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The function of voltage-gated human ether-à-go-gorelated gene ( hERG) K+ channels is critical for both normal cardiac repolarization and suppression of arrhythmias initiated by premature excitation. These important functions are facilitated by their unusual kinetics that combine relatively slow activation and deactivation with rapid and voltage-dependent inactivation and recovery from inactivation. The thermodynamics of these unusual features were examined by exploring the effect of temperature on the activation and inactivation processes of hERG channels expressed in Chinese hamster ovary cells. Increased temperature shifted the voltage dependence of activation in the hyperpolarizing direction but that of inactivation in the depolarizing direction. This increases the relative occupancy of the open state and contributes to the marked temperature sensitivity of hERG current magnitude observed during action potential voltage clamps. The rates of activation and deactivation also increase with higher temperatures, but less markedly than do the rates of inactivation and recovery from inactivation. Our results also emphasize that one cannot extrapolate results obtained at room temperature to 37°C by using a single temperature scale factor.

A transformed line of human embryonic kidney epithelial cells (HEK 293) is commonly used as an expression system for exogenous ion channel genes. Previously, it has been shown that these cells contain mRNAs for a variety of ion channels. Expression of some of these genes has been confirmed at the protein level. Patch-clamp electrophysiology experiments confirm the presence of multiple ion channels and molecular data agree with pharmacological profiles of identified channels. In this work, we show that endogenous voltage-gated potassium channels in HEK cells are a significant source of outward current at positive potentials. We show that both non-transfected HEK cells and HEK cells transfected with hyperpolarization-activated cyclic-nucleotide gated (HCN) channels have a significant amount of voltage-gated potassium (K(V)) current when certain tail current voltage-clamp protocols are used to assay HCN current activation. Specifically, tail current protocols that use a depolarized holding potential of -40 mV followed by hyperpolarizing pulses (-80 to -140 mV) and then a tail pulse potential of +20 mV indicate K(V) channels undergo closed-state inactivation at the more depolarized holding potential of -40 mV, followed by recovery from inactivation (but no activation) at hyperpolarizing potentials and high amount of activation at the positive tail potential. Our results indicate that pulse protocols with positive tail pulses are inaccurate assays for HCN current in certain HEK cells. Surprisingly, HEK-293 cells were found to contain mRNA for HCN2 and HCN3 although we have not detected a significant and consistent endogenous I(f)-like current in these cells.

Plasma levels of serotonin are elevated in primary pulmonary hypertension even after bilateral lung transplantation, suggesting a possible etiologic role. Serotonin is released primarily from the small intestine. Anorectic agents, such as dexfenfluramine, which can cause pulmonary hypertension, are known to inhibit potassium channels in vascular smooth muscle cells. We examined the hypothesis that dexfenfluramine may stimulate release of serotonin from the ileum by inhibition of K+ channels. In an isolated loop of rat ileum perfused with a physiological salt solution, the administration of dexfenfluramine, its major metabolite d-norfenfluramine, the potassium channel blocker 4-aminopyridine (5 mM), and caffeine (30 mM) increased serotonin levels in the venous effluent. Potassium chloride (60 mM) tended to increase serotonin levels. In genetically susceptible individuals, dexfenfluramine may induce pulmonary hypertension by increasing cytosolic calcium in enterochromaffin cells of the small intestine, thus releasing serotonin and causing vasoconstriction. This work indicates that dexfenfluramine and its major metabolite d-norfenfluramine can increase serotonin release from the small intestine.

The main ion channels involved in the propagation of action potentials in cardiac cells are the voltage-gated sodium channels, the inward rectifier potassium channels, and the inter-cellular gap junction channels. Sodium channels have been shown to be localized to the intercalated disks along with Cx43 channels. Recent evidence of reciprocity in the co-localized expression of Kir2.1 and Nav1.5 channels in cardiac myocytes suggest that ionic currents due to these two channels are in some way calibrated to each other. In isolated cells, the fast sodium current is much larger than IK1 resulting in a large depolarization reserve. Using simulations of chains of cardiac cells, we show that the depolarization reserve for conducting action potentials is significantly smaller than in isolated cells. We also studied the changes in the depolarization reserve with variations in gap junction channel density and explored the conditions under which propagation slowed or failed. These insights will allow a better understanding of the effects of Na channel blockers as well as regional differences in action potential conduction in the heart.

