Electrophysiological and proarrhythmic parameters in transmural canine left-ventricular needle biopsies

This study was designed to validate the use of small, transmural, left-ventricular biopsies in the dog for investigations of electrophysiological and proarrhythmic properties of the heart. This technique could facilitate pharmacological in vitro testing in remodelled hearts of both man and animal. Small, transmural, semi-cylindrical, left-ventricular biopsies from dogs with normal sinus rhythm (SR) were characterized electrophysiologically and compared with biopsies from electrically remodelled hearts from dogs with chronic, complete AV-block (CAVB). In at least five biopsy segments recordings were made to determine the action potential duration (APD), the transmural gradient of repolarization, the maximal transmural dispersion (tM_max) and presence of early after-depolarizations (EADs) at different pacing cycle lengths (PCLs) in the absence and presence of a class-III agent, ibutilide (10^–6 M). The biopsies showed stable and normal AP characteristics, a conduction velocity of 0.22±0.05 m/s and normal frequency dependence of the APD. The location of the longest APD varied, thus creating transmural repolarization gradients with differing morphology. Ibutilide prolonged the APD, accentuated repolarization gradients and induced EADs. CAVB biopsies had significantly longer APDs, a larger dispersion of repolarization and showed more EADs in the presence of ibutilide than SR biopsies. We conclude that this biopsy technique provides coherent and valid transmural electrophysiological data in dogs under various conditions.     

Role of Ca2+‐activated Cl− current during proarrhythmic early afterdepolarizations in sheep and human ventricular myocytes

Objectives: The proarrhythmic early afterdepolarizations (EADs) during phase‐2 of the cardiac action potential (phase‐2 EADs) are associated with secondary Ca²⁺‐release of the sarcoplasmic reticulum. This makes it probable that the Ca²⁺‐activated Cl^? current [I_Cl(Ca)] is present during phase‐2 EADs. Activation of I_Cl(Ca) during phase‐2 of the action potential will result in an outwardly directed, repolarizing current and may thus be expected to prevent excessive depolarization of phase‐2 EADs. The present study was designed to test this hypothesis. Methods and Results: The contribution of I_Cl(Ca) during phase‐2 EADs was studied in enzymatically isolated sheep and human ventricular myocytes using the patch‐clamp methodology. EADs were induced by a combination of a low stimulus frequency (0.5 Hz) and exposure to 1 μm noradrenaline. In sheep myocytes, the I_Cl(Ca) blocker 4,4′‐diisothiocyanostilbene‐2,2′‐disulfonic acid (DIDS, 0.5 mm ) abolished phase‐1 repolarization of the action potential in all myocytes tested. This indicates that I_Cl(Ca) is present in all sheep myocytes. However, DIDS had no effect on phase‐2 EAD characteristics. In human myocytes, DIDS neither affected phase‐1 repolarization nor phase‐2 EAD characteristics. Conclusion: In sheep ventricular myocytes, but not in human ventricular myocytes, I_Cl(Ca) contributes to phase‐1 repolarization of the action potential. In both sheep and human myocytes, I_Cl(Ca) plays a limited role during phase‐2 EADs.     

Effects of potassium channel openers in the isolated perfused hypokalaemic murine heart

