Biomedical subjects

W R Giles

Publications and source records attributed to W R Giles.

At least 19 recordsLinked to original sources

PubMed2007

C-type natriuretic peptide activates a non-selective cation current in acutely isolated rat cardiac fibroblasts via natriuretic peptide C receptor-mediated signalling.

In the heart, fibroblasts play an essential role in the deposition of the extracellular matrix and they also secrete a number of hormonal factors. Although natriuretic peptides, including C-type natriuretic peptide (CNP) and brain natriuretic peptide, have antifibrotic effects on cardiac fibroblasts, the effects of CNP on fibroblast electrophysiology have not been examined. In this study, acutely isolated ventricular fibroblasts from the adult rat were used to measure the effects of CNP (2 x 10(-8) M) under whole-cell voltage-clamp conditions. CNP, as well as the natriuretic peptide C receptor (NPR-C) agonist cANF (2 x 10(-8) M), significantly increased an outwardly rectifying non-selective cation current (NSCC). This current has a reversal potential near 0 mV. Activation of this NSCC by cANF was abolished by pre-treating fibroblasts with pertussis toxin, indicating the involvement of G(i) proteins. The cANF-activated NSCC was inhibited by the compounds Gd(3+), SKF 96365 and 2-aminoethoxydiphenyl borate. Quantitative RT-PCR analysis of mRNA from rat ventricular fibroblasts revealed the expression of several transient receptor potential (TRP) channel transcripts. Additional electrophysiological analysis showed that U73122, a phospholipase C antagonist, inhibited the cANF-activated NSCC. Furthermore, the effects of CNP and cANF were mimicked by the diacylglycerol analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG), independently of protein kinase C activity. These are defining characteristics of specific TRPC channels. More detailed molecular analysis confirmed the expression of full-length TRPC2, TRPC3 and TRPC5 transcripts. These data indicate that CNP, acting via the NPR-C receptor, activates a NSCC that is at least partially carried by TRPC channels in cardiac fibroblasts.

Animals↗

PubMed2006

Mathematical simulations of the effects of altered AMP-kinase activity on I and the action potential in rat ventricle.

INTRODUCTION: Alterations in the activity of a so-called "metabolic switch" enzyme, adenosine monophosphate-activated protein kinase (AMP kinase), in mammalian heart contribute to the conduction abnormalities and rhythm disturbances in the settings of Wolff-Parkinson-White syndrome and ventricular pre-excitation. A recent study by Light et al. has shown that augmented AMP kinase activity can alter the biophysical properties of mammalian cardiac sodium currents. These experiments involved an electrophysiological analysis following heterologous expression of human Na(v)1.5 in tsA201 cells. Constitutive activation of AMP kinase followed by co-transfection caused: (i) a hyperpolarizing shift in the activation curve for I(Na), (ii) a small change in the voltage dependence of steady-state inactivation, and (iii) a significant slowing in the rate of inactivation of I(Na). METHODS AND RESULTS: We have attempted to simulate these results using our mathematical model of the membrane action potential of the adult rat ventricular myocyte. The changes in I(Na) produced by AMP kinase activation and/or overexpression can be reconstructed mathematically by altering two rate constants in a Markovian model that governs the I(Na) kinetics. Simulated macroscopic I(Na) records in which a fraction (10-100%) of the Na(+) channels had the appropriate rate constants for two state-dependent transitions increased by a factor of 100-fold exhibited: (i) slowed inactivation, (ii) a shift in steady-state activation to more hyperpolarized membrane potentials, and (iii) a very small change in the voltage dependence of steady-state inactivation. SUMMARY: Thus, straightforward modifications of a previously published kinetic scheme for the time and voltage dependence of mammalian heart I(Na), when incorporated into a mathematical model for the rat ventricular action potential can reproduce the main features of these AMP kinase-induced modifications in I(Na) in mammalian ventricle. Ongoing mathematical simulations are directed toward developing formulations that mimic the molecular mechanisms for the AMP kinase effects, e.g., changes in the kinetics of I(Na) resulting from selective phosphorylation/dephosphorylation of sites on the alpha or beta subunits which comprise human Na(v)1.5. Thereafter, incorporation of these changes into a mathematical model for the action potential of the human ventricular myocyte is planned.

