Pathways of abnormal stress-induced Ca2+ influx into dystrophic mdx cardiomyocytes.

In Duchenne muscular dystrophy, deficiency of the cytoskeletal protein dystrophin leads to well-described defects in skeletal muscle, but also to dilated cardiomyopathy, accounting for about 20% of the mortality. Mechanisms leading to cardiomyocyte cell death and cardiomyopathy are not well understood. One hypothesis suggests that the lack of dystrophin leads to membrane instability during mechanical stress and to activation of Ca2+ entry pathways. Using cardiomyocytes isolated from dystrophic mdx mice we dissected the contribution of various putative Ca2+ influx pathways with pharmacological tools. Cytosolic Ca2+ and Na+ signals as well as uptake of membrane impermeant compounds were monitored with fluorescent indicators using confocal microscopy and photometry. Membrane stress was applied as moderate osmotic challenges while membrane current was quantified using the whole-cell patch-clamp technique. Our findings suggest a major contribution of two primary Ca2+ influx pathways, stretch-activated membrane channels and short-lived microruptures. Furthermore, we found evidence for a secondary Ca2+ influx pathway, the Na+-Ca2+ exchange (NCX), which in cardiac muscle has a large transport capacity. After stress it contributes to Ca2+ entry in exchange for Na+ which had previously entered via primary stress-induced pathways, representing a previously not recognized mechanism contributing to subsequent cellular damage. This complexity needs to be considered when targeting abnormal Ca2+ influx as a treatment option for dystrophy.

Ca2+ release from the sarcoplasmic reticulum activated by the low affinity Ca2+ chelator TPEN in ventricular myocytes.

The Ca2+ content of the sarcoplasmic reticulum (SR) of cardiac myocytes is thought to play a role in the regulation and termination of SR Ca2+ release through the ryanodine receptors (RyRs). Experimentally altering the amount of Ca2+ within the SR with the membrane-permeant low affinity Ca2+ chelator TPEN could improve our understanding of the mechanism(s) by which SR Ca2+ content and SR Ca2+ depletion can influence Ca2+ release sensitivity and termination. We applied laser-scanning confocal microscopy to examine SR Ca2+ release in freshly isolated ventricular myocytes loaded with fluo-3, while simultaneously recording membrane currents using the whole-cell patch-clamp technique. Following application of TPEN, local spontaneous Ca2+ releases increased in frequency and developed into cell-wide Ca2+ waves. SR Ca2+ load after TPEN application was found to be reduced to about 60% of control. Isolated cardiac RyRs reconstituted into lipid bilayers exhibited a two-fold increase of their open probability. At the low concentration used (20-40microTPEN did not significantly inhibit the SR-Ca2+-ATPase in SR vesicles. These results indicate that TPEN, traditionally used as a low affinity Ca2+ chelator in intracellular Ca2+ stores, may also act directly on the RyRs inducing an increase in their open probability. This in turn results in an increased Ca2+ leak from the SR leading to its Ca2+ depletion. Lowering of SR Ca2+ content may be a mechanism underlying the recently reported cardioprotective and antiarrhythmic features of TPEN.

Confocal imaging of CICR events from isolated and immobilized SR vesicles.

The Ca2+ concentration inside the sarcoplasmic reticulum ([Ca2+]SR) is a difficult parameter to measure in ventricular cardiac myocytes. Interference from Ca2+-sensitive dye loading into cellular compartments other than the SR interferes with free Ca2+ measurement. In addition, the composition of the cytosol surrounding the SR in intact cells cannot be easily controlled. We have developed a method to measure localized [Ca2+]SR in immobilized membrane vesicles during rapid solution switches. Ca2+ uptake and release in rat SR membrane vesicles was monitored using confocal microscopy. Vesicles were immobilized on a coverslip using an agarose matrix. Perfusion with a Ca2+-containing solution supplemented with ATP initiated SR Ca2+ uptake, causing a rise in intravesicular fluorescence in vesicles containing the low-affinity Ca2+ indicator fluo-5N. Perfusion with caffeine caused SR Ca2+ release and a decrease in intravesicular flourescence. Although caffeine-dependent release was readily visible with extravesicular Ca2+-green, Ca2+ which leaked from the SR was detected only indirectly as eventless release. We conclude that SR Ca2+ uptake and release can be selectively measured in functional SR vesicles using a confocal microscope. Caffeine-dependent release is directly measurable though SR Ca2+ leak can only be inferred as subresolution events, presumably because channels in separate vesicles were not close enough to result in concerted Ca2+-induced Ca2+ release.

