ATP triggers a robust intracellular [Ca2+ ]-mediated signalling pathway in human synovial fibroblasts.

NEW FINDINGS: What is the central question of this study? What are the main [Ca2+ ]i signalling pathways activated by ATP in human synovial fibroblasts? What is the main finding and its importance? In human synovial fibroblasts ATP acts through a linked G-protein (Gq ) and phospholipase C signalling mechanism to produce IP3 , which then markedly enhances release of Ca2+ from the endoplasmic reticulum. These results provide new information for the detection of early pathophysiology of arthritis. ABSTRACT: In human articular joints, synovial fibroblasts (HSFs) have essential physiological functions that include synthesis and secretion of components of the extracellular matrix and essential articular joint lubricants, as well as release of paracrine substances such as ATP. Although the molecular and cellular processes that lead to a rheumatoid arthritis (RA) phenotype are not fully understood, HSF cells exhibit significant changes during this disease progression. The effects of ATP on HSFs were studied by monitoring changes in intracellular Ca2+ ([Ca2+ ]i ), and measuring electrophysiological properties. ATP application to HSF cell populations that had been enzymatically released from 2-D cell culture revealed that ATP (10-100 µm), or its analogues UTP or ADP, consistently produced a large transient increase in [Ca2+ ]i . These changes (i) were initiated by activation of the P2 Y purinergic receptor family, (ii) required Gq -mediated signal transduction, (iii) did not involve a transmembrane Ca2+ influx, but instead (iv) arose almost entirely from activation of endoplasmic reticulum (ER)-localized inositol 1,4,5-trisphosphate (IP3 ) receptors that triggered Ca2+ release from the ER. Corresponding single cell electrophysiological studies revealed that these ATP effects (i) were insensitive to [Ca2+ ]o removal, (ii) involved an IP3 -mediated intracellular Ca2+ release process, and (iii) strongly turned on Ca2+ -activated K+ current(s) that significantly hyperpolarized these cells. Application of histamine produced very similar effects in these HSF cells. Since ATP is a known paracrine agonist and histamine is released early in the inflammatory response, these findings may contribute to identification of early steps/defects in the initiation and progression of RA.

Intrinsic H(+) ion mobility in the rabbit ventricular myocyte.

The intrinsic mobility of intracellular H(+) ions was investigated by confocally imaging the longitudinal movement of acid inside rabbit ventricular myocytes loaded with the acetoxymethyl ester (AM) form of carboxy-seminaphthorhodafluor-1 (carboxy-SNARF-1). Acid was diffused into one end of the cell through a patch pipette filled with an isotonic KCl solution of pH 3.0. Intracellular H(+) mobility was low, acid taking 20-30 s to move 40 microm down the cell. Inhibiting sarcolemmal Na(+)-H(+) exchange with 1 mM amiloride had no effect on this time delay. Net H(+)(i) movement was associated with a longitudinal intracellular pH (pH(i)) gradient of up to 0.4 pH units. H(+)(i) movement could be modelled using the equations for diffusion, assuming an apparent diffusion coefficient for H(+) ions (D(H)(app)) of 3.78 x 10(-7) cm(2) s(-1), a value more than 300-fold lower than the H(+) diffusion coefficient in a dilute, unbuffered solution. Measurement of the intracellular concentration of SNARF (approximately 400 microM) and its intracellular diffusion coefficient (0.9 x 10(-7) cm(2) s(-1)) indicated that the fluorophore itself exerted an insignificant effect (between 0.6 and 3.3 %) on the longitudinal movement of H(+) equivalents inside the cell. The longitudinal movement of intracellular H(+) is discussed in terms of a diffusive shuttling of H(+) equivalents on high capacity mobile buffers which comprise about half (approximately 11 mM) of the total intrinsic buffering capacity within the myocyte (the other half being fixed buffer sites on low mobility, intracellular proteins). Intrinsic H(+)(i) mobility is consistent with an average diffusion coefficient for the intracellular mobile buffers (D(mob)) of ~9 x 10(-7) cm(2) s(-1).

Facilitation of intracellular H(+) ion mobility by CO(2)/HCO(3)(-) in rabbit ventricular myocytes is regulated by carbonic anhydrase.