Action Potentials↗

PubMed2005

Actions of emigrated neutrophils on Na(+) and K(+) currents in rat ventricular myocytes.

Interactions between neutrophils and the ventricular myocardium can contribute to tissue injury, contractile dysfunction and generation of arrhythmias in acute cardiac inflammation. Many of the molecular events responsible for neutrophil adhesion to ventricular myocytes are well defined; in contrast, the resulting electrophysiological effects and changes in excitation-contraction coupling have not been studied in detail. In the present experiments, rat ventricular myocytes were superfused with either circulating or emigrated neutrophils and whole-cell currents and action potential waveforms were recorded using the nystatin-perforated patch method. Almost immediately after adhering to ventricular myocytes, emigrated neutrophils caused a depolarization of the resting membrane potential and a marked prolongation of myocyte action potential. Voltage clamp experiments demonstrated that following neutrophil adhesion, there was (i) a slowing of the inactivation of a TTX-sensitive Na(+) current, and (ii) a decrease in an inwardly rectifying K(+) current. One cytotoxic effect of neutrophils appears to be initiated by enhanced Na(+) entry into the myocytes. Thus, manoeuvres that precluded activation of Na(+) channels, for example holding the membrane potential at -80 mV, significantly increased the time to cell death or prevented contracture entirely. A mathematical model for the action potential of rat ventricular myocytes has been modified and then utilized to integrate these findings. These simulations demonstrate the marked effects of (50-fold) slowing of the inactivation of 2-4% of the available Na(+) channels on action potential duration and the corresponding intracellular Ca(2+) transient. In ongoing studies using this combination of approaches, are providing significant new insights into some of the fundamental processes that modulate myocyte damage in acute inflammation.

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PubMed2005

K+ currents regulate the resting membrane potential, proliferation, and contractile responses in ventricular fibroblasts and myofibroblasts.

Despite the important roles played by ventricular fibroblasts and myofibroblasts in the formation and maintenance of the extracellular matrix, neither the ionic basis for membrane potential nor the effect of modulating membrane potential on function has been analyzed in detail. In this study, whole cell patch-clamp experiments were done using ventricular fibroblasts and myofibroblasts. Time- and voltage-dependent outward K(+) currents were recorded at depolarized potentials, and an inwardly rectifying K(+) (Kir) current was recorded near the resting membrane potential (RMP) and at more hyperpolarized potentials. The apparent reversal potential of Kir currents shifted to more positive potentials as the external K(+) concentration ([K(+)](o)) was raised, and this Kir current was blocked by 100-300 muM Ba(2+). RT-PCR measurements showed that mRNA for Kir2.1 was expressed. Accordingly, we conclude that Kir current is a primary determinant of RMP in both fibroblasts and myofibroblasts. Changes in [K(+)](o) influenced fibroblast membrane potential as well as proliferation and contractile functions. Recordings made with a voltage-sensitive dye, DiBAC(3)(4), showed that 1.5 mM [K(+)](o) resulted in a hyperpolarization, whereas 20 mM [K(+)](o) produced a depolarization. Low [K(+)](o) (1.5 mM) enhanced myofibroblast number relative to control (5.4 mM [K(+)](o)). In contrast, 20 mM [K(+)](o) resulted in a significant reduction in myofibroblast number. In separate assays, 20 mM [K(+)](o) significantly enhanced contraction of collagen I gels seeded with myofibroblasts compared with control mechanical activity in 5.4 mM [K(+)](o). In combination, these results show that ventricular fibroblasts and myofibroblasts express a variety of K(+) channel alpha-subunits and demonstrate that Kir current can modulate RMP and alter essential physiological functions.

Animals↗

PubMed2005

Skeletal and cardiac muscle defects in a murine model of Emery-Dreifuss muscular dystrophy.

