The distribution of calcium in toad cardiac pacemaker cells during spontaneous firing

Isolated, spontaneously active pacemaker cells from the sinus venosus region of the toad heart were loaded with the calcium indicator fluo-3. The cells were examined with a confocal microscope to investigate the distribution of calcium during spontaneous activity. Three classes of calcium-related signals were present. First, intense, localised, time-invariant signals were detected from structures distributed across the cell interior. Based on the insensitivity to saponin and the distribution in the cell, these signals appear to arise from fluo-3 located in the sarcoplasmic reticulum and the nuclear envelope. Second, spatially uniform signals from the cytoplasm were present at rest and showed spontaneous increases in [Ca2+]i which propagated along the cell. These Ca2+ transients were uniform in intensity across the diameter of the cell and we could detect no significant delay in the middle of the cell compared to the edges. However, within the nucleus the Ca2+ transient showed a clear delay compared to the cytoplasm. Third, localised, transient increases in [Ca2+]i (Ca2+ sparks) which did not propagate were also detectable. These could be detected both near the surface membrane and in the interior of the cell and reduced in magnitude and increased in duration in the presence of ryanodine. The frequency of firing of Ca2+ sparks significantly increased in the 200-ms period preceding a spontaneous Ca2+ transient. These results suggest that pacemaker cells contain sarcoplasmic reticulum which is distributed across the cell. The Ca2+ transient is uniform across the cell indicating that near-synchronous release of Ca2+ from the sarcoplasmic reticulum is achieved. Ca2+ sparks occur in pacemaker cells though their role in pacemaker function remains to be elucidated.     

The role of the calcium and the voltage clocks in sinoatrial node dysfunction

Recent evidence indicates that the voltage clock (cyclic activation and deactivation of membrane ion channels) and Ca(2+) clocks (rhythmic spontaneous sarcoplasmic reticulum Ca(2+) release) jointly regulate sinoatrial node (SAN) automaticity. However, the relative importance of the voltage clock and Ca(2+) clock for pacemaking was not revealed in sick sinus syndrome. Previously, we mapped the intracellular calcium (Ca(i)) and membrane potentials of the normal intact SAN simultaneously using optical mapping in Langendorff-perfused canine right atrium. We demonstrated that the sinus rate increased and the leading pacemaker shifted to the superior SAN with robust late diastolic Ca(i) elevation (LDCAE) during β-adrenergic stimulation. We also showed that the LDCAE was caused by spontaneous diastolic sarcoplasmic reticulum (SR) Ca(2+) release and was closely related to heart rate changes. In contrast, in pacing induced canine atrial fibrillation and SAN dysfunction models, Ca(2+) clock of SAN was unresponsiveness to β-adrenergic stimulation and caffeine. Ryanodine receptor 2 (RyR2) in SAN was down-regulated. Using the prolonged low dose isoproterenol together with funny current block, we produced a tachybradycardia model. In this model, chronically elevated sympathetic tone results in abnormal pacemaking hierarchy in the right atrium, including suppression of the superior SAN and enhanced pacemaking from ectopic sites. Finally, if the LDCAE was too small to trigger an action potential, then it induced only delayed afterdepolarization (DAD)-like diastolic depolarization (DD). The failure of DAD-like DD to consistently trigger a sinus beat is a novel mechanism of atrial arrhythmogenesis. We conclude that dysfunction of both the Ca(2+) clock and the voltage clock are important in sick sinus syndrome.     