Intracellular H(+) mobility was estimated in the rabbit isolated ventricular myocyte by diffusing HCl into the cell from a patch pipette, while imaging pH(i) confocally using intracellular ratiometric SNARF fluorescence. The delay for acid diffusion between two downstream regions approximately 40 microm apart was reduced from approximately 25 s to approximately 6 s by replacing Hepes buffer in the extracellular superfusate with a 5 % CO(2)/HCO(3)(-) buffer system (at constant pH(o) of 7.40). Thus CO(2)/HCO(3)(-) (carbonic) buffer facilitates apparent H(+)(i) mobility. The delay with carbonic buffer was increased again by adding acetazolamide (ATZ), a membrane permeant carbonic anhydrase (CA) inhibitor. Thus facilitation of apparent H(+)(i) mobility by CO(2)/HCO(3)(-) relies on the activity of intracellular CA. By using a mathematical model of diffusion, the apparent intracellular H(+) equivalent diffusion coefficient (D(H)(app)) in CO(2)/HCO(3)(-)-buffered conditions was estimated to be 21.9 x 10(-7) cm(2) s(-1), 5.8 times faster than in the absence of carbonic buffer. Facilitation of H(+)(i) mobility is discussed in terms of an intracellular carbonic buffer shuttle, catalysed by intracellular CA. Turnover of this shuttle is postulated to be faster than that of the intrinsic buffer shuttle. By regulating the carbonic shuttle, CA regulates effective H(+)(i) mobility which, in turn, regulates the spatiotemporal uniformity of pH(i). This is postulated to be a major function of CA in heart.

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.

Early and delayed afterdepolarizations in rabbit heart Purkinje cells viewed by confocal microscopy.

We investigated action potentials and Ca(2+) transients in rabbit Purkinje myocytes using whole cell patch clamp recordings and a confocal microscope. Purkinje cells were loaded with 5 microM Fluo-3/AM for 30min. Action potentials were elicited by application of a stimulus delivered through the recording pipettes. When Purkinje cells were stimulated in 2.0mM Ca(2+), transverse XT line scans revealed a symmetrical 'U'-shaped Ca(2+) transient demonstrating that the transient was initiated at the cell periphery. When Purkinje cells were superfused with 1 microM isoprenaline, both early and delayed afterdepolarizations were induced. XT line scans of cells exhibiting early afterdepolarizations showed a second symmetrical 'U'-shaped transient. This Ca(2+) transient was initiated at the cell periphery suggesting reactivation of the Ca(2+) current. In contrast, in Purkinje cells exhibiting delayed afterdepolarizations and a corresponding transient inward current, XT line scans revealed a heterogenous rise in Ca(2+) at both peripheral and central regions of the cell. Immunofluorescence staining of Purkinje cells with an antibody to ryanodine receptors (RyRs) revealed that RyRs are located at regularly spaced intervals throughout the interior of Purkinje cells. These results suggest that, although RyRs are located throughout Purkinje cells, only peripheral RyRs are activated to produce transients, sparks and early afterdepolarizations. During delayed afterdepolarizations, we observed a heterogenous rise in Ca(2+) at both peripheral and central regions of the cell as well as large central increases in Ca(2+). Although the latter may result from central release, we cannot exclude the possibility that it reflects Ca(2+) diffusion from subsarcolemmal sites.

Transient outward current modulates discontinuous conduction in rabbit ventricular cell pairs.

OBJECTIVE: While several studies have demonstrated that the L-type calcium current maintains discontinuous conduction, the contribution of the transient outward current (I(to)) to conduction remains unclear. This study evaluated the effects of I(to) inhibition on conduction between ventricular myocytes. METHODS: An electronic circuit with a variable resistance (R(j)) was used to electrically couple single epicardial myocytes isolated from rabbit right ventricle. We inhibited I(to) with 4-aminopyridine superfusion, rate-acceleration, or premature stimulation to evaluate the subsequent effects on conduction delay and the critical R(j), which was quantified as the highest R(j) that could be imposed before conduction failed. RESULTS: I(to) inhibition significantly enhanced conduction in all cell pairs (n=23). Pharmacologic inhibition of I(to) resulted in a 32+/-5% decrease in conduction delay and a 36+/-7% increase in critical R(j). Similarly, reduction of the basic cycle length from 2 to 0.5 s resulted in a 31+/-3% decrease in conduction delay and a 31+/-3% increase in critical R(j). Finally, premature action potentials conducted with a 41+/-4% shorter conduction delay and a 73+/-24% higher critical R(j) than basic action potentials. CONCLUSIONS: I(to) inhibition significantly enhanced conduction across high R(j). These results suggest I(to) may contribute to rate-dependent conduction abnormalities.