Previous histological findings, physiological data, and behavioral observations on the A-type lamin knockout mouse (Lmna(-/-)) suggest that important aspects of this model resemble the human Emery-Dreifuss muscular dystrophy (EDMD) phenotype. The main goal of our experiments was to study skeletal and cardiac muscle function in this murine model to obtain the semiquantitative data needed for more detailed comparisons with human EDMD defects. Measurements of the mechanical properties of preparations from two different skeletal muscle groups, the soleus and the diaphragm, were made in vitro. In addition, records of the electrocardiogram, and measurements of heart rate variability were obtained; and phasic contractions (unloaded shortening) of enzymatically isolated ventricular myocytes were monitored. Soleus muscles from Lmna(-/-) mice produced less force and work than control preparations. In contrast, force and work production in strips of diaphragm were not changed significantly. Lead II electrocardiograms from conscious, restrained Lmna(-/-) mice revealed slightly decreased heart rates, with significant prolongations of PQ, QRS, and 'QT' intervals compared with those from control recordings. These ECG changes resemble some aspects of the ECG records from humans with EDMD; however, the cardiac phenotype in this Lmna(-/-) mouse model appears to be less well-defined/developed. Ventricular myocytes isolated from Lmna(-/-) mice exhibited impaired contractile responses, particularly when superfused with the beta-adrenergic agonist, isoproterenol (1 microM). This deficit was more pronounced in myocytes isolated from the left ventricle(s) than in myocytes from the right ventricle(s). In summary, tissues from the Lmna(-/-) mouse exhibit a number of skeletal and cardiac muscle deficiencies, some of which are similar to those which have been reported in studies of human EDMD.

Animals↗

PubMed2003

Inhibition of L-type Ca2+ current by C-type natriuretic peptide in bullfrog atrial myocytes: an NPR-C-mediated effect.

Single atrial myocytes were isolated from the bullfrog heart and studied under current and voltage clamp conditions to determine the electrophysiological effects of the C-type natriuretic peptide (CNP). CNP (10(-8) M) significantly shortened the action potential and reduced its peak amplitude after the application of isoproteronol (10(-7) M). In voltage clamp studies, CNP inhibited isoproteronol-stimulated L-type Ca2+ current (ICa) without any significant effect on the inward rectifier K+ current. The effects of cANF (10(-8) M), a selective agonist of the natriuretic peptide C receptor (NPR-C), were very similar to those of CNP. Moreover, HS-142-1, an antagonist of the guanylyl cyclase-linked NPR-A and NPR-B receptors did not alter the inhibitory effect of CNP on ICa. Inclusion of cAMP in the recording pipette to stimulate ICa at a point downstream from adenylyl cyclase increased ICa, but this effect was not inhibited by cANF. These results provide the first demonstration that CNP can inhibit ICa after binding to NPR-C, and suggest that this inhibition involves a decrease in adenylyl cyclase activity, which leads to reduced intracellular levels of cAMP.

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PubMed2002

Voltage-sensitive dye mapping in Langendorff-perfused rat hearts.

An imaging system suitable for recordings from Langendorff-perfused rat hearts using the voltage-sensitive dye 4-[beta-[2-(di-n-butylamino)-6-naphthyl]vinyl]pyridinium (di-4-ANEPPS) has been developed. Conduction velocity was measured under hyper- and hypokalemic conditions, as well as at physiological and reduced temperature. Elevation of extracellular [K(+)] to 9 mM from 5.9 mM caused a slowing of conduction velocity from 0.66 +/- 0.08 to 0.43 +/- 0.07 mm/ms (35%), and reduction of the temperature to 32 degrees C from 37 degrees C caused a slowing from 0.64 +/- 0.07 to 0.46 +/- 0.05 mm/ms (28%). Ventricular activation patterns in sinus rhythm showed areas of early activation (breakthrough) in both the right and left ventricle, with breakthrough at a site near the apex of the right ventricle usually occurring first. The effects of mechanically immobilizing the preparation to reduce motion artifact were also characterized. Activation patterns in epicardially paced rhythm were insensitive to this procedure over the range of applied force tested. In sinus rhythm, however, a relatively large immobilizing force caused prolonged PQ intervals as well as altered ventricular activation patterns. The time-dependent effects of the dye on the rat heart were characterized and include 1) a transient vasodilation at the onset of dye perfusion and 2) a long-lasting prolongation of the PQ interval of the electrocardiogram, frequently resulting in brief episodes of atrioventricular block.