家兔窦房结的形态学研究

目的 :了解窦房结的形态结构。方法 :取 1 0例家兔心脏 ,常规石蜡包埋整心连续切片 ,HE染色、Masson三色染色和磷钨酸苏木素染色后 ,在光镜下观察。在光镜观察的基础上 ,另取 2例家兔窦房结对其特化的P细胞和T细胞进行透射电镜观察。结果 :家兔窦房结位于界沟上部 ,约 1 / 3跨越腔耳角 ,形态大小变化较大 ,内部有丰富的小血管分布 ,附近有一较大动脉和神经分布 ,未见分层分部现象。P细胞内可见线粒体、电子致密颗粒和高尔基氏器 ,细胞核大而圆可见核仁。T细胞介于P细胞与普通心肌细胞之间。结论 :家兔窦房结的位置较高 ,其细胞发育较为成熟 ,个体差异较大     

Ca2+ signalling in urethral interstitial cells of Cajal

Interstitial cells of Cajal (ICC) in the urethra have been proposed as specialized pacemakers that are involved in the generation of urethral tone and therefore the maintenance of urinary continence. Recent studies on freshly dispersed ICC from the urethra of rabbits have demonstrated that pacemaker activity in urethra ICC is characterized by spontaneous transient depolarizations (STDs) under current clamp and spontaneous transient inward currents (STICs) under voltage clamp. When these events were simultaneously recorded with changes in intracellular Ca²⁺ (using a Nipkow spinning disk confocal microscope) they were found to be associated with global Ca²⁺ oscillations. In this short review we will consider some of these recent findings regarding the contribution of intracellular Ca²⁺ stores and Ca²⁺ influx to the generation of pacemaker activity in urethral ICC with particular emphasis on the contribution of reverse Na⁺/Ca²⁺ exchange (NCX).     

Propagation of excitation-contraction coupling into ventricular myocytes

This paper examines the [Ca²⁺]_i transient in isolated rat heart cells using a laser scanning confocal microscope and the calcium indicator fluo-3. We find that the depolarization-evoked [Ca²⁺]_i transient is activated synchronously near the surface and in the middle of the heart cell with similar kinetics of activation. The time of rise of the transient did not depend on whether the sarcoplasmic reticulum (SR) Ca-release was abolished (by thapsigargin and ryanodine). The synchrony of activation and the similarity of levels of [Ca²⁺]_i at the peripheral and deeper myoplasm (regardless of the availability of SR Ca-release) shows that sarcolemmal Ca channels and SR Ca-release channels are distributed throughout the rat heart cell and that the propagation of the action potential into the interior of the cell is rapid. In addition, the activation of calcium release from the SR by CICR is rapid (2 ms) when compared to the time-course of calcium influx via the sarcolemmal Ca channel.     

The mechanism of increased postnatal heart rate and sinoatrial node pacemaker activity in mice

Heart rate (HR) of mammalian species changes postnatally, i.e., HR of large animals including humans decreases, while HR in small animals such as mice and rats increases. To clarify cellular mechanisms underlying the postnatal HR changes, we performed in vivo HR measurement and electrophysiological analysis on sinoatrial node (SAN) cells in mice. The in vivo HR was ~320 beats min^?1 (bpm) immediately after birth, and increased with age to ~690 bpm at postnatal day 14. Under blockage of autonomic nervous systems, HR remained constant until postnatal day 5 and then increased day by day. The spontaneous beating rate of SAN preparation showed a similar postnatal change. The density of the L-type Ca²⁺ current (LCC) was smaller in neonatal SAN cells than in adult cells, accompanied by a positive shift of voltage-dependent activation. Thus, the postnatal increase in HR is caused by both the increased sympathetic influence and the intrinsic activity of SAN cells. The different conductance and kinetics of LCC may be involved in the postnatal increase in pacemaker activity.     

Relaxation in rabbit and rat cardiac cells: species-dependent differences in cellular mechanisms.