Mechanisms of Na+-Ca2+ exchange inhibition by amphiphiles in cardiac myocytes: importance of transbilayer movement.

The membrane lipid environment and lipid signaling pathways are potentially involved in the modulation of the activity of the cardiac Na(+)-Ca(2+) exchanger (NCX). In the present study biophysical mechanisms of interactions of amphiphiles with the NCX and the functional consequences were examined. For this purpose, intracellular Ca(2+) concentration jumps were generated by laser-flash photolysis of caged Ca(2+) in guinea-pig ventricular myocytes and Na(+)-Ca(2+) exchange currents ( I(Na/Ca)) were recorded in the whole-cell configuration of the patch-clamp technique. The inhibitory effect of amphiphiles increased with the length of the aliphatic chain between C(7) and C(10) and was more potent with cationic or anionic head groups than with uncharged head groups. Long-chain cationic amines (C(12)) exhibited a cut-off in their efficacy in I(Na/Ca) inhibition. Analysis of the time-course, comparison with the Ni(2+)-induced I(Na/Ca) block and confocal laser scanning microscopy experiments with fluorescent lipid analogs (C(6)- and C(12)-NBD-labeled analogs) suggested that amphiphiles need to be incorporated into the membrane. Furthermore, NCX block appears to require transbilayer movement of the amphiphile to the inner leaflet ("flip"). We conclude that both, hydrophobic and electrostatic interactions between the lipids and the NCX may be important factors for the modulation by lipids and could be relevant in cardiac diseases where the lipid metabolism is altered.

L-type Ca2+ current as the predominant pathway of Ca2+ entry during I(Na) activation in beta-stimulated cardiac myocytes.

In the present study Ca2+ entry via different voltage-dependent membrane channels was examined with a fluorescent Ca2+ indicator before and after beta-adrenergic stimulation. To clearly distinguish between Ca2+ influx and Ca2+ release from the sarcoplasmic reticulum the Ca2+ store was blocked with 0.1 microM thapsigargin and 10 microM ryanodine. Omitting Na+ from the pipette filling solution minimized Ca2+ entry via Na+-Ca2+ exchange. Individual guinea-pig ventricular myocytes were voltage clamped in the whole-cell configuration of the patch-clamp technique and different membrane currents were activated using specific voltage protocols. The intracellular Ca2+ concentration was simultaneously recorded with a laser-scanning confocal microscope using fluo-3 as a Ca2+ indicator. Ca2+ entry pathways were discriminated using pharmacological blockers under control conditions and during beta-adrenergic stimulation with 1 microM isoproterenol (isoprenaline) in the bathing solution or 100 microM cAMP in the patch-clamp pipette. Isoproterenol or cAMP potentiated the Ca2+ influx signals recorded during L-type Ca2+ current activation but, more interestingly, also during Na+ current (INa) activation. The Ca2+ influx signal arising from L-type Ca2+ current activation was usually blocked by 50 microM Cd2+. However, the Ca2+ influx signal elicited by the Na+ current activation protocol was only curtailed to 56.4 +/- 28.2 % by 100 microM Ni2+ but was reduced to 17.9 +/- 15.1 % by 50 microM Cd2+ and consistently eliminated by 5 mM Ni2+. The pronounced Cd2+ and moderate Ni2+ sensitivity of the Ca2+ influx signals suggested that the predominant source of Ca2+ influx during the Na+ current activation - before and during beta-adrenergic stimulation - was a spurious activation of the L-type Ca2+ current, presumably due to voltage escape during Na+ current activation. Calculations based on the relationship between Ca2+ current and fluorescence change revealed that, on average, we could reliably detect rapid Ca2+ concentration changes as small as 5.4 +/- 0.7 nM. Thus, we can estimate an upper limit for the Ca2+ permeability of the phosphorylated TTX-sensitive Na+ channels which is less than 0.04:1 for Ca2+ ions flowing through Na+ channels via the proposed 'slip-mode' Ca2+ conductance. Therefore the slip-mode Ca2+ conductance of Na+ channels does not contribute noticeably to the Ca2+ signals observed in our experiments.

