L-type calcium current in right ventricular outflow tract myocytes of rabbit heart

The mechanism of idiopathic ventricular tachycardia originating from the right ventricular outflow tract (RVOT) is not clear. Many clinical reports have suggested a mechanism of triggered activity. However, there are few studies investigating this because of the technical difficulties associated with examining this theory. The L-type calcium current (I _Ca-L), an important inward current of the action potential (AP), plays an important role in arrhythmogenesis. The aim of this study was to explore differences in the APs of right ventricular (RV) and RVOT cardiomyocytes, and differences in electrophysiological characteristics of the ICa-L in these myocytes. Rabbit RVOT and RV myocytes were isolated and their AP and I _Ca-L were investigated using the patch-clamp technique. RVOT cardiomyocytes had a wider range of AP duration (APD) than RV cardiomyocytes, with some markedly prolonged APDs and markedly shortened APDs. The markedly shortened APDs in RVOT myocytes were abolished by treatment with 4-AP, an inhibitor of the transient outward potassium current, but the markedly prolonged APDs remained, with some myocytes with a long AP plateau not repolarizing to resting potential. In addition, early afterdepolarization (EAD) and second plateau responses were seen in RVOT myocytes but not in RV myocytes. RVOT myocytes had a higher current density for I _Ca-L than RV myocytes (RVOT (13.16±0.87) pA pF⁻¹, RV (8.59±1.97) pA pF⁻¹; P<0.05). The I _Ca-L and the prolonged APD were reduced, and the EAD and second plateau response disappeared, after treatment with nifedipine (10 μmol L⁻¹), which blocks the I _Ca-L. In conclusion, there was a wider range of APDs in RVOT myocytes than in RV myocytes, which is one of the basic factors involved in arrhythmogenesis. The higher current density for I _Ca-L is one of the factors causing prolongation of the APD in RVOT myocytes. The combination of EAD with prolonged APD may be one of the mechanisms of RVOT-VT generation.     

Distinctive electrophysiological characteristics of right ventricular out‐flow tract cardiomyocytes

Ventricular arrhythmias commonly originate from the right ventricular out‐flow tract (RVOT). However, the electrophysiological characteristics and Ca²⁺ homoeostasis of RVOT cardiomyocytes remain unclear. Whole‐cell patch clamp and indo‐1 fluorometric ratio techniques were used to investigate action potentials, Ca²⁺ homoeostasis and ionic currents in isolated cardiomyocytes from the rabbit RVOT and right ventricular apex (RVA). Conventional microelectrodes were used to record the electrical activity before and after (KN‐93, a Ca²⁺/calmodulin‐dependent kinase II inhibitor, or ranolazine, a late sodium current inhibitor) treatment in RVOT and RVA tissue preparations under electrical pacing and ouabain (Na⁺/K⁺ ATPase inhibitor) administration. In contrast to RVA cardiomyocytes, RVOT cardiomyocytes were characterized by longer action potential duration measured at 90% and 50% repolarization, larger Ca²⁺ transients, higher Ca²⁺ stores, higher late Na⁺ and transient outward K⁺ currents, but smaller delayed rectifier K⁺, L‐type Ca²⁺ currents and Na⁺‐Ca²⁺ exchanger currents. RVOT cardiomyocytes showed significantly more pacing‐induced delayed afterdepolarizations (22% versus 0%, P < 0.05) and ouabain‐induced ventricular arrhythmias (94% versus 61%, P < 0.05) than RVA cardiomyocytes. Consistently, it took longer time (9 ± 1 versus 4 ± 1 min., P < 0.05) to eliminate ouabain‐induced ventricular arrhythmias after application of KN‐93 (but not ranolazine) in the RVOT in comparison with the RVA. These results indicate that RVOT cardiomyocytes have distinct electrophysiological characteristics with longer AP duration and greater Ca²⁺ content, which could contribute to the high RVOT arrhythmogenic activity.     