Animals↗

PubMed2001

T-tubule localization of the inward-rectifier K(+) channel in mouse ventricular myocytes: a role in K(+) accumulation.

1. The properties of the slow inward 'tail currents' (I(tail)) that followed depolarizing steps in voltage-clamped, isolated mouse ventricular myocytes were examined. Depolarizing steps that produced large outward K(+) currents in these myocytes were followed by a slowly decaying inward I(tail) on repolarization to the holding potential. These currents were produced only by depolarizations: inwardly rectifying K(+) currents, I(K1), produced by steps to potentials negative to the holding potential, were not followed by I(tail). 2. For depolarizations of equal duration, the magnitude of I(tail) increased as the magnitude of outward current at the end of the depolarizing step increased. The apparent reversal potential of I(tail) was dependent upon the duration of the depolarizing step, and the reversal potential shifted to more depolarized potentials as the duration of the depolarization was increased. 3. Removal of external Na(+) and Ca(2+) had no significant effect on the magnitude or time course of I(tail). BaCl(2) (0.25 mM), which had no effect on the magnitude of outward currents, abolished I(tail) and I(K1) simultaneously. 4. Accordingly, I(tail) in mouse ventricular myocytes probably results from K(+) accumulation in a restricted extracellular space such as the transverse tubule system (t-tubules). The efflux of K(+) into the t-tubules during outward currents produced by depolarization shifts the K(+) Nernst potential (E(K)) from its 'resting' value (close to -80 mV) to more depolarized potentials. This suggests that I(tail) is produced by I(K1) in the t-tubules and is inward because of the transiently elevated K(+) concentration and depolarized value of E(K) in the t-tubules. 5. Additional evidence for the localization of I(K1) channels in the t-tubules was provided by confocal microscopy using a specific antibody against Kir2.1 in mouse ventricular myocytes.

Animals↗

PubMed2001

A defect in the Kv channel-interacting protein 2 (KChIP2) gene leads to a complete loss of I(to) and confers susceptibility to ventricular tachycardia.

KChIP2, a gene encoding three auxiliary subunits of Kv4.2 and Kv4.3, is preferentially expressed in the adult heart, and its expression is downregulated in cardiac hypertrophy. Mice deficient for KChIP2 exhibit normal cardiac structure and function but display a prolonged elevation in the ST segment on the electrocardiogram. The KChIP2(-/-) mice are highly susceptible to the induction of cardiac arrhythmias. Single-cell analysis revealed a substrate for arrhythmogenesis, including a complete absence of transient outward potassium current, I(to), and a marked increase in action potential duration. These studies demonstrate that a defect in KChIP2 is sufficient to confer a marked genetic susceptibility to arrhythmias, establishing a novel genetic pathway for ventricular tachycardia via a loss of the transmural gradient of I(to).

Action Potentials↗

PubMed2001

alpha(4)-integrin mediates neutrophil-induced free radical injury to cardiac myocytes.

Previous work has demonstrated that circulating neutrophils (polymorphonuclear leukocytes [PMNs]) adhere to cardiac myocytes via beta(2)-integrins and cause cellular injury via the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase enzyme system. Since PMNs induced to leave the vasculature (emigrated PMNs) express the alpha(4)-integrin, we asked whether (a) these PMNs also induce myocyte injury via NADPH oxidase; (b) beta(2)-integrins (CD18) still signal oxidant production, or if this process is now coupled to the alpha(4)-integrin; and (c) dysfunction is superoxide dependent within the myocyte or at the myocyte-PMN interface. Emigrated PMNs exposed to cardiac myocytes quickly induced significant changes in myocyte function. Myocyte shortening was decreased by 30-50% and rates of contraction and relaxation were reduced by 30% within the first 10 min. Both alpha(4)-integrin antibody (Ab)-treated PMNs and NADPH oxidase-deficient PMNs were unable to reduce myocyte shortening. An increased level of oxidative stress was detected in myocytes within 5 min of PMN adhesion. Addition of an anti-alpha(4)-integrin Ab, but not an anti-CD18 Ab, prevented oxidant production, suggesting that in emigrated PMNs the NADPH oxidase system is uncoupled from CD18 and can be activated via the alpha(4)-integrin. Addition of exogenous superoxide dismutase (SOD) inhibited all parameters of dysfunction measured, whereas overexpression of intracellular SOD within the myocytes did not inhibit the oxidative stress or the myocyte dysfunction caused by the emigrated PMNs. These findings demonstrate that profound molecular changes occur within PMNs as they emigrate, such that CD18 and associated intracellular signaling pathways leading to oxidant production are uncoupled and newly expressed alpha(4)-integrin functions as the ligand that signals oxidant production. The results also provide pathological relevance as the emigrated PMNs have the capacity to injure cardiac myocytes through the alpha(4)-integrin-coupled NADPH oxidase pathway that can be inhibited by extracellular, but not intracellular SOD.