Paradoxical block of the Na+-Ca2+ exchanger by extracellular protons in guinea-pig ventricular myocytes.

1. The Na+-Ca2+ exchange is a major pathway for removal of cytosolic Ca2+ in cardiac myocytes. It is known to be inhibited by changes of intracellular pH that may occur, for example, during ischaemia. In the present study, we examined whether extracellular protons (pHo) can also affect the cardiac exchange. 2. Na+-Ca2+ exchange currents (INa-Ca) were recorded from single adult guinea-pig ventricular myocytes in the whole-cell voltage-clamp configuration while [Ca2+]i was simultaneously imaged with fluo-3 and a laser-scanning confocal microscope. To activate INa-Ca, intracellular Ca2+ concentration jumps were generated by laser flash photolysis of caged Ca2+ (DM-nitrophen). 3. Exposure of the cell to moderately and extremely acidic conditions (pHo 6 and 4) was accompanied by a decrease of the peak INa-Ca to 70 % and less than 10 %, respectively. The peak INa-Ca was also inhibited to about 45 % of its initial value by increasing pHo to 10. The largest INa-Ca was found at pHo approximately 7.6. 4. Simultaneous measurements of [Ca2+]i and INa-Ca during partial proton block of the Na+-Ca2+ exchanger revealed that the exchange current was more inhibited by acidic pHo than the rate of Ca2+ transport. This observation is consistent with a change in the electrogenicity of the Na+-Ca2+ exchange cycle after protonation of the transporter. 5. We conclude that both extracellular alkalinization and acidification affect the Na+-Ca2+ exchanger during changes of pHo that may be present under pathophysiological conditions. During both extreme acidification or alkalinization the Na+-Ca2+ exchanger is strongly inhibited, suggesting that extracellular protons may interact with the Na+-Ca2+ exchanger at multiple sites. In addition, the electrogenicity and stoichiometry of the Na+-Ca2+ exchange may be modified by extracellular protons.

Calcium signalling in cardiac muscle: refractoriness revealed by coherent activation.

Contraction of cardiac myocytes is governed by calcium-ion (Ca2+ )-induced Ca2+ release (CICR) from the sarcoplasmic reticulum through Ca2+-release channels. Ca2+ release occurs by concerted activation of numerous elementary Ca2+ events, 'Ca2+ sparks', that are triggered and locally controlled by Ca2+ influx into the cell through plasmalemmal L-type Ca2+ channels. Because of the positive feedback inherent in CICR, an as-yet-unidentified control mechanism is required to restrain the amplification of Ca2+ signalling and to terminate Ca2+ release from the sarcoplasmic reticulum. Here we use ultraviolet-laser-flash and two-photon photolysis of caged Ca2+ to study spatiotemporal features of the termination and refractoriness of Ca2+ release. Coherent and simultaneous activation of all Ca2+-release sites within a cardiac myocyte unmasked a prominent refractoriness, recovering monotonically within about 1 second. In contrast, selective activation of a few Ca 2+-release sites was not followed by a refractoriness of Ca 2+ release from the sarcoplasmic reticulum. This discrepancy is consistent with the idea that a functional depletion of Ca2+ from the cellular sarcoplasmic-reticulum network may underlie the refractoriness of CICR observed after a whole-cell Ca2+ transient. These results also imply the requirement for further mechanisms to terminate spatially limited subcellular Ca2+-release events such as Ca2+ sparks.

Ni2+ transport by the human Na+/Ca2+ exchanger expressed in Sf9 cells.