HERG (human ether-a-go-go-related gene) encodes a delayed rectifier K + channel vital to normal repolarization of cardiac action potentials. Attenuation of repolarizing K + current caused by mutations in HERG or channel block by common medications prolongs ventricular action potentials and increases the risk of arrhythmia and sudden death. The critical role of HERG in maintenance of normal cardiac electrical activity derives from its unusual gating properties. Opposite to other voltage-gated K + channels, the rate of HERG channel inactivation is faster than activation and appears to be intrinsically voltage dependent. To investigate voltage sensor movement associated with slow activation and fast inactivation, we characterized HERG gating currents. When the cut-open oocyte voltage clamp technique was used, membrane depolarization elicited gating current with fast and slow components that differed 100-fold in their kinetics. Unlike previously studied voltage-gated K + channels, the bulk of charge movement in HERG was protracted, consistent with the slow rate of ionic current activation. Despite similar kinetic features, fast inactivation was not derived from the fast gating component. Analysis of an inactivation-deficient mutant HERG channel and a Markov kinetic model suggest that HERG inactivation is coupled to activation.

Background Low potassium dextran lung preservation solution has reduced primary graft failure in animal and human studies. Though the mechanism of reducing primary graft failure is unknown, low potassium dextran differs most significantly from solutions such as Euro-Collins (EC) and University of Wisconsin in its potassium concentration. The aim of this study was to investigate the impact that potassium concentration in lung preservation solutions had on pulmonary arterial smooth muscle cell depolarization and production of reactive oxygen species. Methods Using isolated pulmonary artery smooth muscle cells from Sprague-Dawley rats, the patch-clamp technique was used to measure resting cellular membrane potential and whole cell potassium current. Measurements were recorded at base line and after exposure to low potassium dextran, EC, and University of Wisconsin solutions. Pulmonary arteries from rats were isolated from the main pulmonary artery to the fourth segmental branch. Arteries were placed into vials containing low potassium dextran, EC, low potassium EC, Celsior, and University of Wisconsin solutions with reactive oxygen species measured by lucigenin-enhanced chemiluminescence. Results Pulmonary artery smooth muscle cell membrane potentials had a significant depolarization when placed in the University of Wisconsin or EC solutions, with changes probably related to inhibition of voltage-gated potassium channels. Low potassium dextran solution did not alter the membrane potential. Production of reactive oxygen species as measured by chemiluminescence was significantly higher when pulmonary arteries were exposed to University of Wisconsin or EC solutions (51,289 ± 5,615 and 35,702 ± 4353 counts/0.1 minute, respectively) compared with low potassium dextran, Celsior, and low potassium EC (12,537 ± 3623, 13,717 ± 3,844 and 15,187 ± 3,792 counts/0.1 minute, respectively). Conclusions Preservation solutions with high potassium concentration are clearly able to depolarize the pulmonary artery smooth muscle cells and increase pulmonary artery reactive oxygen species production. Low potassium preservations solutions may limit reactive oxygen species production and thus reduce the incidence of primary graft failure in lung transplantation.

The opening of voltage-gated sodium, potassium, and calcium ion channels has a steep relationship with voltage. In response to changes in the transmembrane voltage, structural movements of an ion channel that precede channel opening generate a capacitative gating current. The net gating charge displacement due to membrane depolarization is an index of the voltage sensitivity of the ion channel activation process. Understanding the molecular basis of voltage-dependent gating of ion channels requires the measurement and computation of the gating charge, Q. We derive a simple and accurate semianalytic approach to computing the voltage dependence of transient gating charge movement (Q-V relationship) of discrete Markov state models of ion channels using matrix methods. This approach allows rapid computation of Q-V curves for finite and infinite length step depolarizations and is consistent with experimentally measured transient gating charge. This computational approach was applied to Shaker potassium channel gating, including the impact of inactivating particles on potassium channel gating currents.