Characterization of the acetylcholine-sensitive muscarinic K+ channel in isolated feline atrial and ventricular myocytes

M₂-cholinergic receptor activation by acetylcholine (ACh) is known to cause a negative inotropic and chronotropic action in atrial tissues. This effect is still controversial in ventricular tissues. The ACh-sensitive muscarinic K⁺ channel (I _K(ACh)) activity was characterized in isolated feline atrial and ventricular myocytes using the patch-clamp technique. Bath application of ACh (1 m) caused shortening of action potential duration without prior stimulation with catecholamines in atrial and ventricular myocytes. Resting membrane potential was slightly hyperpolarized in both tissues. These effects of ACh were greater in atrium than in ventricle. ACh increased whole-cell membrane current in atrial and ventricular myocytes. The current-voltage (I-V) relationship of the ACh-induced current in ventricle exhibited inward-rectification whose slope conductance was smaller than that in atrium. In single channel recording from cell-attached patches, I _K(ACh) activity was observed when ACh was induced in the pipette solution in both tissues. The channel exhibited a slope conductance of 47 ±1 pS (mean ± sd, n=14) in atrium and 47 ±2 pS (n= 10) in ventricle (not different statistically; ns). The open times were distributed according to a single exponential function with mean open lifetime of 2.0±0.3 msec (n= 14) in atrium and 1.9±0.3 msec (n=10) in ventricle (ns); these conductance and kinetic properties were similar between the two tissues. However, the relationship between the concentration of ACh and single channel activity showed a higher sensitivity to ACh in atrium (IC ₅₀ =0.03 m) than in ventricle (IC ₅₀ =0.15 m). In excised inside-out patches, ventricular I _K(ACh) required higher concentrations of GTP to activate the channel compared to atrial channels. These results suggest a reduced I _K(ACh) channel sensitivity to M₂-cholinergic receptor-linked G protein (G_i) in ventricle compared to atrium in feline heart.     

Activation of inwardly rectifying potassium channels by muscarinic receptor-linked G protein in isolated human ventricular myocytes

Muscarinic receptor-linked G protein, G _i , can directely activate the specific K⁺ channel (I _K(ACh)) in the atrium and in pacemaker tissues in the heart. Coupling of G _i to the K⁺ channel in the ventricle has not been well defined. G protein regulation of K⁺ channels in isolated human ventricular myocytes was examined using the patch-clamp technique. Bath application of 1 μm acetylcholine (ACh) reversibly shortened the action potential duration to 74.4 ± 12.1% of control (at 90% repolarization, mean ±sd, n= 8) and increased the whole-cell membrane current conductance without prior β-adrenergic stimulation in human ventricular myocytes. The ACh effect was reversed by atropine (1 μm). In excised inside-out patch configurations, application of GTPγS (100 μm) to the bath solution (internal surface) caused activation of I _K(ACh) and/or the background inwardly-rectifying K⁺ channel (I _K1) in ventricular cell membranes. I _K(ACh) exhibited rapid gating behavior with a slope conductance of 44 ± 2 pS (n= 25) and a mean open lifetime of 1.8 ± 0.3 msec (n= 21). Single channel activity of GTPγS-activated I _K1 demonstrated long-lasting bursts with a slope conductance of 30 ± 2 pS (n= 16) and a mean open lifetime of 36.4 ± 4.1 msec (n= 12). Unlike I _K(ACh), G protein-activated I _K1 did not require GTP to maintain channel activity, suggesting that these two channels may be controlled by G proteins with different underlying mechanisms. The concentration of GTP at half-maximal channel activation was 0.22 μm in I _K(ACh) and 1.2 μm in I _K1. Myocytes pretreated with pertussis toxin (PTX) prevented GTP from activating these channels, indicating that muscarinic receptor-linked PTX-sensitive G protein, G _i , is essential for activation of both channels. G protein-activated channel characteristics from patients with terminal heart failure did not differ from those without heart failure or guinea pig. These results suggest that ACh can shorten the action potential by activating I _K(ACh) and I _K1 via muscarinic receptor-linked G _i proteins in human ventricular myocytes. Received: 23 September 1996/Revised: 18 December 1996     