The mechanism of Ni2+ block of the Na+/Ca2+ exchanger was examined in Sf 9 cells expressing the human heart Na+/Ca2+ exchanger (NCX1-NACA1). As predicted from the reported actions of Ni2+, its application reduced extracellular Na+-dependent changes in intracellular Ca2+ concentration (measured by fluo 3 fluorescence changes). However, contrary to expectation, the reduced fluorescence was accompanied by measured 63Ni2+ entry. The 63Ni2+ entry was observed in Sf 9 cells expressing the Na+/Ca2+ exchanger but not in control cells. The established sequential transport mechanism of the Na+/Ca2+ exchanger could be compatible with these results if one of the two ion translocation steps is blocked by Ni2+ and the other permits Ni2+ translocation. We conclude that, because Ni2+ entry was inhibited by extracellular Ca2+ and enhanced by extracellular Na+, the Ca2+ translocation step moved Ni2+, whereas the Na+ translocation step was inhibited by Ni2+. A model is presented to discuss these findings.

Functional expression of the human cardiac Na+/Ca2+ exchanger in Sf9 cells: rapid and specific Ni2+ transport.

Although inhibition of the Na+/Ca2+ exchanger normally increases [Ca2+]i in neonatal cardiac myocytes, application of the inhibitor Ni2+ appears to reduce [Ca2+] measured by fluo-3. To investigate how the apparent reduction in [Ca2+]i occurs we examined Ca2+ transport by the human Na+/Ca2+ exchanger expressed in Sf9 cells. Transport of Ca2+ by the Na+/Ca2+ exchanger was examined using a laser-scanning confocal microscope and the fluorescent Ca2+ indicator fluo-3, and the electrogenic function was determined by measuring the Na+/Ca2+ exchange current (INaCa) using patch clamp methods. INaCa was elicited with voltage-clamp steps or flash photolysis of caged Ca2+. We show significant expression of Na+/Ca2+ exchanger function in Sf9 cells infected with a recombinant Baculovirus carrying the Na+/Ca2+ exchanger. In addition to measurements of INaCa, characterization includes Ca2+ transport via the Na+/Ca2+ exchanger and the voltage dependence of Ca2+ transport. Application of Ni2+ blocked INaCa but, contrary to expectation, decreased fluo-3 fluorescence. Experiments with infected Sf9 cells suggested that Ni2+ was transported via the Na+/Ca2+ exchanger at a rate comparable to the Ca2+ transport. Once inside the cells, Ni2+ reduced fluorescence, presumably by quenching fluo-3. We conclude that Ni2+ does indeed block INaCa, but is also rapidly translocated across the cell membrane by the Na+/Ca2+ exchanger itself, most likely via an electroneutral partial reaction of the exchange cycle.

Two-photon and UV-laser flash photolysis of the Ca2+ cage, dimethoxynitrophenyl-EGTA-4.

We report efficient two-photon and UV-laser flash photolysis of dimethoxynitrophenyl-EGTA-4 (DMNPE-4), a newly-developed photolabile Ca(2+)-specific chelator. This compound exhibits good two-photon absorption at 705 nm, has a low Mg2+ affinity (approximately 7 mM), a Kd for Ca2+ of 19 nM, a quantum yield of 0.20 and changes its Ca2+ affinity by 21,000-fold upon photolysis. Two-photon excitation photolysis (TPP) experiments were performed with a Ti:Sapphire laser in solutions containing DMNPE-4 with either 0 or 10 mM Mg2+ and compared to that of the widely used Ca2+ cage, DM-nitrophen (Kd for Ca2+ 5 nM, Kd for Mg2+ 2.5 microM, quantum yield 0.18, affinity change 600,000-fold). The resulting Ca2+ signals were recorded with the fluorescent Ca2+ indicator fluo-3 and a laser-scanning confocal microscope in the line-scan mode. In vitro, photolysis of DMNPE-4:Ca2+ produced Ca(2+)-release signals that had comparable amplitudes and time courses in the presence and absence of Mg2+. However, photorelease of Ca2+ from DM-nitrophen was obviated by the presence of Mg2+. In patch-clamped isolated cardiac myocytes, equivalent TPP results were obtained in analogous experiments. Single-photon excitation of DMNPE-4 by Nd:YAG laser flashes produced Na-Ca exchange currents of comparable amplitude in the absence and presence of Mg2+. However, only very small currents were observed in DM-nitrophen solution containing 10 mM Mg2+. In conclusion, both DMNPE-4 and DM-nitrophen undergo TPP, however, only DMNPE-4 exhibits efficient release of Ca2+ in the presence of Mg2+.