While it is well established that class-I antiarrhythmics block cardiac sodium channels, the mechanism of action of therapeutic levels of these drugs is not well understood. Using a combination of mathematical modeling and in vitro experiments, we studied the failure of activation of action potentials in single ventricular cells and in tissue caused by Na+ channel block. Our computations of block and unblock of sodium channels by a theoretical class-Ib antiarrhythmic agent predict differences in the concentrations required to cause activation failure in single cells as opposed to multicellular preparations. We tested and confirmed these in silico predictions with in vitro experiments on isolated guinea-pig ventricular cells and papillary muscles stimulated at various rates (2–6.67 Hz) and exposed to various concentrations (5 × 10−6 to 500 × 10−6 mol/l) of lidocaine. The most salient result was that whereas large doses (5 × 10−4 mol/l or higher) of lidocaine were required to inhibit action potentials temporarily in single cells, much lower doses (5 × 10−6 mol/l), i.e., therapeutic levels, were sufficient to have the same effect in papillary muscles: a hundredfold difference. Our experimental results and mathematical analysis indicate that the syncytial nature of cardiac tissue explains the effects of clinically relevant doses of Na+ channel blockers.

1. The unusual kinetics of human ether-à-go-go-related gene (HERG) K(+) channels are consistent with a role in the suppression of arrhythmias initiated by premature beats. Action potential clamp protocols were used to investigate the effect of premature stimulation on HERG K(+) channels, transfected in Chinese hamster ovary cells, at 37 degrees C. 2. HERG K(+) channel currents peaked during the terminal repolarization phase of normally paced action potential waveforms. However, the magnitude of the current and the time point at which conductance was maximal depended on the type of action potential waveform used (epicardial, endocardial, Purkinje fibre or atrial). 3. HERG K(+) channel currents recorded during premature action potentials consisted of an early transient outward current followed by a sustained outward current. The magnitude of the transient current component showed a biphasic dependence on the coupling interval between the normally paced and premature action potentials and was maximal at a coupling interval equivalent to 90 % repolarization (APD(90)) for ventricular action potentials. The largest transient current response occurred at shorter coupling intervals for Purkinje fibre (APD(90) - 20 ms) and atrial (APD(90) - 30 ms) action potentials. 4. The magnitude of the sustained current response following premature stimulation was similar to that recorded during the first action potential for ventricular action potential waveforms. However, for Purkinje and atrial action potentials the sustained current response was significantly larger during the premature action potential than during the normally paced action potential. 5. A Markov model that included three closed states, one open and one inactivated state with transitions permitted between the pre-open closed state and the inactivated state, successfully reproduced our results for the effects of premature stimuli, both during square pulse and action potential clamp waveforms. 6. These properties of HERG K(+) channels may help to suppress arrhythmias initiated by early afterdepolarizations and premature beats in the ventricles, Purkinje fibres or atria.

See related article, pages 931–938 Pulmonary vascular tone is largely determined by potassium and calcium currents in the smooth muscle cells (SMCs) of the small resistance arteries. Under normoxic circumstances an outward potassium current ( I K) passes through voltage-gated (Kv), calcium-activated (KCa), and background tandem-pore domain (K2P) potassium channels. The latter (coded by the KCNK family of genes) include the twik-related acid sensitive K+ channel TASK and TASK-like channels. This current keeps the resting membrane potential in the range of −50 to −60 mV and inhibits calcium entry through L-type calcium channels.1 Vasodilator substances such as nitric oxide and prostacyclin, released from the endothelium, increase I K, one of several actions that lead to further vasodilatation.2,3 Vasoconstrictor substances, such as endothelin, can inhibit I K4 and/or release calcium from the sarcoplasmic reticulum.5 In addition to vasoactive effects, an increase in cytosolic potassium inhibits smooth muscle cell apoptosis,6 and an increase in cytosolic calcium promotes cellular proliferation.7 Consequently, the inhibition of I K is important not only in causing membrane depolarization and calcium entry but also in stimulating vascular remodeling and in the development of chronic pulmonary hypertension. Agents that can cause pulmonary hypertension, such as the anorectic drugs and hypoxia, inhibit I K8,9 and also enhance calcium release from the sarcoplasmic reticulum, leading to subsequent repletion of calcium through store-operated channels.10,11,12 The smooth muscle cells in the resistance pulmonary arteries of patients with pulmonary arterial hypertension (PAH) exhibit both reduced I K and decreased expression of Kv channels,13 and also increased expression of the TRPC ( t ransient r eceptor p otential, c anonical) genes that code for store-operated and receptor-operated calcium channels.14 The decreased expression and function of the Kv channels may …