The roles of the sarcoplasmic reticulum (SR) Ca(2+)-ATPase and Na(+)-Ca2+ exchange in Ca2+ removal from cytosol were compared in isolated rabbit and rat ventricular myocytes during caffeine contractures and electrically stimulated twitches. Cell shortening and intracellular calcium concentration ([Ca2+]i) were measured in indo-1-loaded cells. Na(+)-Ca2+ exchange was inhibited by replacement of external Na+ by Li+. To avoid net changes in cell or SR Ca2+ load during a twitch in 0 Na+ solution, intracellular Na+ (Na+i) was depleted using a long pre-perfusion with 0 Na+, 0 Ca2+ solution. SR Ca2+ accumulation was inhibited by caffeine or thapsigargin (TG). Relaxation of steady-state twitches was 2-fold faster in rat than in rabbit (before and after Na+i depletion). In contrast, caffeine contractures (where SR Ca2+ accumulation is inhibited), relaxed faster in rabbit cells. Removal of external Na+ increased the half-time for relaxation of caffeine contractures 15- and 5-fold in rabbit and rat myocytes respectively (and increased contracture amplitude in rabbit cells only). The time course of relaxation in 0 Na+, 0 Ca2+ solution was similar in the two species. Inhibition of the Na(+)-Ca2+ exchange during a twitch increased the [Ca2+]i transient amplitude (delta[Ca2+]i) by 50% and the time constant of [Ca2+]i decline (tau) by 45% in rabbit myocytes. A smaller increase in tau (20%) and no change in delta[Ca2+]i were observed in rat cells in 0 Na+ solution. [Ca2+]i transients remained more rapid in rat cells. Inhibition of the SR Ca(2+)-ATPase during a twitch enhanced delta[Ca2+]i by 25% in both species. The increase in tau after TG exposure was greater in rat (9-fold) than in rabbit myocytes (2-fold), which caused [Ca2+]i decline to be 70% slower in rat compared with rabbit cells. The time course of [Ca2+]i decline during twitch in TG-treated cells was similar to that during caffeine application in control cells. Combined inhibition of these Ca2+ transport systems markedly slowed the time course of [Ca2+]i decline, so that tau was virtually the same in both species and comparable to that during caffeine application in 0 Na+, 0 Ca2+ solution. Thus, the combined participation of slow Ca2+ transport mechanisms (mitochondrial Ca2+ uptake and sarcolemmal Ca(2+)-ATPase) is similar in these species. We conclude that during the decline of the [Ca2+]i transient, the Na(+)-Ca2+ exchange is about 2- to 3-fold faster in rabbit than in rat, whereas the SR Ca(2+)-ATPase is 2- to 3-fold faster in the rat.(ABSTRACT TRUNCATED AT 400 WORDS)     

Relaxation in ferret ventricular myocytes: unusual interplay among calcium transport systems.

Transport systems responsible for removing Ca2+ from the myoplasm during relaxation in isolated ferret ventricular myocytes were studied using caffeine-induced contractures. Internal calcium concentration ([Ca2+]i) was measured with the fluorescent calcium indicator indo-1, and the results were compared with our recent detailed characterizations in rabbit and rat myocytes. Relaxation and [Ca2+]i decline during a twitch in ferret myocytes were fast and similar to that in rat myocytes (i.e. half-time, t 1/2 approximately 100-160 ms). During a caffeine-induced contracture (SR Ca2+ accumulation prevented), relaxation was still relatively fast (t 1/2 = 0.57 s) and similar to relaxation in rabbit supported mainly by a strong Na(+)-Ca2+ exchange. When both the SR Ca2+ uptake and Na(+)-Ca2+ exchange are blocked (by caffeine and 0 Na+, 0 Ca2+ solution) relaxation in the ferret myocyte is remarkably fast (approximately 5-fold) compared with rabbit and rat myocytes. The decline of the Cai2+ transient was also fast under these conditions. These values were similar to those in rat under conditions where relaxation is due primarily to Na(+)-Ca2+ exchange. Additional inhibition of either the sarcolemmal Ca(2+)-ATPase or mitochondrial Ca2+ uptake caused only modest slowing of the relaxation of caffeine-induced contracture in 0 Na+, 0 Ca2+ (t 1/2 increased to approximately 3 s). In rabbit myocytes the relaxation t 1/2 is slowed to 20-30 s by these procedures. Even when the systems responsible for slow relaxation in rabbit ventricular myocytes are inhibited (i.e. sarcolemmal Ca(2+)-ATPase and mitochondrial Ca2+ uptake) along with the SR Ca(2+)-ATPase and Na(+)-Ca2+ exchange, relaxation and [Ca2+]i decline in ferret myocytes remain rapid compared with rabbit myocytes. Ca2+ taken up by mitochondria in rabbit myocytes during a caffeine contracture in 0 Na+, 0 Ca2+ solution gradually returns to the SR after caffeine removal, but this component appears to be much smaller in ferret myocytes under the same conditions. We tested for possible residual Ca2+ transport by each of the four systems which suffice to explain Ca2+ removal from the cytoplasm in rabbit (SR Ca(2+)-ATPase, Na(+)-Ca2+ exchange, sarcolemmal Ca(2+)-ATPase and mitochondrial Ca2+ uptake). We conclude that there is an additional calcium transport system at work in ferret myocytes. For this additional system, our results are most compatible with a trans-sarcolemmal Ca2+ transport, but neither a cation exchanger nor a Ca(2+)-ATPase with characteristics like that in other cardiac cells. This additional system appears able to transport Ca2+ nearly as fast as the Na(+)-Ca2+ exchange in rat ventricular myocytes.     