Animals↗

PubMed2001

Regional expression of the splice variants of Kv4.3 in rat brain and effects of C-terminus deletion on expressed K+ currents.

In situ hybridization and RT-PCR analyses have revealed that, among three Kv4.3 splice variants (a, b, and c) with distinct C-terminal cytoplasmic domains, the mRNA for Kv4.3a is abundant in cerebral cortex, cerebellum, olfactory bulb, and medulla oblongata, whereas the mRNA for Kv4.3c is localized mainly to hippocampus. Three new distinct splice variants of Kv4.3 (Kv4.3d, e and f), which consist of 601, 635, and 628 amino acids, respectively, and have distinct C-terminal cytoplasmic domains, were isolated from rat brain by RT-PCR. Kv4.3b, d, e and f were expressed at much lower levels in brain. Mutagenesis which removed 149 amino acids in C-terminal domain of Kv4.3a significantly slowed its rate of recovery from inactivation as measured in heterologous expression in HEK293 cells. Surprisingly, however, neither the rate of inactivation nor voltage dependence of the activation and inactivation were changed.

Amino Acid Sequence↗

PubMed2001

Location of the initiation site of calcium transients and sparks in rabbit heart Purkinje cells.

1. The distribution and localization of Ca2+ transients and Ca2+ sparks in isolated adult rabbit Purkinje cells were examined using confocal microscopy and the Ca2+ indicator fluo-3. 2. When cells were field stimulated in 2.0 mM Ca2+ buffer, a transverse confocal line scan (500 Hz) showed that the fluorescence intensity was greatest at the cell periphery during the onset of the Ca2+ transient ([Ca2+]i). In contrast, the [Ca2+]i of ventricular cells showed a more uniform pattern of activation across the cell. Staining with di-8-ANEPPS revealed that Purkinje cells lack t-tubules, whereas ventricular cells have an extensive t-tubular system. 3. When we superfused both cell types with a buffer containing 5 mM Ca2+-1 microM isoproterenol (isoprenaline) they produced Ca2+ sparks spontaneously. Ca2+ sparks occurred only at the periphery of Purkinje cells but occurred throughout ventricular cells. Sparks in both cell types could be completely abolished by addition of the SR inhibitor thapsigargin (500 nM). Brief exposure to nifedipine (10 microM) did not reduce the number of spontaneous sparks. 4. Immunofluorescence staining of Purkinje cells with anti-ryanodine antibody revealed that ryanodine receptors (RyRs) are present at both peripheral and central locations. 5.Computer simulations of experiments in which the calcium transient was evoked by voltage clamp depolarizations suggested that the increase in calcium observed in the centre of the cell could be explained by simple buffered diffusion of calcium. These computations suggested that the RyRs deep within the cell do not contribute significantly to the calcium transient. 6. These results provide the first detailed, spatially resolved data describing Ca2+ transients and Ca2+ sparks in rabbit cardiac Purkinje cells. Both types of events are initiated only at subsarcolemmal SR Ca2+ release sites suggesting that in Purkinje cells, Ca2+ sparks only originate where the sarcolemma and sarcoplasmic reticulum form junctions. The role of the centrally located RyRs remains unclear. It is possible that because of the lack of t-tubules these RyRs do not experience a sufficiently large Ca2+ trigger during excitation-contraction (E-C) coupling to become active.