Primary pulmonary hypertension (PPH) is a life-threatening disease that mostly affects young adults, more commonly women than men. It is characterized by vasoconstriction of the small pulmonary arteries, proliferation in all layers of the vessel wall, thrombosis-in situ, and inflammation. Serotonin (5-hydroxytryptamine, 5-HT) is thought to play a role in the pathogenesis of PPH, in part, because it is a pulmonary vasoconstrictor1 and smooth muscle mitogen.2,3 It is synthesized from l-tryptophan, primarily in the enterochromaffin cells of the small intestine. In the blood, 5-HT is mainly transported in the platelets, some are bound to plasma proteins, and only nanomolar concentrations are free in the plasma. The importance of the lungs in the removal of 5-HT from the blood has been known for more than 50 years.4 As much as 95% may be taken up or inactivated.5 5-HT is taken up by the 5-HT transporter (5-HTT) in several cells including platelets, neurons, and pulmonary artery smooth muscle and endothelial cells. 5-HTT belongs to a large 12 transmembrane–domain gene family including the norepinephine transporter, glucose transporters, and transporters of other neurotransmitters. The amino acid sequence of cloned human 5-HTT reveals 6 putative phosphorylation sites, targets for PKA and PKC, which may activate 5-HTT. The Figure shows some of the cellular signaling pathways involving 5-HTT and 5-HT receptors. Schematic representation of serotonin (5-HT) signaling pathways in pulmonary artery smooth muscle cells (PASMCs). Activation of serotonin receptors 5-HT1B, 5-HT2A, and 5-HT2B cause vasoconstriction, although 5-HT2B is also implicated in the mitogenic action of 5-HT. 5-HT1B activates the Gi G-protein, which suppresses the activity of adenylate cyclase (AC). AC normally increases the level of cytosolic cAMP, which activates the protein …

Effects of cell-to-cell coupling conductance on dynamics of sinus node cells are examined. Cell models are biophysically detailed, and are based on the kinetic equations developed by Noble et al. [Neuronal and Cellular Oscillators, edited by J. W. Jacklet, Marcel Deckker, New York (1989).] Resistively coupled cell pairs show five regimes of behavior as a function of coupling conductance: (1) independent oscillation for Gc < 1 pS; (2) primarily quasiperiodic oscillation for 1 ⩽ Gc < 116 pS; (3) windows of periodic behavior which undergo period doubling bifurcation to chaos for 116 ⩽ Gc < 212 pS; (4) frequency entrainment for Gc ⩾ 212 pS; (5) waveform entrainment for Gc ⩾ 50 nS. Thus, only 4–5 gap junction channels are required for frequency entrainment. This is shown to also be the case for large networks of sinus cells modeled on the Connection Machine CM-5. A biophysically detailed two-dimensional network model of the cardiac atrium has also been implemented on the CM-5 supercomputer. The model is used to study effects of spatially localized inhibition of the Na-K pump. Na overloading produced by pump inhibition can induce spontaneous, propagating ectopic beats within the network. At a cell-to-cell coupling value yielding a realistic plane wave conduction velocity of 60cms−1 pump inhibition in small regions of the network containing as few as 1000 cells can induce propagating ectopic beats.

In 1910, James Herrick made the first report of a case of sickle cell anemia.1 He described thin, elongated, sickle-shaped, and crescent-shaped red blood corpuscles. Soon afterward, the observation of sickle cells in the asymptomatic father of a sickle cell anemia patient raised the possibility of an inherited disorder.2 The distinction between symptomatic sickle cell anemia and the asymptomatic sickle cell trait was established in 1933.3 The difference between normal hemoglobin (HbAA) and sickle hemoglobin (HbSS) was recognized in 1959 to be the substitution of a valine residue for a glutamic acid in the β-chain amino terminus.4 Patients who have the sickle cell trait are heterozygotes (HbAS), having an abnormal, as well as a normal, β-globin gene. In sickle cell anemia, sickling may start at an oxygen saturation as high as 85%, while in the trait the desaturation has to be more severe before sickling is induced. Sickling leading to vaso-occlusion and infarction occurs in many organs but was first described in the lungs.5 Initially it was thought that deformation and increased rigidity of the erythrocytes, related to polymerization of HbSS, was sufficient to cause mechanical obstruction. However, the work of Hebbel et al6,7 focused attention on erythrocyte adherence to the endothelium as a mechanism promoting microvascular occlusion. A key observation in this regard is that sickle erythrocytes adhere more readily to microvascular endothelium than to endothelium from conduit vessels.8 If endothelial cells circulating in the blood can be taken as representative of the sedentary population, the endothelium is activated in sickle cell patients, whether in steady state or in an acute crisis.9 This is shown by expression of the adhesion molecules, ICAM-1, VCAM-1, E-selectin, and P-selectin. More recent work has implicated inflammation as a factor in the pathogenesis of vaso-occlusive crises. …