Aim: We explored the anti‐arrhythmic efficacy of K⁺ channel activation in the hypokalaemic murine heart using NS1643 and nicorandil, compounds which augment I_Kr and I_KATP respectively. Methods: Left ventricular epicardial and endocardial monophasic action potentials were compared in normokalaemic and hypokalaemic preparations in the absence and presence of NS1643 (30 μm ) and nicorandil (20 μm ). Results: Spontaneously beating hypokalaemic hearts (3 mm K⁺) all elicited early afterdepolarizations (EADs) and episodes of ventricular tachycardia (VT). Perfusion with NS1643 and nicorandil suppressed EADs and VT in 7 of 13 and five of six hypokalaemic hearts. Provoked arrhythmia studies using programmed electrical stimulation induced VT in all hypokalaemic hearts, but failed to do so in 7 of 13 and five of six hearts perfused with NS1643 and nicorandil respectively. These anti‐arrhythmic effects were accompanied by reductions in action potential duration at 90% repolarization (APD₉₀) and changes in the transmural gradient of repolarization, reflected in ΔAPD₉₀. NS1643 and nicorandil reduced epicardial APD₉₀ from 68.3 ± 1.1 to 56.5 ± 4.1 and 51.5 ± 1.5 ms, respectively, but preserved endocardial APD₉₀ in hypokalaemic hearts. NS1643 and nicorandil thus restored ΔAPD₉₀ from ?9.6 ± 4.3 ms under baseline hypokalaemic conditions to 3.9 ± 4.1 and 9.9 ± 2.1 ms, respectively, close to normokalaemic values. Conclusion: These findings demonstrate, for the first time, the anti‐arrhythmic efficacy of K⁺ channel activation in the setting of hypokalaemia. NS1643 and nicorandil are anti‐arrhythmic through the suppression of EADs, reductions in APD₉₀ and restorations of ΔAPD₉₀.     

Pharmacological separation of early afterdepolarizations from arrhythmogenic substrate in DeltaKPQ Scn5a murine hearts modelling human long QT 3 syndrome

Aim: To perform an empirical, pharmacological, separation of early afterdepolarizations (EADs) and transmural gradients of repolarization in arrhythmogenesis in a genetically modified mouse heart modelling human long QT syndrome (LQT) 3. Methods: Left ventricular endocardial and epicardial monophasic action potentials and arrhythmogenic tendency were compared in isolated wild type (WT) and Scn5a+/Δ hearts perfused with 0.1 and 1 μm propranolol and paced from the right ventricular epicardium. Results: All spontaneously beating bradycardic Scn5a+/Δ hearts displayed EADs, triggered beats and ventricular tachycardia (VT; n = 7), events never seen in WT hearts (n = 5). Perfusion with 0.1 and 1 μm propranolol suppressed all EADs, triggered beats and episodes of VT. In contrast, triggering of VT persisted following programmed electrical stimulation in 6 of 12 (50%), one of eight (12.5%), but six of eight (75%) Scn5a+/Δ hearts perfused with 0, 0.1 and 1 μm propranolol respectively in parallel with corresponding alterations in repolarization gradients, reflected in action potential duration (ΔAPD₉₀) values. Thus 0.1 μm propranolol reduced epicardial but not endocardial APD₉₀ from 54.7 ± 1.6 to 44.0 ± 2.0 ms, restoring ΔAPD₉₀ from ?3.8 ± 1.6 to 3.5 ± 2.5 ms (all n = 5), close to WT values. However, 1 μm propranolol increased epicardial APD₉₀ to 72.5 ± 1.2 ms and decreased endocardial APD₉₀ from 50.9 ± 1.0 to 24.5 ± 0.3 ms, increasing ΔAPD₉₀ to ?48.0 ± 1.2 ms. Conclusion: These findings empirically implicate EADs in potentially initiating spontaneous arrhythmogenic phenomena and transmural repolarization gradients in the re‐entrant substrate that would sustain such activity when provoked by extrasystolic activity in murine hearts modelling human LQT3 syndrome.     

Ca2+‐activated Cl− current reduces transmural electrical heterogeneity within the rabbit left ventricle

Objective: Various cationic membrane channels contribute to the heterogeneity of action potential configuration between the transmural layers of the left ventricle. The role of anionic membrane channels is less intensively studied. We investigated the role of the Ca²⁺‐activated Cl^? current, I_Cl(Ca), in transmural electrical heterogeneity. Methods and Results: We determined the density of I_Cl(Ca) and its physiological role in subepicardial and subendocardial ventricular myocytes of rabbit using the patch‐clamp technique. I_Cl(Ca) was measured as the 4,4′diisothiocyanatostilbene‐2,2′‐disulphonic acid (DIDS) sensitive current. The current–voltage relationships and the densities of I_Cl(Ca) were similar in subepicardial and subendocardial myocytes. However, the functional role of I_Cl(Ca) exhibited striking differences. In subendocardial myocytes, blockade of I_Cl(Ca) by DIDS increased action potential duration (APD) significantly at all measured stimulus frequencies (3.33–0.2 Hz). In subepicardial myocytes, I_Cl(Ca) blockade increased APD only at 3.33 Hz, but not at the lower stimulus frequencies. At 1 Hz, I_Cl(Ca) blockade in subepicardial myocytes only caused an APD increase when the transient outward K⁺ current, I_to1, was blocked. Conclusions: The densities and gating properties of I_Cl(Ca) are similar in subepicardial and subendocardial myocytes. I_Cl(Ca) contributes to APD shortening in subendocardial, but not in subepicardial myocytes except at 3.33 Hz. These differences in functional expression of I_Cl(Ca) reduce the electrical heterogeneity in rabbit left ventricle.     