Localized intracellular calcium signaling in muscle: calcium sparks and calcium quarks.

Subcellularly localized Ca2+ signals in cardiac and skeletal muscle have recently been identified as elementary Ca2+ signaling events. The signals, termed Ca2+ sparks and Ca2+ quarks, represent openings of Ca2+ release channels located in the membrane of the sarcoplasmic reticulum (SR). In cardiac muscle, the revolutionary discovery of Ca2+ sparks has allowed the development of a fundamentally different concept for the amplification of Ca2+ signals by Ca(2+)-induced Ca2+ release. In such a system, a graded amplification of the triggering Ca2+ signal entering the myocyte via L-type Ca2+ channels is accomplished by a recruitment process whereby individual SR Ca2+ release units are locally controlled by L-type Ca2+ channels. In skeletal muscle, the initial SR Ca2+ release is governed by voltage-sensors but subsequently activates additional Ca2+ sparks by Ca(2+)-induced Ca2+ release from the SR. Results from studies on elementary Ca2+ release events will improve our knowledge of muscle Ca2+ signaling at all levels of complexity, from the molecule to normal cellular function, and from the regulation of cardiac and skeletal muscle force to the pathophysiology of excitation-contraction coupling.

Erythropoietin modulates intracellular calcium in a human neuroblastoma cell line.

1. Recent investigations have shown that the glycoprotein erythropoietin (Epo) and its specific receptor (EpoR) are present in the mammalian brain including human, monkey and mouse. These findings suggest a local action of Epo in the nervous system. The aim of this study was to elucidate a possible functional interaction of Epo with neuronal cells. 2. To examine the influence of externally applied Epo on Ca2+ homeostasis the human neuroblastoma cell line SK-N-MC was chosen as a suitable in vitro model for undifferentiated neuronal cells. 3. Expression of the EpoR in SK-N-MC cells was detected by reverse transcription-PCR, Western blot and immunofluorescence analysis. 4. Patch-clamp studies of SK-N-MC cells confirmed the expression of T-type Ca2+ channels, whose peak macroscopic current was increased by the addition of recombinant human Epo (rhEpo) to the bathing medium. 5. Confocal laser scanning microscopy analysis of SK-N-MC cells confirmed a transient increase in intracellular free [Ca2+] in response to externally applied rhEpo. 6. The transient response to Epo was dependent on external Ca2+ and remained even after depletion of internal Ca2+ stores by caffeine or thapsigargin. However, after depletion the response to Epo was absent when cells were superfused with the T-type Ca2+ channel blocker flunarizine. 7. This study demonstrates that Epo can interact with neuronal cells by affecting Ca2+ homeostasis through an increase in Ca2+ influx via plasma membrane T-type voltage-dependent Ca2+ channels.

Confocal near-membrane detection of calcium in cardiac myocytes.