Summary Models of the electrical activity in heart cells have tended to focus on the transmembrane potential and the non linear nature of ionic currents in the cell membrane. This can be attributed to the success of the Hodgkin-Huxley paradigm used in modeling cellular electrical activity. However, it is well known that the small size of heart cells makes small concentration changes have important effects on the overall functioning of the cells. In particular, it has been shown in experiment and theory that inhibition of the sodium-potassium pump can cause a significant build-up of internal sodium which in turn causes subcellular oscillations of calcium concentration. In this talk, I will look at the effects of these subcellular oscillations on the transmembrane potential in the DiFrancesco-Noble model of electrical activity in mammalian cardiac Purkinje fiber. It will be shown that the subcellular oscillations interferes with the normal pacemaking mechanism of the Purkinje fiber. Numerical continuation methods will be used to construct bifurcation diagrams which predict a dramatic change in the structure of periodic orbits of the cell electrical activity with pump inhibition.

A minimal model of the dynamics of internal calcium concentration of the mammalian cardiac Purkinje fiber is examined in order to identify the cause of certain arrhythmias of the heart. The effect of inhibition of the sodium/potassium pump is modeled by an elevated value of internal sodium cocentration. Effects of pump inhibition are examined at different clamp voltages. Such conditions mimic those which have been examined experimentally and which are known to cause oscillatory calcium release [W.J. Lederer, PhD thesis, Yale University, New Haven, CT (1976), 168 pp.; R.S. Kass, W.J. Lederer, R.W. Tsien and R. Weingart, J. Physiol. (London) 281 (1978) 187–208]. System dynamics are investigated using numerical continuation methods. Results of these analyses predict the existence of stable periodic oscillations of internal calcium over a range of voltage-clamp values. The emergence of these oscillations depends on the intracellular sodium concentration.

Computational methods for simulating biophysically detailed, large-scale models of mammalian cardiac sinus and atrial networks on the massively parallel Connection Machine CM-2, and techniques for visualization of simulation data, are presented. Individual cells are modeled using the formulations of Noble et al. Models incorporate properties of voltage-dependent membrane currents, ion pumps and exchangers, and internal calcium sequestering and release mechanisms. Network models are used to investigate factors determining the site of generation and direction of propagation of the pacemaker potential. Models of the isolated sinus node are used to show that very few gap junction channels are required to support frequency entrainment. When cell membrane properties in the isolated sinus node models are modified to reproduce regional differences in oscillation properties, as described by the data of Kodama and Boyett, an excitatory wave is generated in the node periphery which propagates towards the node center. This agrees with activation patterns measured in the isolated sinus node by Kirchoff. When the model sinus node is surrounded by a region of atrial cells, the site of pacemaker potential generation is shifted away from the periphery towards the node center. This is in agreement with activation patterns measured by Kirchoff in the intact sinus node of the rabbit heart, and demonstrates the importance of sinus node boundary conditions on shaping the site of generation and direction of propagation of the pacemaker potential.

The hyperpolarization-activated cyclic nucleotide-gated (HCN) channels modulate and regulate cardiac rhythm and rate. It has been suggested that, unlike the HCN1 and HCN2 channels, the slower HCN4 channel may not exhibit voltage-dependent hysteresis. We studied the electrophysiological properties of human HCN4 (hHCN4) channels and its modulation by cAMP to determine whether hHCN4 exhibits hysteresis, by using single-cell patch-clamp in HEK293 cells stably transfected with hHCN4. Quantitative real-time RT-PCR was also used to determine levels of expression of HCNs in human cardiac tissue. Voltage-clamp analysis revealed that hHCN4 current (I(h)) activation shifted in the depolarizing direction with more hyperpolarized holding potentials. Triangular ramp and action potential clamp protocols also revealed hHCN4 hysteresis. cAMP enhanced I(h) and shifted activation in the depolarizing direction, thus modifying the intrinsic hHCN4 hysteresis behavior. Quantitative PCR analysis of human sinoatrial node (SAN) tissue showed that HCN4 accounts for 75% of the HCNs in human SAN while HCN1 (21%), HCN2 (3%), and HCN3 (0.7%) constitute the remainder. Our data suggest that HCN4 is the predominant HCN subtype in the human SAN and that I(h) exhibits voltage-dependent hysteresis behavior that can be modified by cAMP. Therefore, hHCN4 hysteresis potentially plays a crucial role in human SAN pacemaking activity.