兔左室肥厚心肌跨室壁复极不均一性及其膜离子流变化的实验研究

目的:探讨兔左心室肥厚心肌心外膜下、中层、心内膜下3层心肌细胞动作电位及膜离子流变化的不均一性。方法: 心肌肥厚组以腹主动脉缩窄术复制兔压力超负荷心肌肥厚模型,并设正常对照组以作比较。胶原酶两步消化法分离获取兔单个心室肌细胞,其中用植皮刀分离左室游离壁内膜下、中层、外膜下3层心肌。以全细胞膜片钳记录单细胞跨膜动作电位和离子电流。结果: 肥厚组3层心肌细胞动作电位时程(APD₉₀)较对照组3层心肌细胞APD₉₀均分别有明显延长,而以中层心肌细胞APD₉₀延长最为明显(延长比例:中层26.0%±2.7%,外膜14.0%±1.6%,内膜10.0%±1.1%),使肥厚心肌跨室壁复极不均一性明显大于对照组。肥厚组各层心肌细胞瞬时外向钾电流(I_to)和缓慢激活的延迟整流钾电流(I_Ks)密度均低于对照组,且均以中层细胞下降的幅度最大。肥厚组各层心肌细胞L型钙电流(I_Ca,L)与快速激活的延迟整流钾电流(I_Kr) 密度与对照组均无明显差异。肥厚组各层心肌细胞内向整流钾电流(I_K1) 均明显低于对照组,但各层变低的幅度无明显差异。 结论:兔肥厚心肌跨室壁复极不均一性明显增大,I_to及I_Ks的跨室壁不均一性下降可能是其主要原因。     

Beta-adrenergic stimulation reverses the I Kr–I Ks dominant pattern during cardiac action potential

β-Adrenergic stimulation differentially modulates different K⁺ channels and thus fine-tunes cardiac action potential (AP) repolarization. However, it remains unclear how the proportion of I _Ks, I _Kr, and I _K1 currents in the same cell would be altered by β-adrenergic stimulation, which would change the relative contribution of individual K⁺ current to the total repolarization reserve. In this study, we used an innovative AP-clamp sequential dissection technique to directly record the dynamic I _Ks, I _Kr, and I _K1 currents during the AP in guinea pig ventricular myocytes under physiologically relevant conditions. Our data provide quantitative measures of the magnitude and time course of I _Ks, I _Kr, and I _K1 currents in the same cell under its own steady-state AP, in a physiological milieu, and with preserved Ca²⁺ homeostasis. We found that isoproterenol treatment significantly enhanced I _Ks, moderately increased I _K1, but slightly decreased I _Kr in a dose-dependent manner. The dominance pattern of the K⁺ currents was I _Kr?>?I _K1?>?I _Ks at the control condition, but reversed to I _Kr?I _K1?I _Ks following β-adrenergic stimulation. We systematically determined the changes in the relative contribution of I _Ks, I _Kr, and I _K1 to cardiac repolarization during AP at different adrenergic states. In conclusion, the β-adrenergic stimulation fine-tunes the cardiac AP morphology by shifting the power of different K⁺ currents in a dose-dependent manner. This knowledge is important for designing antiarrhythmic drug strategies to treat hearts exposed to various sympathetic tones.     