Caffeine-induced myocardial injury in calcium-free perfused rat hearts.

Hearts depleted of extracellular calcium become susceptible to injury caused by repletion of extracellular calcium (calcium paradox). It has been suggested that calcium-free perfusion causes weakening of intercalated disks and that the physical stress of contracture may cause sarcolemmal membrane rupture and creatine kinase (CK) release. To further investigate this hypothesis, the effects of caffeine on contracture, cellular morphology, and CK release were studied in control and calcium-free perfused isolated rat hearts. Control hearts perfused with 2.5 mM calcium retained normal ultrastructure for long periods of perfusion. Calcium-free hearts perfused for 12 minutes developed separations of fascia adherens portions of intercalated disks but retained intact nexus junctions. Hearts subjected to 5-minute calcium-free perfusion, followed by calcium repletion, developed a massive CK release and extensive contraction band necrosis (calcium paradox). Ten millimolar caffeine, which causes rapid calcium release from sarcoplasmic reticulum (SR), produced contracture, but not CK release, from control hearts perfused with medium containing 2.5 mM calcium. In calcium-free perfused hearts, caffeine caused sudden CK release accompanied by contracture, development of contraction bands, wide separations of cells at intercalated disks, and sarcolemmal membrane injury. Caffeine-induced injury occurred despite 3 mM amobarbital inhibition of mitochondrial respiration. Hearts perfused with caffeine in the presence of calcium relaxed when made calcium-free and did not release CK. Addition of caffeine following calcium-free perfusion at 22 C, which protects the heart from the calcium paradox, produced a rapid, transient contracture. These results are compatible with the hypothesis that myocardial cell injury in calcium-free hearts is not dependent on repletion of extracellular calcium or mitochondrial function, but can result from contracture following caffeine-induced release of intracellular calcium from the SR.     

Carboxyeosin decreases the rate of decay of the [Ca2+]i transient in uterine smooth muscle cells isolated from pregnant rats