Animals↗

PubMed2001

A mathematical model of action potential heterogeneity in adult rat left ventricular myocytes.

Mathematical models were developed to reconstruct the action potentials (AP) recorded in epicardial and endocardial myocytes isolated from the adult rat left ventricle. The main goal was to obtain additional insight into the ionic mechanisms responsible for the transmural AP heterogeneity. The simulation results support the hypothesis that the smaller density and the slower reactivation kinetics of the Ca(2+)-independent transient outward K(+) current (I(t)) in the endocardial myocytes can account for the longer action potential duration (APD), and more prominent rate dependence in that cell type. The larger density of the Na(+) current (I(Na)) in the endocardial myocytes results in a faster upstroke (dV/dt(max)). This, in addition to the smaller magnitude of I(t), is responsible for the larger peak overshoot of the simulated endocardial AP. The prolonged APD in the endocardial cell also leads to an enhanced amplitude of the sustained K(+) current (I(ss)), and a larger influx of Ca(2+) ions via the L-type Ca(2+) current (I(CaL)). The latter results in an increased sarcoplasmic reticulum (SR) load, which is mainly responsible for the higher peak systolic value of the Ca(2+) transient [Ca(2+)](i), and the resultant increase in the Na(+)-Ca(2+) exchanger (I(NaCa)) activity, associated with the simulated endocardial AP. In combination, these calculations provide novel, quantitative insights into the repolarization process and its naturally occurring transmural variations in the rat left ventricle.

Action Potentials↗

PubMed2000

Electrophysiological effects of ibutilide on the delayed rectifier K(+) current in rabbit sinoatrial and atrioventricular node cells.

Biophysical and pharmacological characteristics of the delayed rectifier K(+) current (I(K)) of rabbit sinoatrial (SA) node and atrioventricular (AV) node cells have been studied using the whole-cell patch clamp technique together with a recently developed antiarrhythmic agent, ibutilide. Ibutilide is a potent blocker of the rapid delayed rectifier K(+) current, I(Kr). Superfusion with ibutilide (10(-7) M) caused a decrease in the spontaneous firing frequency, depolarization of the maximal diastolic potential and prolongation of the action potential duration in both SA and AV node cells. In whole cell voltage clamp experiments done on myocytes from SA node, ibutilide (10(-7) M) blocked I(K) strongly (40%) and had smaller effects on Ca(2+) current (10%) and hyperpolarization-activated inward current, I(f) (11%). In AV node cells, the corresponding reductions were I(K) (68%), I(Ca) (13%) and I(f) (10%), respectively. A 10-fold increase in the concentration of ibutilide further decreased I(K) in SA node cells (67+/-8%), and blocked I(K) almost completely in AV node cells. These results are consistent with the hypothesis that the delayed rectifier K(+) current in SA node cell is generated by both I(Kr) and I(Ks), whereas I(Kr) predominates in AV node cells. Knowledge of the differences in the distribution of I(Kr), as well as the different sensitivity to blockers of I(Kr) in nodal cells, is important for understanding modifications of the automaticity, conduction velocity, and refractoriness by class III antiarrhythmic agents.

Action Potentials↗

PubMed2000

A novel genetic pathway for sudden cardiac death via defects in the transition between ventricular and conduction system cell lineages.

HF-1 b, an SP1 -related transcription factor, is preferentially expressed in the cardiac conduction system and ventricular myocytes in the heart. Mice deficient for HF-1 b survive to term and exhibit normal cardiac structure and function but display sudden cardiac death and a complete penetrance of conduction system defects, including spontaneous ventricular tachycardia and a high incidence of AV block. Continuous electrocardiographic recordings clearly documented cardiac arrhythmogenesis as the cause of death. Single-cell analysis revealed an anatomic substrate for arrhythmogenesis, including a decrease and mislocalization of connexins and a marked increase in action potential heterogeneity. Two independent markers reveal defects in the formation of ventricular Purkinje fibers. These studies identify a novel genetic pathway for sudden cardiac death via defects in the transition between ventricular and conduction system cell lineages.