Modeling gender effects on electrical activity of single ventricular myocytes

In this study we investigate the mechanisms underlying gender differences in the generation of arrhythmias in the long QT and Brugada syndromes. Simulations were conducted at the single myocyte level using a detailed mathematical model of human ventricular myocytes. Given the scarce human data on the gender-related differences in single cardiac cells, we assumed gender-related differences in five ionic-current systems: fast sodium current (I_Na), slowly inactivating late sodium current (I_Nal), transient outward potassium current (I_to), slow delayed rectifier potassium current (I_Ks), and calcium current through the L-type channel (I_Ca(L)), based on experimental results obtained in canine myocytes. Our modeling results suggest that in left ventricular myocytes, enhanced I_Nal under conditions of reduced repolarization reserve results in sex-dependent development of early afterdepolarizations (EADs) in the post-pause action potentials (APs). Moreover, this modeling study demonstrates increased propensity for the development of the loss of the AP dome in male epicardial myocytes of the right ventricle compared with other types of myocytes from the left and right ventricles. Finally, we also found a slight effect of I_Nal on gender-dependent loss of AP dome in epicardial right ventricular myocytes. In conclusion, at the cellular level, gender differences in the development of EADs and the propensity to develop the loss of the AP dome can be attributed to male/female related differences in I_Na, I_Nal, I_to, I_Ks, and I_Ca(L).     

Transfection by eukaryotic expression vector pcDNA3-HERG inhibits the cultured neonatal rabbit ventricular myocyte hypertrophy induced by phenylephrine

ObjectiveProlonged action potential and decreased outward K⁺ currents are consistent findings in hypertrophic myocardium. The relation between action potential prolongation and myocyte hypertrophy has remained unclear. The present study investigated the temporal relation between action potential prolongation and myocyte hypertrophy, and the effect of enhancing repolarization on myocyte hypertrophy induced by phenylephrine.MethodsNeonatal rabbit ventricular myocytes were cultured and treated with 10 μmol/l phenylephrine. At 6 and 48 h after phenylephrine stimulation, myocyte hypertrophic parameters (including myocyte volume, total protein content, and membrane capacitance), action potential duration (APD), and calcineurin activity were measured; meanwhile, the effect of human-ether-a-go-go–related gene (HERG; encoding the αsubunit of rapidly activating delayed rectifier potassium channel) transfection on the above parameters at 48 h of phenylephrine stimulation was also measured.ResultsAt 6 h after phenylephrine treatment, APD at 90% repolarization of neonatal rabbit ventricular myocytes was prolonged by 14.3% (P<.05), but myocyte hypertrophy was not found. At 48 h after phenylephrine stimulation, APD at 90% repolarization of neonatal rabbit ventricular myocytes was furthermore prolonged by 18.8% (P<.05); at the same time, myocyte volume, total protein content, membrane capacitance, and calcineurin activity were increased by 40.0%, 41.8%, 36.4%, and 124.1%, respectively (P<.01). Neonatal rabbit ventricular myocytes transfected by pcDNA3-HERG overexpressed I_HERG,tail current, which was about fourfold higher than I_Kr (rapidly activating delayed rectifier K⁺ current) of neonatal rabbit ventricular myocytes without transfection of HERG. HERG overexpression could accelerate repolarization and shorten APD at 90% repolarization prolonged by phenylephrine and partially inhibit myocyte hypertrophy and calcineurin activation.ConclusionsDuring the myocyte hypertrophy induced by phenylephrine, prolongation of APD at 90% repolarization is not secondary to but precedes myocyte hypertrophy. HERG overexpression could enhance the repolarization and inhibit the calcineurin activation and myocyte hypertrophy induced by phenylephrine.     