The Venom of Ornithoctonus huwena affect the electrophysiological stability of neonatal rat ventricular myocytes by inhibiting sodium,potassium and calcium current

Spider venoms are known to contain various toxins that are used as an effective means to capture their prey or to defend themselves against predators. An investigation of the properties of Ornithoctonus huwena (O.huwena) crude venom found that the venom can block neuromuscular transmission of isolated mouse phrenic nerve-diaphragm and sciatic nerve-sartorius preparations. However, little is known about its electrophysiological effects on cardiac myocytes. In this study, electrophysiological activities of ventricular myocytes were detected by 100 μg/mL venom of O.huwena, and whole cell patch-clamp technique was used to study the acute effects of the venom on action potential (AP), sodium current (I_Na), potassium currents (I_Kr, I_Ks, I_to1 and I_K1) and L-type calcium current (I_CaL). The results indicated that the venom prolongs APD₉₀ in a frequency-dependent manner in isolated neonatal rat ventricular myocytes. 100 μg/mL venom inhibited 72.3 ± 3.6% I_Na current, 58.3 ± 4.2% summit current and 54 ± 6.1% the end current of I_Kr, and 65 ± 3.3% I_CaL current, yet, didn't have obvious effect on I_Ks, I_to1 and I_K1 currents. In conclusion, the O.huwena venom represented a multifaceted pharmacological profile. It contains abundant of cardiac channel antagonists and might be valuable tools for investigation of both channels and anti- arrhythmic therapy development.     

Voltage-dependence of contraction in streptozotocin-induced diabetic myocytes

Cardiac contractile dysfunction is frequently reported in human patients and experimental animals with type-1 diabetes mellitus. The aim of this study was to investigate the voltage-dependence of contraction in ventricular myocytes from the streptozotocin (STZ)-induced diabetic rat. STZ-induced diabetes was characterised by hyperglycaemia and hypoinsulinaemia. Other characteristics included reduced body and heart weight and raised blood osmolarity. Isolated ventricular myocytes were patched in whole cell, voltage-clamp mode after correcting for membrane capacitance and series resistance. From a holding membrane potential of –40 mV, test pulses were applied at potentials between –30 and +50 mV in 10 mV increments. L-type Ca²⁺ current (I _Ca,L) density and contraction were measured simultaneously using a video-edge detection system. Membrane capacitance was not significantly altered between control and STZ-induced diabetic myocytes. The I _Ca,L density was significantly (p < 0.05) reduced throughout voltage ranges (–10 mV to +10 mV) in myocytes from STZ-treated rats compared to age-matched controls. Moreover, the amplitude of contraction was significantly reduced (p < 0.05) in myocytes from STZ-treated rats at all test potentials between –20 mV and +30 mV. However, in electrically field-stimulated (1 Hz) myocytes, the amplitude of contraction was not altered by STZ-treatment. It is suggested that in field-stimulated myocytes taken from STZ-induced diabetic hearts, prolonged action potential duration may promote increased Ca²⁺ influx via the sodium-calcium exchanger (NCX), which may compensate for a reduction in Ca²⁺ trigger through L-type-Ca²⁺-channels and lead to normalised contraction. (Mol Cell Biochem 261: 235–243, 2004)     

Mode of inhibition of transient outward current by 1-benzyl-1,2,3,4-tetrahydroisoquinoline in rat ventricular cells

In this study, we examined the effects of 1-benzyl-1,2,3,4-tetrahydroisoquinoline (S49) on a transient outward current (I_to) in rat ventricular myocytes using the whole-cell patch-clamp technique. Depolarization of ventricular myocytes not only activated I_to, but sustained outward currents as well. S49 dose-dependently inhibited the amplitude or integral of I_to, with a 50% inhibitory concentration (IC₅₀) of 4.3 and 2.7 µM, respectively. The inhibition of I_to by S49 was associated with an acceleration of I_to decay. However, S49 had no effect on half inactivation voltage (V_0.5) and slope factor of the voltage-dependent steady-state inactivation curve of I_to. Time-course analysis revealed that S49 developed a block during the depolarizing voltage-clamp step in a monoexponential manner. The rate and magnitude of block were concentration dependent. The equilibrium dissociation constant (K_d) used to inhibit I_to induced by S49, as calculated from the time constant of developing block, was 3.4 µM. The time constant of recovery of I_to from the inactivation state was prolonged by S49. Following treatment with quinidine, the process of I_to recovery was divided into rapid and extremely slow recovery components. Also, the relief from quinidine- or S49-induced block was assessed by comparing the recovery processes of I_to with or without drugs. That comparison revealed the relief from the block of I_to channels by S49 to be more rapid. In summary, the inhibition of I_to by S49 was dose dependent, time dependent but voltage independent. The mechanisms of action might be an open-state block.     