Distinct Phenotypes in hERG Pore Domain Mutations. Introduction: Mutations in the pore domain of the human ether-a-go-go-related gene (hERG) potassium channel are associated with higher risk of sudden death. However, in many kindreds clinical presentation is variable, making it hard to predict risk. We hypothesized that in vitro phenotyping of the intrinsic severity of individual mutations can assist with risk stratification. Methods and Results: We analyzed 2 hERG pore domain mutations, G572S and G584S. Similar to 90% of hERG missense mutations, G572S-hERG subunits did not traffic to the plasma membrane but could coassemble with WT subunits and resulted in a dominant negative suppression of hERG current density. The G584S-hERG subunits traffic normally but have abnormal inactivation gating. Computer models of human ventricular myocyte action potentials (AP), incorporating Markov models of the hERG mutants, indicate that G572S-hERG channels would cause more severe AP prolongation than that seen with G584ShERG channels. Conclusions: hERG-G572S and -G584S are 2 pore domain mutations that involve the same change in sidechain but have very different in vitro phenotypes; G572S causes a dominant negative trafficking defect, whereas G584S is the first hERG missense mutation where the cause of disease can be exclusively attributed to enhanced inactivation. The G572S mutation is intrinsically more severe than the G584S mutation, consistent with the overall clinical presentation in the 2 small kindreds studied here. Further investigation, involving a larger number of cohorts, to test the hypothesis that in vitro phenotyping of the intrinsic severity of a given mutation will assist with risk stratification is therefore warranted.

Background— At birth, the increase in oxygen causes contraction of the ductus arteriosus, thus diverting blood flow to the lungs. Although this contraction is modulated by substances such as endothelin and dilator prostaglandins, normoxic contraction is an intrinsic property of ductus smooth muscle. Normoxic inhibition of potassium channels causes membrane depolarization and calcium entry through L-type calcium channels. However, the studies reported here show that after inhibition of this pathway there is still substantial normoxic contraction, indicating the involvement of additional mechanisms. Methods and Results— Using ductus ring experiments, calcium imaging, reverse-transcription polymerase chain reaction, Western blot, and cellular electrophysiology, we find that this depolarization-independent contraction is caused by release of calcium from the IP 3 -sensitive store in the sarcoplasmic reticulum, by subsequent calcium entry through store-operated channels, and by increased calcium sensitization of actin-myosin filaments, involving Rho-kinase. Conclusions— Much of the normoxic contraction of the ductus arteriosus at birth is related to calcium entry through store-operated channels, encoded by the transient receptor potential superfamily of genes, and to increased calcium sensitization. A clearer understanding of the mechanisms involved in normoxic contraction of the ductus will permit the development of better therapy to close the patent ductus arteriosus, which constitutes ≈10% of all congenital heart disease and is especially common in premature infants.

This study sought to evaluate mutations in genes encoding the slow component of the cardiac delayed rectifier K+ current (I(Ks)) channel in familial atrial fibrillation (AF).Although AF can have a genetic etiology, links between inherited gene defects and acquired factors such as atrial stretch have not been explored.Mutation screening of the KCNQ1, KCNE1, KCNE2, and KCNE3 genes was performed in 50 families with AF. The effects of mutant protein on cardiac I(Ks) activation were evaluated using electrophysiological studies and human atrial action potential modeling.One missense KCNQ1 mutation, R14C, was identified in 1 family with a high prevalence of hypertension. Atrial fibrillation was present only in older individuals who had developed atrial dilation and who were genotype positive. Patch-clamp studies of wild-type or R14C KCNQ1 expressed with KCNE1 in CHO cells showed no statistically significant differences between wild-type and mutant channel kinetics at baseline, or after activation of adenylate cyclase with forskolin. After exposure to hypotonic solution to elicit cell swelling/stretch, mutant channels showed a marked increase in current, a leftward shift in the voltage dependence of activation, altered channel kinetics, and shortening of the modeled atrial action potential duration.These data suggest that the R14C KCNQ1 mutation alone is insufficient to cause AF. Rather, we suggest a model in which a "second hit", such as an environmental factor like hypertension, which promotes atrial stretch and thereby unmasks an inherited defect in ion channel kinetics (the "first hit"), is required for AF to be manifested. Such a model would also account for the age-related increase in AF development.