Mechanisms responsible for the altered cardiac repolarization dispersion in experimental hypothyroidism

Aims: To identify the causes for the inhomogeneity of ventricular repolarization and increased QT dispersion in hypothyroid mice. Methods: We studied the effects of 5‐propyl‐2‐thiouracil‐induced hypothyroidism on the ECG, action potential (AP) and current density of the repolarizing potassium currents I_to,fast, I_to,slow, I_K,slow and I_ss in enzymatically isolated myocytes from three different regions of mouse heart: right ventricle (RV), epicardium of the left ventricle (Epi‐LV) and interventricular septum. K⁺ currents were recorded with the patch‐clamp technique. Membranes from isolated ventricular myocytes were extracted by centrifugation. Kv4.2, Kv4.3, KChIP and Na/Ca exchanger proteins were visualized by Western blot. Results: The frequency or conduction velocity was not changed by hypothyroidism, but QTc was prolonged. Neither resting membrane potential nor AP amplitude was modified. The action potential duration (APD)₉₀ increased in the RV and Epi‐LV, but not in the septum. Hypothyroid status has no effect either on I_to,slow, I_k,slow or I_ss in any of the regions analysed. However, I_to,fast was significantly reduced in the Epi‐LV and in the RV, whereas it was not altered in cells from the septum. Western blot analysis reveals a reduction in Kv4.2 and Kv4.3 protein levels in both the Epi‐LV and the RV and an increase in Na/Ca exchanger. Conclusion: From these results we suggest that the regional differences in APD lengthening, and thus in repolarization inhomogeneity, induced by experimental hypothyroidism are at least partially explained by the uneven decrease in I_to,fast and the differences in the relative contribution of the depolarization‐activated outward currents to the repolarization process.     

Arrhythmogenic mechanisms in the isolated perfused hypokalaemic murine heart

Aim: Hypokalaemia is associated with a lethal form of ventricular tachycardia (VT), torsade de pointes, through pathophysiological mechanisms requiring clarification. Methods: Left ventricular endocardial and epicardial monophasic action potentials were compared in isolated mouse hearts paced from the right ventricular epicardium perfused with hypokalaemic (3 and 4 mm [K⁺]_o) solutions. Corresponding K⁺ currents were compared in whole‐cell patch‐clamped epicardial and endocardial myocytes. Results: Hypokalaemia prolonged epicardial action potential durations (APD) from mean APD₉₀s of 37.2 ± 1.7 ms (n = 7) to 58.4 ± 4.1 ms (n =7) and 66.7 ± 2.1 ms (n = 11) at 5.2, 4 and 3 mm [K⁺]_o respectively. Endocardial APD₉₀s correspondingly increased from 51.6 ± 1.9 ms (n = 7) to 62.8 ± 2.8 ms (n = 7) and 62.9 ± 5.9 ms (n = 11) giving reductions in endocardial–epicardial differences, ΔAPD₉₀, from 14.4 ± 2.6 to 4.4 ± 5.0 and ?3.4 ± 6.0 ms respectively. Early afterdepolarizations (EADs) occurred in epicardia in three of seven spontaneously beating hearts at 4 mm [K⁺]_o with triggered beats followed by episodes of non‐sustained VT in nine of 11 preparations at 3 mm . Programmed electrical stimulation never induced arrhythmic events in preparations perfused with normokalemic solutions yet induced VT in two of seven and nine of 11 preparations at 4 and 3 mm [K⁺]_o respectively. Early outward K⁺ current correspondingly fell from 73.46 ± 8.45 to 61.16±6.14 pA/pF in isolated epicardial but not endocardial myocytes (n = 9) (3 mm [K⁺]_o). Conclusions: Hypokalaemic mouse hearts recapitulate the clinical arrhythmogenic phenotype, demonstrating EADs and triggered beats that might initiate VT on the one hand and reduced transmural dispersion of repolarization reflected in ΔAPD₉₀ suggesting arrhythmogenic substrate on the other.     