Role of kinases and G-proteins in the hyposmotic stimulation of cardiac IKs

Exposure of cardiac myocytes to hyposmotic solution stimulates slowly-activating delayed-rectifying K⁺ current (I_Ks) via unknown mechanisms. In the present study, I_Ks was measured in guinea-pig ventricular myocytes that were pretreated with modulators of cell signaling processes, and then exposed to hyposmotic solution. Pretreatment with compounds that (i) inhibit serine/threonine kinase activity (10-100 μM H89; 200 μM H8; 50 μM H7; 1 μM bisindolylmaleimide I; 10 μM LY294002; 50 μM PD98059), (ii) stimulate serine/threonine kinase activity (1-5 μM forskolin; 0.1 μM phorbol-12-myristate-13-acetate; 10 μM acetylcholine; 0.1 μM angiotensin II; 20 μM ATP), (iii) suppress G-protein activation (10 mM GDPβS), or (iv) disrupt the cytoskeleton (10 μM cytochalasin D), had little effect on the stimulation of I_Ks by hyposmotic solution. In marked contrast, pretreatment with tyrosine kinase inhibitor tyrphostin A25 (20 μM) strongly attenuated both the hyposmotic stimulation of I_Ks in myocytes and the hyposmotic stimulation of current in BHK cells co-expressing Ks channel subunits KCNQ1 and KCNE1. Since attenuation of hyposmotic stimulation was not observed in myocytes and cells pretreated with inactive tyrphostin A1, we conclude that TK has an important role in the response of cardiac Ks channels to hyposmotic solution.     

Effects of pressure overload-induced hypertrophy on TTX-sensitive inward currents in guinea pig left ventricle

We investigated the effects of pressure overload hypertrophy on inward sodium (I _Na) and calcium currents (I _Ca) in single left ventricular myocytes to determine whether changes in these current systems could account for the observed prolongation of the action potential. Hypertrophy was induced by pressure overload caused by banding of the abdominal aorta. Whole-cell patch clamp experiments were used to measure tetrodotoxin (TTX)-sensitive inward currents. The main findings were that I _Ca density was unchanged whereas I _Na density after stepping from –80 to –30 mV was decreased by 30% (–9.0 ± 1.16 pA pF^–1 in control and –6.31 ± 0.67 pA pF^–1 in hypertrophy, p < 0.05, n= 6). Steady-state activation/inactivation variables of I _Na, determined by using double-pulse protocols, were similar in control and hypertrophied myocytes, whereas the time course of fast inactivation of I _Na was slowed (p < 0.05) in hypertrophied myocytes. In addition, action potential clamp experiments were carried out in the absence and presence of TTX under conditions where only Ca²⁺ was likely to enter the cell via TTX-sensitive channels. We show for the first time that a TTX-sensitive inward current was present during the plateau phase of the action potential in hypertrophied but not control myocytes. The observed decrease in I _Na density is likely to abbreviate rather than prolong the action potential. Delayed fast inactivation of Na⁺ channels was not sustained throughout the voltage pulse and may therefore merely counteract the effect of decreased I _Na density so that net Na⁺ influx remains unaltered. Changes in the fast I _Na do not therefore appear to contribute to lengthening of the action potential in this model of hypertrophy. However, the presence of a TTX-sensitive current during the plateau could potentially contribute to the prolongation of the action potential in hypertrophied cardiac muscle. (Mol Cell Biochem 261: 217–226, 2004)     