The sodium pump modulates the influence of I(Na) on [Ca2+]i transients in mouse ventricular myocytes.

To investigate whether activity of the sarcolemmal Na pump modulates the influence of sodium current on excitation-contraction (E-C) coupling, we measured [Ca(2+)](i) transients (fluo-3) in single voltage-clamped mouse ventricular myocytes ([Na+](pip) = 15 or 0 mM) when the Na pump was activated (4.4 mM K(+)(o)) and during abrupt inhibition of the pump by exposure to 0 K with a rapid solution-switcher device. After induction of steady state [Ca2+](i) transients by conditioning voltage pulses (0.25 Hz), inhibition of the Na pump for 1.5 s immediately before and continuing during a voltage pulse (200 ms, -80 to 0 mV) caused a significant increase (15 +/- 2%; n = 16; p < 0.01) in peak systolic [Ca2+](i) when [Na+](pip) was 15 mM. In the absence of sodium current (I(Na), which was blocked by 60 microM tetrodotoxin (TTX)), inhibition of the Na pump immediately before and during a voltage pulse did not result in an increase in peak systolic [Ca2+](i). Abrupt blockade of I(Na) during a single test pulse with TTX caused a slight decrease in peak [Ca2+](i), whether the pump was active (9%) or inhibited (10%). With the reverse-mode Na/Ca exchange inhibited by KB-R 7943, inhibition of the Na pump failed to increase the magnitude of the peak systolic [Ca2+](i) (4 +/- 1%; p = NS) when [Na+](pip) was 15 mM. When [Na+](pip) was 0 mM, the amplitude of the peak systolic [Ca2+](i) was not altered by abrupt inhibition of the Na pump immediately before and during a voltage pulse. These findings in adult mouse ventricular myocytes indicate the Na pump can modulate the influence of I(Na) on E-C coupling in a single beat and provide additional evidence for the existence of Na fuzzy space, where [Na+] can significantly modulate Ca2+ influx via reverse Na/Ca exchange.

Electrotonic suppression of early afterdepolarizations in isolated rabbit Purkinje myocytes.

Many studies suggest that early afterdepolarizations (EADs) arising from Purkinje fibers initiate triggered arrhythmias under pathological conditions. However, electrotonic interactions between Purkinje and ventricular myocytes may either facilitate or suppress EAD formation at the Purkinje-ventricular interface. To determine conditions that facilitated or suppressed EADs during Purkinje-ventricular interactions, we coupled single Purkinje myocytes and aggregates isolated from rabbit hearts to a passive model cell via an electronic circuit with junctional resistance (R(j)). The model cell had input resistance (R(m,v)) of 50 M Omega, capacitance of 39 pF, and a variable rest potential (V(rest,v)). EADs were induced in Purkinje myocytes during superfusion with 1 microM isoproterenol. Coupling at high R(j) to normally polarized V(rest,v) established a repolarizing coupling current during all phases of the Purkinje action potential. This coupling current preferentially suppressed EADs in single cells with mean membrane resistance (R(m,p)) of 297 M Omega, whereas EAD suppression in larger aggregates with mean R(m,p) of 80 M Omega required larger coupling currents. In contrast, coupling to elevated V(rest,v) established a depolarizing coupling current during late phase 2, phase 3, and phase 4 that facilitated EAD formation and induced spontaneous activity in single Purkinje myocytes and aggregates. These results have important implications for arrhythmogenesis in the infarcted heart when reduction of the ventricular mass due to scarring alters the R(m,p)-to-R(m,v) ratio and in the ischemic heart when injury currents are established during coupling between polarized Purkinje myocytes and depolarized ventricular myocytes.

Generation of intracellular pH gradients in single cardiac myocytes with a microperfusion system.

