left/right atria. Both dofetilide (an IKr blocker) and anemone toxin (a specific INa-L enhancer) amplified QT/APD rate adaptation preferentially in the left ventricle and therefore exacerbated interregional dispersion of repolarization. On the other hand, the atria exhibited blunted responses to both QT-prolonging agents. Mexiletine with an INa-L blockade effect blunted QT/APD rate adaptation preferentially in the ventricles and significantly reduced bradycardia-dependent interregional dispersion of repolarization. Our results demonstrate that heterogeneous distribution of INa-L contributes importantly to the interregional heterogeneity of repolarization in response to rate changes and APD/QT-prolonging agents.

Intracellular Calcium Alters IKs Amplitude and Kinetics in Rabbit Myocytes

The slowly activating delayed rectifier K+ current (IKs) contributes to repolarization of the cardiac action potential (AP). Intracellular Ca2+ ([Ca2+]i) can modulate IKs, but details of this potentially important regulation are largely unknown. Here, we aimed at assessing [Ca2+]i regulation of IKs and how it governs myocyte AP duration. IKs was recorded from freshly isolated rabbit ventricular myocytes using the patch clamp technique with intracellular pipette solutions that buffered [Ca2+]i to 0, 100, 300, and 500 nM. From a holding potential of −50 mV, IKs was recorded by implementing step-like pulses from −40 to 50 mV in 10 mV increments for 3 s, followed by a tail-pulse to −50 mV for 3 s. When the [Ca2+]i was increased to 300 nM, the maximally activated tail IKs (IMAX) was more than 2-fold greater compared to 0 nM [Ca2+]i (IMAX ∼0.8 pA/pF vs. 0.3 pA/pF). Importantly, when the pipette solution contained 500 nM [Ca2+]i (IMAX ∼1.0 pA/pF), IMAX was 3-fold greater than 0 nM and more than 2-fold greater than 100 nM [Ca2+]i (IMAX ∼0.4 pA/pF). The potential of half-maximal activation (V½) was not different for any situations (∼15 mV). However, deactivation kinetics of tail IKs were slower for cells recorded with 300 and 500 nM [Ca2+]i compared to 0 and 100 nM [Ca2+]i (τdeact ∼1200 ms vs. 800 ms). These results indicate that a rise in [Ca2+]i increases IKs amplitude in rabbits, without altering the voltage dependence of activation, and slows IKs deactivation. Computational modeling suggests these [Ca2+]i-dependent changes might contribute to ventricular AP duration alterations, especially in the presence of adrenergic activation.

Ventricular hypertrophy amplifies transmural dispersion of repolarization by preferentially increasing the late sodium current in endocardium

BackgroundThe late sodium current (INa-L) contributes importantly to rate-dependent change in action potential duration (APD) and transmural dispersion of repolarization (TDR). However, little is known about the mechanisms of increased APD rate-dependence and amplified TDR in left ventricular hypertrophy (LVH) and failure. The purpose of this study was to investigate the role of INa-L in rate-adaptation of transmural APD heterogeneity. MethodsAPD, its rate-dependence and INa-L current were examined in myocytes isolated from the endocardium and epicardium of the control and LVH rabbits. AP was recorded using the standard microelectrode technique, and INa-L was recorded using the whole-cell patch clamp technique. ResultsEarly afterdepolarizations (EADs) were frequently recorded in the isolated myocytes of the LVH rabbits but not in those of controls. LVH prolonged APD more significantly in the endocardial myocytes than in the epicardium (31.7±3.4 vs. 21.6±1.5% n=6, p<0.05), leading to a marked increase in TDR. LVH endocardial myocytes exhibited a greater rate-dependent change in APD compared to the epicardial myocytes. INa-L densities were significantly increased in both LVH endocardium and epicardium. However, LVH increased the INa-L density preferentially in the endocardial myocytes compared to the epicardial myocytes (54.5±4.8% vs. 39.2±3.3%, n=6, p<0.05). ConclusionsOur results demonstrate that LVH increased the INa-L preferentially in the endocardium over the epicardium, which contributes importantly to the stronger rate-dependent change in repolarization and longer APD in the endocardium. This results in an amplified TDR capable of initiating EAD and ventricular arrhythmias.

A modified local control model for Ca2 + transients in cardiomyocytes: Junctional flux is accompanied by release from adjacent non-junctional RyRs

Excitation–contraction coupling in cardiomyocytes requires Ca2+ influx through dihydropyridine receptors in the sarcolemma, which gates Ca2+ release through sarcoplasmic ryanodine receptors (RyRs). Ca2+ influx, release and diffusion produce a cytosolic Ca2+ transient. Here, we investigated the relationship between Ca2+ transients and the spatial arrangement of the sarcolemma including the transverse tubular system (t-system). To accomplish this, we studied isolated ventricular myocytes of rabbit, which exhibit a heterogeneously distributed t-system. We developed protocols for fluorescent labeling and triggered two-dimensional confocal microscopic imaging with high spatiotemporal resolution. From sequences of microscopic images, we measured maximal upstroke velocities and onset times of local Ca2+ transients together with their distance from the sarcolemma. Analyses indicate that not only sarcolemmal release sites, but also those that are within 1μm of the sarcolemma actively release Ca2+. Our data also suggest that release does not occur at sites further than 2.5μm from the sarcolemma. The experimental data are in agreement with results from a mathematical model of Ca2+ release and diffusion. Our findings can be explained by a modified local control model, which constrains the region of regenerative activation of non-junctional RyR clusters. We believe that this model will be useful for describing excitation–contraction coupling in cardiac myocytes with a sparse t-system, which includes those from diseased heart tissue as well as atrial myocytes of some species.

Mechano-Chemotransduction in the Single Cardiac Myocyte Contracting in 3D Elastic Gel

Objective: Mechanical stress under pathological conditions such as hypertension, infarction and fibrosis can cause heart failure and arrhythmias. However, little is known about the mechanotransduction mechanisms that underlie heart disease development due to previous lack of practical techniques to control the mechanical stress on single myocytes necessary for investigating at cellular and molecular levels. Here we use this system to study the mechanical load effects on modulating myocyte Ca2+ signaling and contraction dynamics. Methods: Recently, we developed a novel Cell-in-Gel system that allows control of mechanical load on single rabbit myocytes during excitation-contraction coupling in a 3D elastic gel matrix composed of polyvinyl alcohol (PVA) and tetravalent boronate-PEG crosslinker. The mechanical load can be controlled by adjusting gel stiffness with different mixing ratios of PVA and crosslinker. Results: Myocyte contraction and calcium transients were measured in-gel and compared with load-free cells. Contracting cells in-gel showed a significantly lower fractional shortening (12.6 ± 0.9 in-gel vs. 18.2 ± 0.9 load-free, p<0.001), demonstrating a “knock-down factor” of 31% when the myocyte is pulling mechanical load. Contraction departure and return velocities were significantly slower in-gel than in the load-free state as expected. However, the systolic calcium transient was greater in-gel than load-free (Fura-2 fluorescence ratio peak height 1.47 ± 0.09 in-gel vs. 0.87 ± 0.04 load-free, p<0.0001), revealing the mechano-chemotransduction that translates external stress to intracellular Ca2+ increase. The calcium transient departure and return velocities were also significantly higher in-gel than load-free. Conclusions: Our newly-developed versatile Cell-in-Gel system provides a novel experimental method to control mechanical stress at the single cell level for investigating mechano-chemotransduction pathways in intact myocytes. The above experimental results are consistent with our modeling predictions, demonstrating the mechanical load effects on altering myocyte Ca2+ handling and contraction dynamics.