Near-membrane [Ca2+] may differ significantly from bulk cytosolic [Ca2+], particularly during rapid Ca2+ signalling events related to cardiac muscle excitation-contraction coupling. We used the lipophilic membrane-associated Ca2+ indicator Ca(2+)-Green C-18 (C-18) and laser-scanning confocal microscopy to detect extracellular [Ca2+] and changes of t-tubular [Ca2+] in cultured neonatal rat myocytes and in freshly isolated adult guinea pig ventricular myocytes. Changes of extracellular [Ca2+] were readily detected by the C-18 located in the cell membrane. Control experiments were carried out with 100 mM extracellular nickel to rapidly quench the fluorescent indicator accessible form the extracellular space. After exposure to Ni2+, C-18 fluorescence was lower than measured in Ca(2+)-free conditions indicating that C-18 was located in the outer leaflet of the cell membrane. In contrast, the lipophilic derivative of Indo-1 (FIP-18) was significantly internalized, as visualized using two-photon excitation of FIP-18. Surprisingly, in low extracellular [Ca2+], C-18 located in the outer leaflet of the cell membrane also reported transient elevations of intracellular [Ca2+] during application of 10 mM caffeine. In the absence of extracellular Na+ to inhibit Ca2+ removal via Na/Ca exchange, the intracellular Ca2+ signals evoked by caffeine were prolonged, as recorded with Fura-Red. However, the near-membrane Ca2+ signal simultaneously detected by C-18 did not increase during caffeine stimulation in the absence of extracellular Na+. These results suggest that the C-18 signal reports extrusion of cytosolic Ca2+ from the subsarcolemmal space mediated by Na/Ca exchange. C-18 was also used to analyze the extracellular accessibility of the t-tubular lumen in isolated guinea pig ventricular myocytes. After stepwise increases of [Ca2+]o with a rapid superfusion device, a wave-like Ca2+ gradient travelled along the t-tubules at a velocity of 3.4-16.3 microns/s. The solution change within the t-tubules was delayed by 0.63-2.3 s and wash-out of Ca2+ from the t-tubules slowed from t1/2 = 0.9 s at the surface to 1.7 s in deeper regions of the t-tubular system. This slow exchange of the solution within the t-tubules, lasting several seconds, may give rise to spatially inhomogeneous accumulation and/or depletion resulting from ion fluxes across the t-tubular membrane during physiological activity.

Fundamental calcium release events revealed by two-photon excitation photolysis of caged calcium in Guinea-pig cardiac myocytes.

1. In cardiac muscle, 'Ca2+ sparks' have been proposed to underlie Ca2+-induced Ca2+ release (CICR), and to result from openings of clusters of Ca2+ channels (ryanodine receptors; RyRs) located in the sarcoplasmic reticulum membrane. 2. To investigate the elementary nature of these Ca2+ signals directly, a diffraction-limited point source of Ca2+ was created in single cardiac myocytes by two-photon excitation photolysis of caged Ca2+. Simultaneously, concentration profiles of released Ca2+ were imaged at high temporal and spatial resolution with a laser-scanning confocal microscope. 3. This approach enabled us to generate and detect photolytic Ca2+ signals that closely resembled the Ca2+ sparks occurring naturally, not only in amplitude and size, but also in their ability to trigger additional Ca2+ sparks or Ca2+ waves. 4. Surprisingly, at low photolytic power minuscule events with estimated Ca2+ release fluxes 20-40 times smaller than those calculated for a typical Ca2+ spark were directly resolved. These events appeared to arise from the opening of a more limited number of RyRs (possibly one) or from RyRs exhibiting a different gating mode and may correspond to the elusive 'Ca2+ quark'. 5. The Ca2+ quark represents the fundamental Ca2+ release event of excitable cells implementing hierarchical Ca2+ signalling systems with Ca2+ release events of various but distinct amplitude levels (i.e. Ca2+ quarks, Ca2+ sparks and full cellular Ca2+ transients). 6. A graded recruitment of nanoscopic Ca2+ release domains (i.e. Ca2+ quarks) exhibiting variable degrees of spatial coherence and coupling may then build up intermediate Ca2+ signalling events (i.e. Ca2+ sparks). This mechanism suggests the existence of Ca2+ sparks caused by gating of a variable fraction of RyRs from within an individual cluster. Additional mobilization of a variable number of these Ca2+ sparks enables cardiac cells to show graded cellular Ca2+ transients. Similar recruitment processes may underlie regulation of Ca2+ signalling on the cellular level in general.

[Insufficient understanding of second tumors after childhood neoplasms in Switzerland]. / Ungenügende Erfassung von Zweittumoren nach Neoplasien im Kindesalter in der Schweiz.