Separation of early afterdepolarizations from arrhythmogenic substrate in the isolated perfused hypokalaemic murine heart through modifiers of calcium homeostasis

Aims: We resolved roles for early afterdepolarizations (EADs) and transmural gradients of repolarization in arrhythmogenesis in Langendorff‐perfused hypokalaemic murine hearts paced from the right ventricular epicardium. Methods: Left ventricular epicardial and endocardial monophasic action potentials (MAPs) and arrhythmogenic tendency were compared in the presence and absence of the L‐type Ca²⁺ channel blocker nifedipine (10 nm –1 μm ) and the calmodulin kinase type II inhibitor KN‐93 (2 μm ). Results: All the hypokalaemic hearts studied showed prolonged epicardial and endocardial MAPs, decreased epicardial‐endocardial APD₉₀ difference, EADs, triggered beats and ventricular tachycardia (VT) (n = 6). In all spontaneously beating hearts, 100 (but not 10) nm nifedipine reduced both the incidence of EADs and triggered beats from 66.9 ± 15.7% to 28.3 ± 8.7% and episodes of VT from 10.8 ± 6.3% to 1.2 ± 0.7% of MAPs (n = 6 hearts, P < 0.05); 1 μm nifedipine abolished all these phenomena (n = 6). In contrast programmed electrical stimulation (PES) still triggered VT in six of six hearts with 0, 10 and 100 nm but not 1 μm nifedipine. 1 μm nifedipine selectively reduced epicardial (from 66.1 ± 3.4 to 46.2 ± 2.5 ms) but not endocardial APD₉₀, thereby restoring ΔAPD₉₀ from ?5.9 ± 2.5 to 15.5 ± 3.2 ms, close to normokalaemic values. KN‐93 similarly reduced EADs, triggered beats and VT in spontaneously beating hearts to 29.6 ± 8.9% and 1.7 ± 1.1% respectively (n = 6) yet permitted PES‐induced VT (n = 6), in the presence of a persistently negative ΔAPD₉₀. Conclusions: These findings empirically implicate both EADs and triggered beats alongside arrhythmogenic substrate of ΔAPD₉₀ in VT pathogenesis at the whole heart level.     

Role of slow delayed rectifier K+-current in QT prolongation in the alloxan-induced diabetic rabbit heart

Aim: In diabetes mellitus, several cardiac electrophysiological parameters are known to be affected. In rodent experimental diabetes models, changes in these parameters were reported, but only limited relevant information is available in other species, having cardiac electrophysiological properties more resembling the human, including the rabbit. The present study was designed to analyse the effects of experimental type 1 diabetes on ventricular repolarization and the underlying transmembrane potassium currents in rabbit hearts. Methods: Diabetes was induced by a single injection of alloxan (145 mg kg⁻¹ i.v.). After the development of diabetes (3 weeks), electrophysiological studies were performed using whole cell voltage clamp and ECG measurements. Results: The QT_c interval in diabetic rabbits was moderately but statistically significantly longer than measured in the control animals (155 ± 1.8 ms vs. 145 ± 2.8 ms, respectively, n = 9–10, P < 0.05). This QT_c-lengthening effect of diabetes was accompanied by a significant reduction in the density of the slow delayed rectifier K⁺ current, I_Ks (from 1.48 ± 0.35 to 0.86 ± 0.17 pA pF⁻¹ at +50 mV, n = 19–21, P < 0.05) without changes in current kinetics. No differences were observed either in the density or in the kinetics of the inward rectifier K⁺ current (I_K1), the rapid delayed rectifier K⁺ current (I_Kr), the transient outward current (I_to) and the L-type calcium current (I_CaL) between the control and alloxan-treated rabbits. Conclusion: It is concluded that type 1 diabetes mellitus, although only moderately, lengthens ventricular repolarization. Diabetes attenuates the repolarization reserve by decreasing the density of I_Ks current, and thereby may enhance the risk of sudden cardiac death.     

Phosphatidylinositol 4,5-bisphosphate interactions with the HERG K+ channel

Regulation of ion channel activity plays a central role in controlling heart rate, rhythm, and contractility responses to cardiovascular demands. Dynamic beat-to-beat regulation of ion channels is precisely adjusted by autonomic stimulation of cardiac G protein-coupled receptors. The rapidly activating delayed rectifier K⁺ current (I _Kr) is produced by the channel that is encoded by human ether-a-gogo-related gene (HERG) and is essential for the proper repolarization of the cardiac myocyte at the end of each action potential. Reduction of I _Kr via HERG mutations or drug block can lead to lethal cardiac tachyarrhythmias. Autonomic regulation of HERG channels is an area of active investigation with the emerging picture of a complex interplay of signal transduction events, including kinases, second messengers, and protein–protein interactions. A recently described pathway for regulation of HERG is through channel interaction with the phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2). Changes in cellular PIP2 concentrations may occur with Gq-coupled receptor activation. Here, we review the evidence for PIP2–HERG interactions, its potential biological significance, and unfilled gaps in our understanding of this regulatory mechanism.     