Premature ventricular contractions of the right ventricular outflow tract: is there an incipient underlying disease? New insights from a speckle tracking echocardiography study

ContextPremature ventricular contractions (PVCs) originating in the right ventricular outflow tract (RVOT) are traditionally considered idiopathic and benign. Echocardiographic conventional measurements are typically normal.AimsTo assess whether right ventricle longitudinal strain, determined by two-dimensional speckle tracking echocardiography, differ between RVOT PVCs patients (treated with catheter ablation) and healthy controls.MethodsWe retrospectively selected patients with PVCs from the RVOT who underwent electrophysiological study and catheter ablation between 2016 and 2019. Patients with documented structural heart disease were excluded. Transthoracic echocardiography was performed and right ventricle global longitudinal strain (RV-GLS), free wall longitudinal strain (RVFW-LS) and left ventricle global longitudinal strain (LV-GLS) were determined as well as conventional ultrasound measurements of RV and LV function.ResultsWe studied 21 patients with RVOT PVCs and 13 controls. Patients with PVCs from the RVOT had lower values of RV-GLS and RVFW-LS compared with the control group (?19.4% versus ?22.5%, P = 0.015 and ?22.1% versus ?25.5, P = 0.041, respectively). They also had lower values of LV-GLS, although still within the normal range (?19.1% versus ?20.9%, P = 0.047). Regarding RVOT PVCs patients only, RV-GLS and RVFW-LS had no correlation with the PVCs burden prior to catheter ablation and they did not differ between the patients in whom the catheter ablation was successful and those in whom it was not. RV-GLS also had a positive correlation with RVOT proximal diameter (r = 0.487, P = 0.025).ConclusionsIn this group of RVOT PVCs patients, we found worse RV longitudinal strain values (and therefore sub-clinical myocardial dysfunction) when compared to healthy controls.     

兔右室流出道室速对L型钙通道α1c蛋白表达的影响

目的：通过建立右室流出道室速（RVOT-VT）的动物模型,以L型钙通道α1c蛋白作为观察指标,观察RVOT-VT时对L型钙通道α1c蛋白表达的影响,旨在探讨L型钙通道在RVOT-VT中的作用。方法：健康新西兰大耳白兔30只,随机分三组,分别为对照组（10只）、室速组（10只）、室速加维拉帕米干预组（10只）。采用免疫组织化学的方法对三组实验动物的右室流出道心肌组织进行L型钙通道cdc蛋白表达的检测。结果：1、高频刺激主动脉与肺动脉交界处均诱发了起源于右室流出道部位的室速,且室速持续时间均大于4小时。2、室速组L型钙通道α1c蛋白表达量明显下降;干预组L型钙通道α1cc蛋白的表达下降,但与对照组比较无显著差异。结论：1、室速组的心肌L型钙通道α1c蛋白表达发生了重构。2、维拉帕米可以改善心肌L型钙通道α1c蛋白的重构。3、L型钙通道在RVOT-VT发生、持续中起重要作用。     

Calcium-Activated Potassium Current Modulates Ventricular Repolarization in Chronic Heart Failure

The role of I_KCa in cardiac repolarization remains controversial and varies across species. The relevance of the current as a therapeutic target is therefore undefined. We examined the cellular electrophysiologic effects of I_KCa blockade in controls, chronic heart failure (HF) and HF with sustained atrial fibrillation. We used perforated patch action potential recordings to maintain intrinsic calcium cycling. The I_KCa blocker (apamin 100 nM) was used to examine the role of the current in atrial and ventricular myocytes. A canine tachypacing induced model of HF (1 and 4 months, n = 5 per group) was used, and compared to a group of 4 month HF with 6 weeks of superimposed atrial fibrillation (n = 7). A group of age-matched canine controls were used (n = 8). Human atrial and ventricular myocytes were isolated from explanted end-stage failing hearts which were obtained from transplant recipients, and studied in parallel. Atrial myocyte action potentials were unchanged by I_KCa blockade in all of the groups studied. I_KCa blockade did not affect ventricular myocyte repolarization in controls. HF caused prolongation of ventricular myocyte action potential repolarization. I_KCa blockade caused further prolongation of ventricular repolarization in HF and also caused repolarization instability and early afterdepolarizations. SK2 and SK3 expression in the atria and SK3 in the ventricle were increased in canine heart failure. We conclude that during HF, I_KCa blockade in ventricular myocytes results in cellular arrhythmias. Furthermore, our data suggest an important role for I_KCa in the maintenance of ventricular repolarization stability during chronic heart failure. Our findings suggest that novel antiarrhythmic therapies should have safety and efficacy evaluated in both atria and ventricles.     