Action Potentials↗

PubMed2000

Na(+)-Ca(2+)-K(+) currents measured in insect cells transfected with the retinal cone or rod Na(+)-Ca(2+)-K(+) exchanger cDNA.

The recently cloned retinal cone Na(+)-Ca(2+)-K(+) exchanger (NCKX) was expressed in cultured insect cells, and whole-cell patch clamp was used to measure transmembrane currents generated by this transcript and compare them with currents generated by retinal rod NCKX or by a deletion mutant rod NCKX from which the two large hydrophilic loops were removed. We have characterized the ionic currents generated by both the forward (Ca(2+) extrusion) and reverse (Ca(2+) influx) modes of all three NCKX proteins. Reverse NCKX exchange generated outward current that required the simultaneous presence of both external Ca(2+) and external K(+). Forward NCKX exchange carried inward current with Na(+), but not with Li(+) in the bath solution. The cation dependencies of the three NCKX tested (external K(+), external Na(+), internal Ca(2+)) were very similar to each other and to those reported previously for the in situ rod NCKX. These findings provide the first electrophysiological characterization of cone NCKX and the first electrophysiological characterization of potassium-dependent Na(+)-Ca(+) exchangers in heterologous systems. Our results demonstrate the feasibility of combining heterologous expression and biophysical measurements for detailed NCKX structure/function studies.

Amino Acid Sequence↗

PubMed2000

Mathematical simulation of slowing of cardiac conduction velocity by elevated extracellular.

We have studied the dependence of conduction velocity (theta) on extracellular potassium concentration ([K+]o) in a model of one-dimensional conduction using an idealized strand of human atrial cells. Elevated [K+]o in the 5-20 mM range shifts the resting potential (Vrest) in the depolarizing direction and reduces input resistance (Rin) by increasing an inwardly rectifying K+ conductance, I(Kl). Our results show that in this model: (1) theta depends on [K+]o in a "biphasic" fashion. Moderate elevations of [K+]o (to less than 8 mM) result in a small increase in theta, whereas at higher [K+]o (8-16 mM) theta is reduced. (2) This biphasic relationship can be attributed to the competing effects of (i) the smaller depolarization needed to reach the excitation threshold (Vthresh-Vrest) and (ii) reduced availability (increased inactivation) of sodium current, INa, as the cell depolarizes progressively. (3) Decreasing Rin reduces theta due to the increased electrical load on surrounding cells. (4) The effect on theta of [K+]o-induced changes in Rin in the atrium (as well as other high-Rin tissue, such as that of the Purkinje system or nodes) is likely to be small. This effect could be substantial, however, under conditions in which Rin is comparable in size to gap junction resistance and membrane resistance (inverse slope of the whole-cell current-voltage relationship) when sodium channels are open, which is likely to be the case in ventricular tissue.

Action Potentials↗

PubMed2000

Voltage-sensitive dye mapping of activation and conduction in adult mouse hearts.

A custom-made apparatus based on a charge-coupled-device camera has been used to monitor changes in fluorescence from Langendorff-perfused adult mouse hearts stained with a voltage-sensitive dye, di-4-ANEPPS. With this approach it is possible to monitor activation of the ventricles at high temporal (375 micros/frame) and spatial resolution (72 x 78 pixels, 100 x 100 microm/pixel). In sinus rhythm, activation occurred with a complicated breakthrough pattern on both ventricles, and a total activation time of 3.51+/-0.16 ms (32 degrees C). A stimulus applied near the apex of the left ventricle resulted in a single activation wave front with a total activation time of 8.18+/-0.25 ms. Pacing from a site near the middle of the left ventricular epicardial surface revealed anisotropic conduction, indicating that conduction occurs preferentially in the direction of the predominant fiber orientation. The total activation time in this configuration was 5.44+/-0.24 ms. The difference in total activation time between sinus rhythm and epicardial stimulation suggests an important role for transmural conduction (the Purkinje system) in the mouse heart. These findings provide much of the necessary background needed for studying conduction abnormalities in genetically altered mice and suggest that the comparison of sinus rhythm and epicardial pacing can be used to reveal transmural conduction abnormalities.

Animals↗