 In myometrial smooth muscle cells the rate of decline of intracellular calcium ([Ca²⁺]_i) is determined by Ca²⁺ extrusion from the cell and uptake into intracellular stores. The relative quantitative contribution of these processes however, has not been established. We therefore examined the effect of the sarcolemmal Ca²⁺ pump inhibitor, carboxyeosin, on the rate of the [Ca²⁺]_i transient decline in myocytes isolated from pregnant rat uterus. Indo-1 was used in conjunction with the whole-cell patch-clamp technique to measure [Ca²⁺]_i simultaneously with transmembrane calcium current (I _Ca). [Ca²⁺]_i transients were elicited by repetitive membrane depolarization to simulate the natural pattern of uterine electrical activity. The rate of [Ca²⁺]_i removal was calculated from the falling phase of the [Ca²⁺]_i transient. Pre-treatment of the cells with 2 μM carboxyeosin led to a marked decrease in the rate of [Ca²⁺]_i transient decay, suggesting that the sarcolemmal Ca²⁺ pump is involved in the calcium extrusion process. Removal of the extracellular Na also decreased the rate of [Ca²⁺]_i decay, indicating an important role for the Na⁺/Ca²⁺ exchange. When both the sarcolemmal Ca²⁺ pump and Na⁺/Ca²⁺ exchange were inhibited the cell failed to restore [Ca²⁺]_i after the stimulation. Comparison of the rate constants of [Ca²⁺]_i decay in control conditions and after carboxyeosin treatment shows that approximately 30% of [Ca²⁺]_i decay is due to the sarcolemmal calcium pump activity. The remaining 70% can be attributed to the activity of Na⁺/Ca²⁺ exchanger and the intracellular calcium stores. Received: 17 July 1998 / Received after revision: 23 September 1998 / Accepted: 25 September 1998     

Role of the Transverse-Axial Tubule System in Generating Calcium Sparks and Calcium Transients in Rat Atrial Myocytes

Cardiac atrial cells lack a regular system of transverse tubules like that in cardiac ventricular cells. Nevertheless, many atrial cells do possess an irregular internal transverse-axial tubular system (TATS). To investigate the possible role of the TATS in excitation-contraction coupling in atrial myocytes, we visualized the TATS (labelled with the fluorescent indicator, Di-8-ANEPPS) simultaneously with Ca²⁺ transients and/or Ca²⁺ sparks (fluo-4). In confocal transverse linescan images of field-stimulated cells, whole-cell Ca²⁺ transients had two morphologies: 'U-shaped' transients and irregular or 'W-shaped' transients with a varying number of points of origin of the Ca²⁺ transient. About half (54 %, n =289 cells, 13 animals) of the cells had a TATS. Cells with TATS had a larger mean diameter (13.2 ± 2.8 μm) than cells without TATS (11.7 ± 2.0 μm) and were more common in the left atrium ( n = 206 cells; left atrium: 76 with TATS, 30 without TATS; right atrium: 42 with TATS, 58 without TATS). Simultaneous measurement of Ca²⁺ sparks and sarcolemmal structures showed that cells without TATS had U-shaped transients that started at the cell periphery, and cells with TATS had W-shaped transients that began simultaneously at the cell periphery and the TATS. Most (82 out of 102 from 31 cells) 'spontaneous' (non-depolarized) Ca²⁺ sparks occurred within 1 μm of a sarcolemmal structure (cell periphery or TATS), and 33 % occurred within 1 pixel (0.125 μm). We conclude that the presence of a sarcolemmal membrane either at the cell periphery or in the TATS in close apposition to the sarcoplasmic reticulum is required for the initiation of an evoked Ca²⁺ transient and for spontaneous Ca²⁺ sparks.     

Role of sarcolemmal ATP-sensitive K+ channels in the regulation of sinoatrial node automaticity: an evaluation using Kir6.2-deficient mice