Ionic currents in cardiac myocytes of squid, Alloteuthis subulata

This paper provides the first study of voltage-sensitive membrane currents present in heart myocytes from cephalopods. Whole cell patch clamp recordings have revealed six different ionic currents in myocytes freshly dissociated from squid cardiac tissues (branchial and systemic hearts). Three types of outward potassium currents were identified: first, a transient outward voltage-activated A-current (I_A), blocked by 4-aminopyridine, and inactivated by holding the cells at a potential of −40 mV; second, an outward, voltage-activated, delayed rectifier current with a sustained time course (I_K); and third, an outward, calcium-dependent, potassium current (I_K(Ca)) sensitive to Co²⁺ and apamin, and with the characteristic N-shaped current voltage relationship. Three inward voltage-activated currents were also identified. First, a rapidly activating and inactivating, sodium current (I_Na), blocked by tetrodotoxin, inactivated at holding potentials more positive than −40 mV, and abolished when external sodium was replaced by choline. Second, an L-type calcium current (I_Ca,L) with a sustained time course, suppressed by nifedipine or Co²⁺, and enhanced by substituting Ca²⁺ for Ba²⁺ in the external medium. The third inward current was also carried by calcium ions, but could be distinguished from the L-type current by differences in its voltage dependence. It also had a more transient time course, was activated at more negative potentials, and resembled the previously described low-voltage-activated, T-type calcium current. Accepted: 24 September 1999     

Suppression of Calcium Sparks in Rat Ventricular Myocytes and Direct Inhibition of Sheep Cardiac RyR Channels by EPA,DHA and Oleic Acid

The anti-arrhythmic effects of long-chain polyunsaturated fatty acids (PUFAs) may be related to their ability to alter calcium handling in cardiac myocytes. We investigated the effect of eicosapentanoic acid (EPA) and docosahexaenoic acid (DHA) on calcium sparks in rat cardiac myocytes and the effects of these PUFAs and the monounsaturated oleic acid on cardiac calcium release channels (RyRs). Visualization of subcellular calcium concentrations in single rat ventricular myocytes showed that intensity of calcium sparks was reduced in the presence of EPA and DHA (15 µM). It was also found that calcium sparks decayed more quickly in the presence of EPA but not DHA. Sarcoplasmic vesicles containing RyRs were prepared from sheep hearts and RyR activity was determined by either [³H]ryanodine binding or by single-channel recording. Bilayers were formed from phosphatidylethanolamine and phosphatidylcholine dissolved in either n-decane or n-tetradecane. EPA inhibited [³H]ryanodine binding to RyRs in SR vesicles with K _I = 40 µM. Poly- and mono-unsaturated free fatty acids inhibited RyR activity in lipid bilayers. EPA (cytosolic or luminal) inhibited RyRs with K _I =32 µM and Hill coefficient, n ₁ = 3.8. Inhibition was independent of the n-alkane solvent and whether RyRs were activated by ATP or Ca²⁺. DHA and oleic acid also inhibited RyRs, suggesting that free fatty acids generally inhibit RyRs at micromolar concentrations.     