Identification of <Emphasis Type="Italic">I</Emphasis><Subscript>Kr</Subscript> Kinetics and Drug Binding in Native Myocytes

Determining the effect of a compound on I _Kr is a standard screen for drug safety. Often the effect is described using a single IC₅₀ value, which is unable to capture complex effects of a drug. Using verapamil as an example, we present a method for using recordings from native myocytes at several drug doses along with qualitative features of I _Kr from published studies of HERG current to estimate parameters in a mathematical model of the drug effect on I _Kr. I _Kr was recorded from canine left ventricular myocytes using ruptured patch techniques. A voltage command protocol was used to record tail currents at voltages from −70 to −20 mV, following activating pulses over a wide range of voltages and pulse durations. Model equations were taken from a published I _Kr Markov model and the drug was modeled as binding to the open state. Parameters were estimated using a combined global and local optimization algorithm based on collected data with two additional constraints on I _Kr I–V relation and I _Kr inactivation. The method produced models that quantitatively reproduce both the control I _Kr kinetics and dose dependent changes in the current. In addition, the model exhibited use and rate dependence. The results suggest that: (1) the technique proposed here has the practical potential to develop data-driven models that quantitatively reproduce channel behavior in native myocytes; (2) the method can capture important drug effects that cannot be reproduced by the IC₅₀ method. Although the method was developed for I _Kr, the same strategy can be applied to other ion channels, once appropriate channel-specific voltage protocols and qualitative features are identified. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Intracellular calcium and electrical restitution in mammalian cardiac cells

The role of calcium current and changes in intracellular calcium concentration ([Ca²⁺]_i) in regulation of action potential duration (APD) during electrical restitution process was studied in mammalian ventricular preparations. Properly timed action potentials were recorded from multicellular preparations and isolated cardiomyocytes using conventional microelectrodes and EGTA-containing patch pipettes. APD increased monotonically in canine and guinea pig ventricular preparations with increasing diastolic interval (DI), while in rabbit papillary muscles the restitution process was biphasic: APD first lengthened, then shortened as the DI increased. When the restitution process was studied in single cardiomyocytes using EGTA-containing patch pipettes, the restitution pattern was similar in the three species studied. Similarly, no difference was observed in the recovery time constant of calcium current (I_Ca-L) measured under these conditions in voltage clamped myocytes. Loading the myocytes with the [Ca²⁺]_i-chelator BAPTA-AM had adverse effects in rabbit and canine cells. In rabbit myocytes steady-state APD lengthened and the late shortening component of restitution was abolished in BAPTA-loaded cells. In canine myocytes BAPTA-load shortened steady-state APD markedly, and during restitution, APD decreased with increasing DI. The late shortening component of restitution, observed in untreated rabbit preparations, was greatly reduced after nifedipine treatment, but remained preserved in the presence of 4-aminopyridine or nicorandil. Beat to beat changes in APD, peak I_Ca-L and [Ca²⁺]_i, measured using the fluorescent dye, Fura-2, were monitored in rabbit ventricular myocytes after a 1-min period of rest. In these cells, the shortening of APD was accompanied by a gradual reduction of the peak I_Ca-L and elevation of diastolic [Ca²⁺]_i during the initial eight post-rest action potentials. It is concluded that elevation of [Ca²⁺]_i shortens, while reduction of [Ca²⁺]_i lengthens APD in rabbit, but not in canine ventricular myocytes. These differences may probably be related to different distributions of [Ca²⁺]_i-dependent ion currents and/or to differences in calcium handling between the two species.     