The role of cardiac sarcolemmal ATP-sensitive K⁺ (K_ATP) channels in the regulation of sinoatrial node (SAN) automaticity is not well defined. Using mice with homozygous knockout (KO) of the Kir6.2 (a pore-forming subunit of cardiac K_ATP channel) gene, we investigated the pathophysiological role of K_ATP channels in SAN cells during hypoxia. Langendorff-perfused mouse hearts were exposed to hypoxic and glucose-free conditions (hypoxia). After 5 min of hypoxia, sinus cycle length (CL) was prolonged from 207 ± 10 to 613 ± 84 ms ( P < 0.001) in wild-type (WT) hearts. In Kir6.2 KO hearts, CL was slightly prolonged from 198 ± 17 to 265 ± 32 ms. The CL of spontaneous action potentials of WT SAN cells, recorded in the current-clamp mode, was markedly prolonged from 410 ± 56 to 605 ± 108 ms ( n = 6, P < 0.05) with a decrease of the slope of the diastolic depolarization (SDD) after the application of the K⁺ channel opener pinacidil (100 μ m ). Pinacidil induced a glibenclamide (1 μ m )-sensitive outward current, which was recorded in the voltage-clamp mode, only in WT SAN cells. During metabolic inhibition by 2,4-dinitrophenol, CL was prolonged from 292 ± 38 to 585 ± 91 ms ( P < 0.05) with a decrease of SDD in WT SAN cells but not in Kir6.2 KO SAN cells. Diastolic Ca²⁺ concentration, measured by fluo-3 fluorescence, was decreased in WT SAN cells but increased in Kir6.2 KO SAN cells after short-term metabolic inhibition. In conclusion, the present study using Kir6.2 KO mice indicates that, during hypoxia, activation of sarcolemmal K_ATP channels in SAN cells inhibits SAN automaticity, which is important for the protection of SAN cells.     

What is the hair cell transduction channel?

Rhythmic electrical activity is a feature of most smooth muscles but the mechanical consequences can vary from regular rapid phasic contractions to sustained contracture. For many years it was thought that spontaneous electrical activity originated in smooth muscle cells but recently it has become apparent that there are specialized pacemaker cells in many organs that are morphologically and functionally distinct from smooth muscle and that the former cells are the source of spontaneous electrical activity. Such a pacemaker function is well documented for the ICC of the gastrointestinal tract but evidence is accumulating that ICC-like cells play a similar role in other types of smooth muscle. We have recently shown that there are specialized pacemaking cells in the rabbit urethra which are spontaneously active when freshly isolated, readily distinguishable from smooth muscle cells under bright field illumination and relatively easy to study using patch-clamp and confocal imaging techniques. Recent results suggest that calcium oscillations in isolated rabbit urethral interstitial cells are initiated by calcium release from ryanodine sensitive intracellular stores, that oscillation frequency is very sensitive to the external calcium concentration and that conversion of the primary oscillation to a propagated calcium wave depends upon IP₃-induced calcium release.     

Pacemaker mechanism of porcine sino-atrial node cells.

In cardiac sino-atrial node (SAN) cells, time- and voltage-dependent changes in the gating of various ionic currents provide spontaneous, stable and repetitive firing of action potentials. To address the ionic nature of the species-dependent heart rate, action potentials and membrane currents were recorded in single cells dissociated from the porcine SAN, and compared with those from SAN cells of rabbits, guinea-pigs and mice. The porcine SAN cells exhibited spontaneous activity with a frequency of 60-80 min(-1), which was much slower than that of rabbit SAN cells. Under voltage clamp conditions, depolarization activated the L-type Ca2+ current (I(CaL)) followed by a gradual activation of the delayed rectifier K+ current (I(K)) while hyperpolarization activated the hyperpolarization-activated cation current (I(h)). It was found that the major component of I(K) in porcine SAN is the slowly activating I(K) (I(Ks)), in contrast to SAN cells of the rabbit and other species in which the rapid I(K) (I(Kr)) plays an active role in repolarization and the subsequent pacemaker depolarization. Replacement of rabbit I(Kr) with porcine I(Ks) and a slight modification in the gating parameters and amplitudes of other current systems in the 'Kyoto Model' gave an adequate reconstruction of spontaneous action potentials as well as of the voltage clamp recordings. We conclude that the density and the kinetics of I(K) contribute, in part, to the different heart rates of various species.     