Changes in oxygen tension are sensed by a variety of tissues, such as the carotid body, ductus arteriosus (DA), and pulmonary vasculature. In the fetus, hypoxia keeps the DA open and the pulmonary vessels constricted. After birth, hypoxic pulmonary vasoconstriction (HPV) helps to match ventilation and perfusion. HPV is induced by three general mechanisms—influx of calcium into the smooth muscle cells through L-type channels and store-operated channels, release of calcium from the sarcoplasmic reticulum, and sensitization of actin/myosin to a given level of calcium. We show that reducing agents mimic the effects of hypoxia by reducing potassium current, causing membrane depolarization and increasing calcium influx in pulmonary artery smooth muscle cells (PASMCs), while doing exactly the opposite in the DA. On the other hand, oxidizing agents mimic normoxia by increasing potassium current, causing membrane hyperpolarization and reducing cytosolic calcium in PASMCs. They again do the opposite in the DA. As these redox agents elicit the same responses as shifts in oxygen tension, we consider that changes in oxygen may be signaled by changes in redox status.

1. We studied the gating kinetics of Kv4.2 channels, the molecular substrate of neuronal somatodendritic A-type currents. For this purpose wild-type and mutant channels were transiently expressed in the human embryonic kidney (HEK) 293 cell line and currents were measured in the whole-cell patch-clamp configuration. 2. Kv4.2 channels inactivated from pre-open closed state(s) with a mean time constant of 959 ms at -50 mV. This closed-state inactivation was not affected by a deletion of the Kv4.2 N-terminus (Delta2-40). 3. Kv4.2 currents at +40 mV inactivated with triple-exponential kinetics. A fast component (tau = 11 ms) accounted for 73 %, an intermediate component (tau = 50 ms) for 23 % and a slow component (tau = 668 ms) for 4 % of the total decay. 4. Both the fast and the intermediate components of inactivation were slowed by a deletion of the Kv4.2 N-terminus (tau = 35 and 111 ms) and accounted for 33 and 56 %, respectively, of the total decay. The slow component was moderately accelerated by the truncation (tau = 346 ms) and accounted for 11 % of the total Kv4.2 current inactivation. 5. Recovery from open-state inactivation and recovery from closed-state inactivation occurred with similar kinetics in a strongly voltage-dependent manner. Neither recovery reaction was affected by the N-terminal truncation. 6. Kv4.2 Delta2-40 channels displayed slowed deactivation kinetics, suggesting that the N-terminal truncation leads to a stabilization of the open state. 7. Simulations with an allosteric model of inactivation, supported by the experimental data, suggested that, in response to membrane depolarization, Kv4.2 channels accumulate in the closed-inactivated state(s), from which they directly recover, bypassing the open state.

HERG is believed to encode the major sub-unit of the cardiac 'rapid' delayed rectifier K channel (I(Kr)). Both I(Kr) and HERG exhibit marked inward rectification at positive membrane potentials due to rapid inactivation and this is thought to influence significantly the contribution of the current to cardiac action potential (AP) repolarisation. We investigated directly the role played by rapid inactivation, by measuring current activated by a ventricular AP waveform, from Chinese Hamster Ovary cells transfected with HERG cDNA with a point-mutation (S631A) in the pore region. Square command pulses elicited HERG-S631A current which increased progressively in magnitude with test potential up to +30/+40 mV (n=6). During test pulses to +40mV, HERG-S631A exhibited little inactivation compared to wildtype HERG. During an action potential command, WT-HERG current developed progressively during the AP plateau and slow repolarisation phase, showing maximal current between -30mV and -40 mV (n=10). In contrast, HERG-S631A current increased earlier during the AP plateau, with a maximal amplitude near +30mV (n=7). Current then declined as the AP proceeded, giving rise to a 'bow'- or 'inverted-U-' shaped current profile. A mathematical model with inactivation removed from the HERG current reproduced the I-V profile of HERG-S631A. These data provide a direct demonstration that rapid inactivation normally plays a critical role in determining both time-course and voltage dependence of HERG/I(Kr) -current during the cardiac ventricular AP.