Role of external Ca2+ and K+ in gating of cardiac delayed rectifier K+ currents

We sought to determine whether extracellular Ca²⁺ (Ca _e ²⁺ ) and K⁺ (K _e ⁺ ) play essential roles in the normal functioning of cardiac K⁺ channels. Reports by others have shown that removal of Ca _e ²⁺ and K _e ⁺ alters the gating properties of neural delayed rectifier (I _K) and A-type K⁺ currents, resulting in a loss of normal cation selectivity and voltage-dependent gating. We found that removal of Ca _e ²⁺ and K _e ⁺ from the solution bathing guinea pig ventricular myocytes often induced a leak conductance, but did not affect the ionic selectivity or time-dependent activation and deactivation properties of I _K. The effect of [K⁺]_e on the magnitude of the two components of cardiac I _K was also examined. I _K in guinea pig myocytes is comprised of two distinct types of currents: I _Kr (rapidly activating, rectifying) and I _Ks (slowly activating). The differential effect of Ca _e ²⁺ on the two components of I _K (previously shown to shift the voltage dependence of activation of the two currents in opposite directions) was exploited to determine the role of K _e ⁺ on the magnitude of I _Ks and I _Kr. Lowering [K⁺]_e from 4 to 0 mM increased I _Ks, as expected from the change in driving force for K⁺, but decreased I _Kr. The differential effect of [K⁺]_e on the two components of cardiac I _K may explain the reported discrepancies regarding modulation of cardiac I _K conductance by this cation.     

Computer simulation of wild-type and mutant human cardiac Na+ current

Long QT syndrome (LQTS) and Brugada syndrome (BrS) are inherited diseases predisposing to ventricular arrhythmias and sudden death. Genetic studies linked LQTS and BrS to mutations in genes encoding for cardiac ion channels. Recently, two novel missense mutations at the same codon in the gene encoding the cardiac Na⁺ channel (SCN5A) have been identified: Y1795C (causing the LQTS phenotype) and Y1795H (causing the BrS phenotype). Functional studies in HEK293 cells showed that both mutations alter the inactivation of Na⁺ current and cause a sustained Na⁺ current upon depolarisation. In this paper, a nine state Markov model was used to simulate the Na⁺ current in wild-type Na⁺ cardiac channel and the current alterations observed in Y1795C and Y1795H mutant channels. The model includes three distinct closed states, a conducting open state and five inactivation states (one fast-, two intermediate- and two closed-inactivation). Transition rates between these states were identified on the basis of previously published voltage-clamp experiments. The model was able to reproduce the experimental Na⁺ current in mutant channels just by altering the assignment of model parameters with respect to wild-type case. Parameter assignment was validated by performing action potential clamp experiments and comparing experimental and simulated I _Na current. The Markov model was subsequently introduced in the Luo–Rudy model of ventricular myocyte to investigate “in silico” the consequences on the ventricular cell action potential of the two mutations. Coherently with their phenotypes, the Y1795C mutation prolongs the action potential, while the Y1795H mutation causes only negligible changes in action potential morphology.     

The effects of over-expression of the FK506-binding protein FKBP12.6 on K+ currents in adult rabbit ventricular myocytes

This study examines the effects of the intracellular protein FKBP12.6 on action potential and associated K⁺ currents in isolated adult rabbit ventricular cardiomyocytes. FKBP12.6 was over-expressed by ~6 times using a recombinant adenovirus coding for human FKBP12.6. This over-expression caused prolongation of action potential duration (APD) by ~30%. The amplitude of the transient outward current (I _to) was unchanged, but rate of inactivation at potentials positive to +40 mV was increased. FKBP12.6 over-expression decreased the amplitude of the inward rectifier current (I _K1) by ~25% in the voltage range −70 to −30 mV, an effect prevented by FK506 or lowering intracellular [Ca²⁺] below 1 nM. Over-expression of an FKBP12.6 mutant, which cannot bind calcineurin, prolonged APD and affected I _to and I _K1 in a similar manner to wild-type protein. These data suggest that FKBP12.6 can modulate APD via changes in I _K1 independently of calcineurin binding, suggesting that FKBP12.6 may affect APD by direct interaction with I _K1.