A rabbit ventricular action potential model replicating cardiac dynamics at rapid heart rates

Mathematical modeling of the cardiac action potential has proven to be a powerful tool for illuminating various aspects of cardiac function, including cardiac arrhythmias. However, no currently available detailed action potential model accurately reproduces the dynamics of the cardiac action potential and intracellular calcium (Ca_i) cycling at rapid heart rates relevant to ventricular tachycardia and fibrillation. The aim of this study was to develop such a model. Using an existing rabbit ventricular action potential model, we modified the L-type calcium (Ca) current (I_Ca,L) and Ca_i cycling formulations based on new experimental patch-clamp data obtained in isolated rabbit ventricular myocytes, using the perforated patch configuration at 35-37°C. Incorporating a minimal seven-state Markovian model of I_Ca,L that reproduced Ca- and voltage-dependent kinetics in combination with our previously published dynamic Ca_i cycling model, the new model replicates experimentally observed action potential duration and Ca_i transient alternans at rapid heart rates, and accurately reproduces experimental action potential duration restitution curves obtained by either dynamic or S1S2 pacing.     

Lysophosphatidic Acid Increases the Electrophysiological Instability of Adult Rabbit Ventricular Myocardium by Augmenting L-Type Calcium Current

Lysophosphatidic acid (LPA) has diverse actions on the cardiovascular system and is widely reported to modulate multiple ion currents in some cell types. However, little is known about its electrophysiological effects on cardiac myocytes. This study investigated whether LPA has electrophysiological effects on isolated rabbit myocardial preparations. The results indicate that LPA prolongs action potential duration at 90% repolarization (APD₉₀) in a concentration- and frequency-dependent manner in isolated rabbit ventricular myocytes. The application of extracellular LPA significantly increases the coefficient of APD₉₀ variability. LPA increased L-type calcium current (I_Ca,L) density without altering its activation or deactivation properties. In contrast, LPA has no effect on two other ventricular repolarizing currents, the transient outward potassium current (I_to) and the delayed rectifier potassium current (I_K). In arterially perfused rabbit left ventricular wedge preparations, the monophasic action potential duration, QT interval, and Tpeak-end are prolonged by LPA. LPA treatment also significantly increases the incidence of ventricular tachycardia induced by S₁S₂ stimulation. Notably, the effects of LPA on action potentials and I_Ca,L are PTX-sensitive, suggesting LPA action requires a G_i-type G protein. In conclusion, LPA prolongs APD and increases electrophysiological instability in isolated rabbit myocardial preparations by increasing I_Ca,L in a G_i protein-dependent manner.     

Adrenergic Stimulation of Na/K Pump Current in Adult Rat Cardiac Myocytes in Short-term Culture

Passive membrane properties, steady-state Na/K pump current (I _p) and modulation of I _p by adrenergic agonists were studied with patch-clamp techniques in adult rat ventricular myocytes that were freshly isolated or maintained in culture for 1–4 days. Freshly isolated (day 0) myocytes had a 1.7–1.8 times smaller specific membrane resistance compared with that of cells on any day in culture. From day 0 to 4 there was a progressive decrease in cell capacitance (−17.6 ± 0.8 pF/day) without a parallel decline in cell dimensions. The pump current density (1.55 pA/pF) was stable over the 0–4 days in culture. In rod-shaped myocytes norepinephrine (NE) and isoproterenol (ISO) stimulated I _p in a dose-dependent manner, with an apparent affinity of 36 ± 8 and 1.5 ± 0.4 nm, and maximum stimulation of 0.65 ± 0.02 and 0.57 ± 0.02 pA/pF, respectively. Nadolol suppressed this effect, suggesting that it was mediated by β-adrenergic receptor activation. An inverse relationship was found between steady-state I _p and the stimulation of I _p by NE. In contrast to what was shown in guinea pig cardiac myocytes, in rat myocytes isoproterenol stimulation of I _p was not increased by intracellular [Ca] and it did not change the I _p -membrane potential relation. These results show that in adult rat cardiac myocytes NE and ISO are potent stimulators of Na/K pump activity, and this effect may be studied using rat myocytes maintained in short-term culture. Received: 12 November 1997/Revised: 5 March 1998     

Increased intracellular calcium concentration causes electrical turbulence in guinea pig ventricular myocytes