In this paper we show the presence of a latent conservation principle in the formulation of ionic currents in cardiac cells and examine its effect on a formulation of the sodium–calcium exchange current appearing in the Noble model of the sinoatrial node cell in the mammalian heart [see Noble et al . (1989) or Winslow et al . (1991)]. Our objective is to show that this formulation, if not corrected, will result in a serious instability in this cardiac cell model. In particular, under certain initial conditions, the solutions of the model equations will blow-up in finite time. We also propose a correction to the model equation of the sodium–calcium exchange current, and we show that the modified model agrees favorably with the original model. These phenomena also occur in the other cardiac cell models, such as those modeling the Purkinje fiber, the atrial cell and the ventricular cell. The changes proposed in this paper can be applied directly to these models as well.

A model of the electrical activity in mammalian cardiac Purkinje fiber is examined to study the effects of subcellular oscillations on the transmembrane potential. Inhibition of the sodium/potassium pump causes oscillations in the internal calcium concentration and these oscillations are shown to interfere with the normal pacemaking mechanism of the Purkinje fiber. Bifurcation diagrams were constructed using numerical continuation methods. These results predict a dramatic change in the structure of periodic orbits of the cell electrical activity with pump inhibition and elevated levels of internal sodium concentration.

A biophysically detailed two-dimensional network model of the cardiac atrium has been implemented on the Thinking Machines massively parallel CM-5 supercomputer. The model is used to study the effects of spatially localized inhibition of the Na--K pump. Na overloading produced by pump inhibition can induce spontaneous, propagating ectopic beats within the network. At a cell-to-cell coupling value yielding a realistic plane wave conduction velocity of 0.6 m s$^{-1}$, pump inhibition in roughly 1000 cells can induce propagating ectopic beats in a 512 $\times $ 512 lattice of cells.

The human ether a-go-go related gene (hERG) encodes the pore forming subunit of the cardiac delayed rectifier potassium channel. Mutations in hERG that disrupt gating can cause Long QT syndrome type 2, a disorder of cardiac repolarisation characterised by prolongation of the QT interval and risk of sudden cardiac death due to ventricular arrhythmias. There is therefore considerable interest in developing accurate mathematical descriptions of hERG gating in order to simulate the effect of hERG mutants on cardiac action potentials at both the cellular and tissue levels.In the first part of this study we have used a 5 state Markov model (1) minimised to in vitro electrophysiological data to describe hERG ionic currents associated with two clinically relevant mutants: G584S and G572S. These Markov descriptions were incorporated into models of the human ventricular action potential (2) and used to simulate the effects of these mutants on action potential waveforms and responses to premature stimuli. The results of our simulations for each mutant correlated well with the reported clinical phenotypes and were able to show how compensatory currents can contribute to phenotypic variability between families carrying the same hERG mutation.In the second part of this study we compare the abilities of several Markov schemes to reproduce hERG currents. In doing so we evaluate their suitability for our goal of incorporating mathematical descriptions of hERG into whole heart simulations as a way of assessing how altered hERG function predisposes to the development of arrhythmias triggered by premature beats.Our results demonstrate how in silico techniques lend an additional level of insight into understanding and characterising clinical mutations.1) Lu et al., J. Physiol. (2001), 537, 843-851.2) ten Tusscher KH, Panfilov AV., Am. J. Physiol. (2006) 291, H1088-100.

We characterized the epicardial activation sequence during a norepinephrine (NE)-induced ventricular arrhythmia in anesthetized pigs and studied factors that modulated it. Subepicardial NE infusion caused the QRS complex to invert within a single beat ( n = 35 animals, 101 observations), and the earliest epicardial activation consistently shifted to the randomly located infusion site ( n = 14). This preceded right atrial activation, whereas the total ventricular epicardial activation time increased from 20 ± 4 to 50 ± 9 ms ( P < 0.01). These events were accompanied by a ventricular tachycardia and a drop in left ventricular pressure, which were fully reversed after the infusion was stopped. Epicardial pacing at the infusion site mimicked all electrical and hemodynamic changes induced by NE. The arrhythmia was prevented by propranolol and abolished by cardiac sympathetic or vagal nerve stimulation. Focal automaticity was computationally reconstructed using a two-dimensional sheet of 256 × 256 resistively coupled ventricular cells, where calcium handling was abnormally high in the central region. We conclude that adrenergic stimulation to a small region of the ventricle elicits triggered automaticity and that computational reconstruction implicates calcium overload. Interventions that reduce spatial inhomogeneities of intracellular calcium may prevent this type of arrhythmia.