This study describes the use of a microperfusion system to create rapid, large regional changes in intracellular pH (pH(i)) within single ventricular myocytes. The spatial distribution of pH(i) in single myocytes was measured with seminaphthorhodafluor-1 fluorescence using confocal imaging. Changes in pH(i) were induced by local external application of NH(4)Cl, CO(2), or sodium propionate. Local application was achieved by simultaneously directing two parallel square microstreams, each 275 microm wide, over a single myocyte oriented perpendicular to the direction of flow. One stream contained the control solution, and the other contained a weak acid or base. End-to-end, stable pH(i) gradients as large as 1 pH unit were readily created with this technique. This result indicates that pH within a single cardiac cell may not always be spatially uniform, particularly when weak acid or base gradients are present, which can occur, for example, in regional myocardial ischemia. The microperfusion method should be useful for studying the effects of localized acidosis on myocyte function, estimating intracellular ion diffusion rates, and, possibly, inducing regional changes in other important intracellular ions.

Beat-to-beat repolarization variability in ventricular myocytes and its suppression by electrical coupling.

Single ventricular myocytes paced at a constant rate and held at a constant temperature exhibit beat-to-beat variations in action potential duration (APD). In this study we sought to quantify this variability, assess its mechanism, and determine its responsiveness to electrotonic interactions with another myocyte. Interbeat APD(90) (90% repolarization) of single cells was normally distributed. We thus quantified APD(90) variability as the coefficient of variability, CV = (SD/mean APD(90)) x 100. The mean +/- SD of the CV in normal solution was 2.3 +/- 0.9 (132 cells). Extracellular TTX (13 microM) and intracellular EGTA (14 mM) both significantly reduced the CV by 44 and 26%, respectively. When applied in combination the CV fell by 54%. In contrast, inhibition of the rapid delayed rectifier current with L-691,121 (100 nM) increased the CV by 300%. The CV was also significantly reduced by 35% when two normal myocytes were electrically connected with a junctional resistance (R(j)) of 100 MOmega. Electrical coupling (R(j) = 100 MOmega) of a normal myocyte to one producing early afterdepolarization (EAD) completely blocked EAD formation. These results indicate that beat-to-beat APD variability is likely mediated by stochastic behavior of ion channels and that electrotonic interactions act to limit temporal dispersion of refractoriness, a major contributor to arrhythmogenesis.

Caffeine-induced Ca(2+) sparks in mouse ventricular myocytes.

Ca(2+) sparks are spatially localized intracellular Ca(2+) release events that were first described in 1993. Sparks have been ascribed to sarcoplasmic reticulum Ca(2+) release channel (ryanodine receptor, RyR) opening induced by Ca(2+) influx via L-type Ca(2+) channels or by spontaneous RyR openings and have been thought to reflect Ca(2+) release from a cluster of RyR. Here we describe a pharmacological approach to study sparks by exposing ventricular myocytes to caffeine with a rapid solution-switcher device. Sparks under these conditions have properties similar to naturally occurring sparks in terms of size and intracellular Ca(2+) concentration ([Ca(2+)](i)) amplitude. However, after the diffusion of caffeine, sparks first appear close to the cell surface membrane before coalescing to produce a whole cell transient. Our results support the idea that a whole cell [Ca(2+)](i) transient consists of the summation of sparks and that Ca(2+) sparks consist of the opening of a cluster of RyR and confirm that characteristics of the cluster rather than the L-type Ca(2+) channel-RyR relation determine spark properties.

Quantitation of Na/Ca exchanger function in single ventricular myocytes.

A Na/Ca exchange current can be elicited in voltage clamped single ventricular myocytes by the abrupt removal of extracellular Na+ by means of a rapid switcher device. We measured this reverse Na/Ca exchange current in isolated mouse ventricular myocytes from wild-type mice, and from transgenic mice with hearts overexpressing the Na/Ca exchanger. In mouse ventricular myocytes, the current was sensitive to nickel, and was eliminated by removal of intracellular Na+. It was not influenced by 3 m m ouabain, and thus not contaminated by Na pump currents. The magnitude of the current reached a plateau within 10-15 min after obtaining a whole cell patch with the pipettes containing EGTA, to buffer [Ca2+]i and in zero extracellular K+ concentration to completely inhibit the Na pump, and allow equilibration of pipette Na+ with subsarcolemmal [Na+]. The magnitude of the current increased with increases in pipette [Na+]. Comparison of the current magnitudes in wild-type and transgenic myocytes showed a 2.5 and 2.7 fold increase in the current in transgenic myocytes at pipette [Na+] of 10 and 20 m m. The magnitude of this increase in Na/Ca exchanger currents in single transgenic myocytes compares well with the reported 2.5 fold increase in Na+-dependent 45Ca2+ uptake measured in ventricular sarcolemmal vesicles obtained from transgenic animals. With this approach, we found variation in exchanger current densities in different species, with values for mouse>rat>rabbit>dog>human. This technique should also be useful in quantifying changes in Na/Ca exchanger current density as a consequence of pathologic processes, and exposure to drugs.