Dysregulation of intracellular Ca²⁺ homeostasis is associated with various pathological conditions and arrhythmogenesis of the heart. The objective of this study was to investigate the effects of an acute increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) on the electrophysiology of ventricular myocytes by mimicking intracellular Ca²⁺ overload. The [Ca²⁺]_i was clamped to either a controlled (65–100 nmol L⁻¹) or increased (1 μmol L⁻¹) level. The transmembrane action potentials and ionic currents were recorded using whole-cell patch clamp techniques. We found that the acute increase in [Ca²⁺]_i shortened the action potential duration, reduced the action potential amplitude, maximum depolarization velocity and resting membrane potential, caused delayed after-depolarizations (DADs), and triggered activity—compared with these parameters in the control. The increased [Ca²⁺]_i augmented late I _Na in a time-dependent manner, reduced I _CaL and I _K1, and increased I _Kr but not I _Ks. The results of this study can be used to explain calcium overload-induced ventricular arrhythmias.     

Cardiac calcium dysregulation in mice with chronic kidney disease

Cardiovascular complications are leading causes of morbidity and mortality in patients with chronic kidney disease (CKD). CKD significantly affects cardiac calcium (Ca²⁺) regulation, but the underlying mechanisms are not clear. The present study investigated the modulation of Ca²⁺ homeostasis in CKD mice. Echocardiography revealed impaired fractional shortening (FS) and stroke volume (SV) in CKD mice. Electrocardiography showed that CKD mice exhibited longer QT interval, corrected QT (QTc) prolongation, faster spontaneous activities, shorter action potential duration (APD) and increased ventricle arrhythmogenesis, and ranolazine (10 µmol/L) blocked these effects. Conventional microelectrodes and the Fluo-3 fluorometric ratio techniques indicated that CKD ventricular cardiomyocytes exhibited higher Ca²⁺ decay time, Ca²⁺ sparks, and Ca²⁺ leakage but lower [Ca²⁺]_i transients and sarcoplasmic reticulum Ca²⁺ contents. The CaMKII inhibitor KN93 and ranolazine (RAN; late sodium current inhibitor) reversed the deterioration in Ca²⁺ handling. Western blots revealed that CKD ventricles exhibited higher phosphorylated RyR2 and CaMKII and reduced phosphorylated SERCA2 and SERCA2 and the ratio of PLB-Thr17 to PLB. In conclusions, the modulation of CaMKII, PLB and late Na⁺ current in CKD significantly altered cardiac Ca²⁺ regulation and electrophysiological characteristics. These findings may apply on future clinical therapies.     

Role of Na+–H+ exchange in the modulation of L-type Ca2+ current during fluid pressure in rat ventricular myocytes

Application of fluid pressure (FP) using pressurized fluid flow suppresses the L-type Ca²⁺ current through both enhancement of Ca²⁺ release and intracellular acidosis in ventricular myocytes. As FP-induced intracellular acidosis is more severe during the inhibition of Na⁺–H⁺ exchange (NHE), we examined the possible role of NHE in the regulation of I_Ca during FP exposure using HOE642 (cariporide), a specific NHE inhibitor. A flow of pressurized (∼16 dyn/cm²) fluid was applied onto single rat ventricular myocytes, and the I_Ca was monitored using a whole-cell patch-clamp under HEPES-buffered conditions. In cells pre-exposed to FP, additional treatment with HOE642 dose-dependently suppressed the I_Ca (IC₅₀ = 0.97 ± 0.12 μM) without altering current–voltage relationships and inactivation time constants. In contrast, the I_Ca in control cells was not altered by HOE642. The HOE642 induced a left shift in the steady-state inactivation curve. The suppressive effect of HOE642 on the I_Ca under FP was not altered by intracellular high Ca²⁺ buffering. Replacement of external Cl⁻ with aspartate to inhibit the Cl⁻-dependent acid loader eliminated the inhibitory effect of HOE642 on I_Ca. These results suggest that NHE may attenuate FP-induced I_Ca suppression by preventing intracellular H⁺ accumulation in rat ventricular myocytes and that NHE activity may not be involved in the Ca²⁺-dependent inhibition of the I_Ca during FP exposure.