Inhomogeneous distribution of action potential characteristics in the rabbit sino-atrial node revealed by voltage imaging

The sino-atrial node (SAN) is the natural pacemaker of the heart. Mechanisms of the leading pacemaker site generation and dynamic pacemaker shifts in the SAN have been so far studied with an electrophysiological technique, but the detailed spatial distribution of action potential characteristics in the SAN has not been analyzed due to the limited number of simultaneously recorded sites in microelectrode recording. To elucidate the mechanism of leading pacemaker site generation in the SAN, we applied a voltage imaging technique and analyzed the spatial distribution of action potential characteristics in the rabbit SAN. Action potential parameters, i.e., action potential duration at 50% repolarization level, the slope of upstroke, and the slope of the linearly depolarizing early phase of pacemaker activity (phase-4), were calculated from optical signals. Action potential parameter values derived from intracellular recording with a microelectrode and those from optical recording were significantly correlated. The leading pacemaker site occurred in the region of either globally or locally maximum phase-4 slope in 7 of 12 preparations, however, it did not coincide with the region of the early maximum phase-4 slope in the other 5 preparations. Carbenoxolone, a gap junction blocker, changed action potential properties and caused pacemaker shifts. Model simulation, assuming an inhomogeneous distribution of intrinsic properties of SAN cells, reproduced the experimental results. We conclude that the functional structure of the SAN is more inhomogeneous than that dictated by previous models. Besides intrinsic cellular properties, cell-to-cell interaction through gap junctions influences action potential characteristics and leading pacemaker site generation.     

Caffeine stimulates the reverse mode of Na/Ca2+ exchanger in ferret ventricular muscle.

This study investigated the effect of caffeine on the sarcolemmal mechanisms involved in intracellular calcium control. Ferret cardiac preparations were treated with ryanodine and thapsigargin in order to eliminate the sarcoplasmic reticulum (SR) function. This treatment abolished caffeine contracture irreversibly in normal solution. The perfusion with K-free medium that blocked the Na+--K+ pump resulted in a recovery of slow relaxing caffeine contractures similar to Na-free contractures. The amplitude of caffeine contractures was dependent on the bathing [caffeine]o and [Ca2+]o. Divalent cations Ni2+ and Cd2+, which have an inhibitory effect on the Na+/Ca2+ exchanger, produced dose-dependent inhibition of caffeine responses with apparent Ki of 780 +/- 19 and 132 +/- 5 microM, respectively. Caffeine also caused dose-dependent inhibition of Na-free contractures (Ki=4.62 +/- 1.5 mM), and the reduction or removal of [Na+]o exerted an inhibitory effect on caffeine contractures (Ki=73.5 +/- 17.12 mM). These experiments indicate that the increase in resting tension following exposure to caffeine was mediated by Na+/Ca2+ exchanger, which represents an additional element of complexity in caffeine action on cardiac muscle.     

Phase response characteristics of sinoatrial node cells

In this work, the dynamic response of the sinoatrial node (SAN), the natural pacemaker of the heart, to short external stimuli is investigated using the Zhang et al. model. The model equations are solved twice for the central cell and for the peripheral cell. A short current pulse is applied to reset the spontaneous rhythmic activity of the single sinoatrial node cell. Depending on the stimulus timing either a delay or an advance in the occurrence of next action potential is produced. This resetting behavior is quantified in terms of phase transition curves (PTCs) for short electrical current pulses of varying amplitude which span the whole period. For low stimulus amplitudes the transition from advance to delay is smooth, while at higher amplitudes abrupt changes and discontinuities are observed in PTCs. Such discontinuities reveal critical stimuli, the application of which can result in annihilation of activity in central SAN cells. The detailed analysis of the ionic mechanisms involved in its resetting behavior of sinoatrial node cell models provides new insight into the dynamics and physiology of excitation of the sinoatrial node of the heart.     