Modulation of repolarization in rabbit Purkinje and ventricular myocytes coupled by a variable resistance.

Purkinje-ventricular junctions (PVJs) have been implicated as potential sites of arrhythmogenesis, in part because of the dispersion of action potential duration (APD) between Purkinje (P) and ventricular (V) myocytes. To characterize electrotonic modulation of APD as a function of junctional resistance (Rj), we coupled single isolated rabbit P and V myocytes with an electronic circuit. In seven of eight PV myocyte pairs, both APDs shortened on coupling at Rj = 50 MOmega. This was in contrast to modulation of APD in paired ventricular myocytes, which demonstrated APD shortening of the intrinsically longer action potential and APD prolongation of the intrinsically shorter action potential. Companion computer simulations, performed to suggest possible mechanisms for the paradoxical shortening of the V action potential in paired P and V myocytes, showed that the difference in intrinsic peak plateau potentials (Vpp) of the P and V myocytes determined whether the V action potential shortened or prolonged on coupling. This difference in Vpp caused a large, repolarizing coupling current to flow to the V myocyte, contributing to early inactivation of the L-type calcium current and early activation of the inward rectifier current. These results suggest that intrinsic differences in phase 1 repolarization could yield differing patterns of APD shortening or prolongation in the network of subendocardial PVJs, leaving some PVJs vulnerable to conduction of premature stimuli while other PVJs remain refractory.

Effect of ANG II on pHi, [Ca2+]i, and contraction in rabbit ventricular myocytes from infarcted hearts.

In this study we examined Na+/H+ exchange activity, Ca2+ transients, and contractility in rabbit ventricular myocytes isolated from normal and chronically (8-12 wk) infarcted left ventricles. Myocytes from infarcted hearts (post-MI myocytes) were isolated from the peri-infarcted region of the left ventricle. Intracellular pH (pHi) and Ca2+ concentration ([Ca2+]i) were measured with the fluorescent pH indicators seminaphthorhodafluor 1 and fluo 3, respectively, and contractility was assessed from changes in cell shortening during field stimulation. Experiments were performed at extracellular pH 7. 4 in the presence and absence (HEPES buffer) of CO2 and HCO-3. Our findings demonstrate that 1) myocytes after myocardial infarction (post-MI) were significantly larger than normal, 2) post-MI hypertrophy was not accompanied by changes in non-CO2 intracellular buffering power, 3) post-MI hypertrophy did not significantly affect the ability of Na+/H+ exchange to mediate pHi recovery from intracellular acidosis, 4) the stimulatory effect of ANG II (100 nM) on Na+/H+ exchange was significantly reduced in post-MI myocytes, 5) in HCO-3-buffered solutions, ANG II did not significantly stimulate pHi recovery from acidosis in post-MI myocytes, 6) the angiotensin AT1 receptor mediates the stimulatory action of ANG II on Na+/H+ exchange in normal and post-MI myocytes, and 7) the stimulatory effect of ANG II on the Ca2+ transient and contraction was blunted in post-MI myocytes bathed in HEPES-buffered solution. A suppressed ventricular responsiveness to ANG II may be beneficial in the intact myocardium by attenuating ATP consumption and by reducing intracellular Na+ accumulation during ischemia-reperfusion.

Abnormal myocyte Ca2+ homeostasis in rabbits with pacing-induced heart failure.