Morphological study of sarcoplasmic reticulum in the atrioventricular node and bundle cells in guinea pig hearts

The osmium-ferrocyanide method for staining of the sarcoplasmic reticulum (SR) was used for a morphological investigation of the various components of the SR in the atrioventricular node and bundle (AVNB) cells of guinea pig hearts. On the basis of light microscopic observations, the AVNB tissue in guinea pig hearts can be divided into five regions: atrionodal junction, midnode, proximal bundle, distal bundle, and bundle branches. Electron microscopic observations revealed two types of junctional SR (j-SR) saccules in the cells from all the regions of AVNB tissue. One is similar to that seen in the working cardiac cells, i. e., flattened saccules with junctonal granules. The second type is dilated and contains electrondense granular material throughout its lumen. The flattened type is seen more often than the dilated type in atrionodal junctional cells and midnode cells, whereas the dilated type occurs more often in distal bundle cells and bundle branch cells. In most cells from the atrionodal junction and midnode regions, the j-SR saccules are apposed more often to sarcolemmal areas associated with nonspecialized regions of intercellular junctions than to other sarcolemmal areas. This distribution was not found in the distal bundle and bundle branch cells. Free SR tubules around the myofilament bundles are poorly developed in the midnode cells, generally in accord with the extent of development of myofibrils. Z-tubules are found in cells from all regions but are poorly developed in midnode cells. Corbular SR vesicles are found in cells from all the regions of AVNB tissues but are rare in midnode cells. Thus, each of the regions in the AVNB tissue has a different, characteristic distribution of SR components. Because of their possible relationship to the regulation of the intracellular concentrations of calcium, these differences in SR morphology may contribute to the diverse physiological properties of the different regions of the AV node and bundle.     

Migration of the true pacemaker within the sinoatrial cell aggregate in man

The functional anatomy of the sinoatrial node (SAN) in man is first reviewed, together with its possible anatomical substructure. The true group pacemaker (PM) shift under autonomic drive is then related to a continuous competition between the intrinsic auto-firing period hierarchy and the autonomic topological susceptibility hierarchy. Accordingly, the PM ‘skip’ to both lower and higher periods following an abrupt and consistent acetylcholine (ACh) release at the SAN periphery, and the PM ‘slip’ towards a relocation of the next period, as a possible response to a slower and smaller ACh release, are considered. The PM ‘skip’ and ‘slip’ as boundaries of the true PM excursion within the SAN during the respiratory cycle, and their statistical properties, are then examined. Under current heart rate control menus in normals, the PM skip appears to follow central influence, whereas the conservative or slipping PMs suggest peripheral control. Finally, interpretation of the PM skip as a salutary sign of functional reserve is proposed, and a method of alleviating the PM skip which confounds electrophysiological testing of the SAN function in patients is devised.     

Modulation of rabbit sinoatrial node activation sequence by acetylcholine and isoproterenol investigated with optical mapping technique

Aims: Changes in the rabbit sinoatrial node (SAN) activation sequence with the cholinergic and adrenergic factors were studied. The correlation between the sinus rhythm rate and the leading pacemaker site shift was determined. The hypothesis concerning the cholinergic suppression of nodal cell excitability as one of the mechanisms associated with pacemaker shift was tested. Methods: A high‐resolution optical mapping technique was used to register beat‐to‐beat changes in the SAN activation pattern under the influence of the cholinergic and adrenergic factors. Results: Acetylcholine (10 μm ) and strong intramural parasympathetic nerve stimulation caused a pacemaker shift as well as rhythmic slowing and the formation of an inexcitable region in the central part of SAN. In this region the generation of action potentials was suppressed. The slowing of the sinus rhythm (which exceeded 12.8 ± 3.1% of the rhythm control rate) always accompanied the pacemaker shift. Isoproterenol (10, 100 nm , 1 μm ) and sympathetic postganglionic nerve stimulation also evoked a pacemaker shift but without formation of an inexcitable zone. The acceleration of the sinus rhythm, which exceeded 10.5 ± 1.3% of the control rate of the rhythm, always accompanied the shift. Conclusions: Both cholinergic and adrenergic factors cause pacemaker shifts in the rabbit SAN. While modest changes in the sinus rhythm do not coincide with the pacemaker shift, greater changes always accompany the shift and may be caused by it, according to one hypothesis. The formation of an inexcitable zone at the place where the leading pacemaker is situated is one of the mechanisms associated with pacemaker shift.