To determine whether there are abnormalities in myocyte excitation-contraction coupling and intracellular Ca2+ concentration ([Ca2+]i) homeostasis in pacing-induced heart failure (PF), we measured L-type Ca2+ current (ICa,L) and Na+/Ca2+ exchanger current (INa/Ca) with voltage clamp and measured intracellular Na+ concentration ([Na+]i) and [Ca2+]i with the use of sodium-binding benzofuran isophthalate (SBFI) and fluo 3 in ventricular myocytes isolated from control and paced rabbits. The peak systolic and diastolic levels and the amplitude of electrically stimulated [Ca2+]i transients (0.25 Hz, extracellular Ca2+ concentration = 1.08 mM) were significantly less in PF myocytes. Also, there was prolongation of the times to peak and decline of [Ca2+]i transients. ICa,L density was markedly decreased in PF myocytes. INa/Ca at -40 mV elicited by rapid exposure to 0 Na+ solution with a rapid solution switcher was significantly reduced in PF myocytes, suggesting that the function of the Na+/Ca2+ exchanger is impaired in these myocytes. In PF myocytes the decline of the [Ca2+]i transient when the Na+/Ca2+ exchanger was abruptly disabled was markedly prolonged compared with the decline in control myocytes, consistent with depressed sarcoplasmic reticulum (SR) Ca2+-ATPase function. RNase protection assay showed decreased levels of Na+/Ca2+ exchanger and SR Ca2+-ATPase mRNA in PF hearts, consistent with the function studies. We conclude that the functions of L-type Ca2+ channels, Na+/Ca2+ exchanger, and SR Ca2+-ATPase are impaired in myocytes from rabbit hearts with failure induced by rapid pacing. These abnormalities result in reduced [Ca2+]i transients and systolic and diastolic dysfunction and appear to account for the abnormal ventricular function observed.

[Electrophysiological heterogeneity of rabbit atrioventricular node cells: possible relationship to fast and slow pathways].

Catheter ablation has been applied for the therapy of atrioventricular nodal re-entrant tachycardia for several years. Although this procedure is quite successful, the cellular electrophysiological mechanisms underlying fast and slow conductions remain unknown. Therefore, the characteristics of the action potential waveform of rabbit atrioventricular node (AVN) cells obtained from different regions within Koch's triangle were studied using nystatin-permeabilized patch methods, and 64 AVN cells were used for this study. Based on gross morphology, AVN cells were classified into three groups: ovoid cells, rod shaped cells, and cells with an intermediate shape. Results obtained by measuring the maximum velocity dV/dt of action potentials fell into four subgroups: Group I (dV/dt < 5 V/sec, n = 23), Group II (dV/dt > 5 but < 10 V/sec, n = 20), Group III (dV/dt > 10 but < 20 V/sec, n = 13), and Group IV (dV/dt > 20 V/sec, n = 8). Ovoid cells had the smallest dV/dt, whereas the rod shaped cells had the highest dV/dt. The maximum diastolic potential was more negative in Groups III and IV than in Groups I and II. A notch in the phase II of the action potential was observed in 23% of Group III cells and 56% of those in Group IV, but was not present in Groups I and II. These findings provide further evidence that AVN cells are heterogeneous both in morphology and electrophysiological characteristics. Provided that values of dV/dt can be related to conduction velocity, our findings suggest that cells in Groups III and IV may contribute to fast conduction pathways whereas those in Groups I and II are responsible for slow conduction pathways.

Repolarizing K+ currents in rabbit heart Purkinje cells.

1. Electrophysiological experiments on single myocytes obtained from Purkinje fibres and ventricular tissue of adult rabbit hearts were done to compare the contributions of three potassium (K+) currents to the action potentials in these two tissues. 2. In Purkinje cells reductions in extracellular potassium, [K+]o, from normal (5.4 mM) to 2.0 mM resulted in a large hyperpolarization and marked lengthening of the action potential. In ventricular myocytes, these changes were much less pronounced. Voltage clamp measurements demonstrated that these differences were mainly due to a much smaller inward rectifier K+ current, IK1, in Purkinje cells than in ventricular myocytes. 3. Application of 4-aminopyridine (4-AP, 2 mM) showed that all Purkinje cells exhibited a very substantial Ca2+-independent transient K+ outward current, It. 4-AP significantly broadened the early, rapid repolarization phase of the action potential. 4. Selective inhibitors of the fast component, IK, r (MK-499, 200 nM) and the slow component IK,s (L-735821 (propenamide), 20 nM) of the delayed rectifier K+ currents both significantly lengthened the action potential, suggesting that these conductances are present, but very small (< 20 pA) in Purkinje cells. Attempts to identify time- and voltage-dependent delayed rectifier K+ current(s) in Purkinje cells failed, although a slow delayed rectifier was observed in ventricular myocytes. 5. These results demonstrate significant differences in action potential waveform, and underlying K+ currents in rabbit Purkinje and ventricular myocytes. Purkinje cells express a much smaller IK1, and a larger It than ventricular myocytes. These differences in current densities can explain some of the most important electrophysiological properties of these two tissues.

Conduction between isolated rabbit Purkinje and ventricular myocytes coupled by a variable resistance.

Conduction at the Purkinje-ventricular junction (PVJ) demonstrates unidirectional block under both physiological and pathophysiological conditions. Although this block is typically attributed to multidimensional electrotonic interactions, we examined possible membrane-level contributions using single, isolated rabbit Purkinje (P) and ventricular (V) myocytes coupled by an electronic circuit. When we varied the junctional resistance (Rj) between paired V myocytes, conduction block occurred at lower Rj values during conduction from the smaller to larger myocyte (115 +/- 59 M omega) than from the larger to smaller myocyte (201 +/- 51 M omega). In Purkinje-ventricular myocyte pairs, however, block occurred at lower Rj values during P-to-V conduction (85 +/- 39 M omega) than during V-to-P conduction (912 +/- 175 M omega), although there was little difference in the mean cell size. Companion computer simulations, performed to examine how the early platea currents affected conduction, showed that P-to-V block occurred at lower Rj values when the transient outward current was increased or the calcium current was decreased in the model P cell. These results suggest that intrinsic differences in phase 1 repolarization can contribute to unidirectional block at the PVJ.

Complications associated with rapid caffeine application to cardiac myocytes that are not voltage clamped.

The rapid application of caffeine to cardiac myocytes is commonly used to assess changes in the Ca2+ content of the sarcoplasmic reticulum (SR) and to study other parameters of intracellular Ca2+ regulation. Here we examined the effects of rapid caffeine application on membrane potential, intracellular Ca2+, and cell shortening in ventricular myocytes (rat, rabbit, guinea pig, dog) and atrial myocytes (rabbit) that were not voltage clamped. Conditioning pacing was used to achieve a steady-state level of SR Ca2+ loading prior to caffeine (10 mM) application. Caffeine transiently depolarized myocytes as expected from activation of forward Na+-Ca2+ exchange. However, we also found in each species (50% rat, 36% rabbit ventricular, 53% rabbit atrial, 56% guinea pig, 31% dog) that the caffeine-induced depolarization could also trigger an action potential. Caffeine-triggered potentials were completely blocked by thapsigargin (1 microM). The Ca2+ transient and contraction that accompanied caffeine-triggered action potentials had a larger magnitude and slower rate of decline (or relaxation) than occurred during caffeine-induced subthreshold depolarizations. Thus, the use of rapid caffeine application to study SR function and [Ca2+]i regulation in myocytes that are not voltage clamped can yield erroneous results.

Electrotonic modulation of electrical activity in rabbit atrioventricular node myocytes.

Electrotonic effects of electrically coupling atrioventricular (AV) nodal cells to each other and to real and passive models of atrial and ventricular cells were studied using a technique that does not require functional gap junctions. Membrane potential was measured in each cell using suction pipettes. Mutual entrainment of two spontaneously firing AV nodal cells was achieved with a junctional resistance (Rj) of 500 M omega, which corresponds to only 39 junctional channels, assuming a single-channel conductance of 50 pS. Coupling of AV nodal and atrial cells at Rj of 50 M omega caused hyperpolarization of the nodal cell, decreasing its action potential duration and either slowing or blocking diastolic depolarization in the AV node myocyte. Opposite changes occurred in the atrial action potential. When AV nodal and ventricular cells were coupled at Rj of 50 M omega, nodal diastolic potential was markedly hyperpolarized and diastolic depolarization was completely blocked with little change in ventricular diastolic potential. However, coupling did elicit marked changes in the action potential duration of both cells, with prolongation in the nodal cell and shortening in the ventricular cell. Nodal maximum upstroke velocity was increased by both atrial and ventricular coupling, as expected from the hyperpolarization that occurred. With an Rj of 50 M omega, spontaneous firing was blocked in all single AV nodal pacemaker cells during coupling to a real or passive model of an atrial or ventricular cell. These results demonstrate that action potential formation and waveform in a single AV nodal cell is significantly affected by electrical coupling to other myocytes.