Second malignancy after childhood neoplasms is a well-known complication. However, frequency differs considerably according to the types of primary neoplasm and the specifics of therapy. Ten patients with a second malignancy after being cured of the primary tumor are described. There were 2 patients with acute lymphoblastic leukemia, one with non-Hodgkin's lymphoma, and one with breast cancer after Hodgkin's disease. Two patients with heritable retinoblastoma developed osteosarcomas in the irradiation field after a latent period of 7 and 14 years respectively. There was another osteosarcoma in a Wilms' tumor survivor. One patient with acute lymphoblastic leukemia developed a secondary AML 10 years after achieving initial remission, and a meningioma was diagnosed in another patient with cured acute lymphoblastic leukemia. One patient died of peritoneal sarcomatosis of unknown origin 20 years after the diagnosis of acute myeloid leukemia. All patients received radiotherapy for the primary neoplasms. Secondary neoplasms in other patients were probably missed because they occurred in adulthood when the patients were transferred to other medical centres. It is impossible to trace these patients because central registration of patients with neoplasms is lacking. It is therefore important to establish a central cancer registry for the whole of Switzerland. Second malignancy after childhood cancer is not a rare event and requires long-term follow-up of patients with neoplasms.

Imaging the hierarchical Ca2+ signalling system in HeLa cells.

1. Confocal microscopy was used to investigate hormone-induced subcellular Ca2+ release signals from the endoplasmic reticulum (ER) in a prototype non-excitable cell line (HeLa cells). 2. Histamine application evoked two types of elementary Ca2+ signals: (i) Ca2+ blips arising from single ER Ca2+ release channels (amplitude, 30 nM; lateral spreading, 1.3 microns); (ii) Ca2+ puffs resulting from the concerted activation of several Ca2+ blips (amplitude, 170 nM; spreading, 4 microns). 3. Ca2+ waves in the HeLa cells arose from a variable number of initiation sites, but for individual cells, the number and subcellular location of the initiation sites were constant. The kinetics and amplitude of global Ca2+ signals were directly proportional to the number of initiation sites recruited. 4. Reduction of the feedback inherent in intracellular Ca2+ release caused saltatoric Ca2+ waves, revealing the two principal steps underlying wave propagation: diffusion and regeneration. Threshold stimulation evoked abortive Ca2+ waves, caused by the limited recruitment of Ca2+ puffs. 5. The hierarchy of Ca2+ signalling events, from fundamental levels (blips) to intermediate levels (puffs) to Ca2+ waves, is a prototype for Ca2+ signal transduction for non-excitable cells, and is also analogous to the Ca2+ quarks, Ca2+ sparks and Ca2+ waves in cardiac muscle cells.

Spatially non-uniform Ca2+ signals induced by the reduction of transverse tubules in citrate-loaded guinea-pig ventricular myocytes in culture.

1. Ratiometric confocal microscopy and the whole-cell patch clamp technique were used to simultaneously record intracellular Ca2+ transients and membrane currents from guinea-pig ventricular myocytes. Intracellular dialysis with the low-affinity Ca2+ buffer citrate enabled us to record and analyse Ca2+ transients caused by Ca2+ influx alone and by additional Ca2+ release from the sarcoplasmic reticulum (SR) in the same cell. 2. In freshly isolated adult myocytes (used within 1-4 h of isolation) both types of Ca2+ transients ('Ca2+ entry' and 'Ca2+ release' transients) were spatially uniform regardless of the Ca2+ current (ICa) duration. In contrast, Ca2+ transients in short-term cultured (1-2 days) myocytes exhibited marked spatial inhomogeneities. ICa frequently evoked Ca2+ waves that propagated from either or both ends of the cardiac myocyte. Reduction of the ICa duration caused Ca2+ release that was restricted to one of the two halves of the cell. 3. Analysis of the Ca2+ entry signals in freshly isolated and short-term cultured myocytes indicated that the spatial properties of the Ca2+ influx signal were responsible for the spatial properties of the triggered Ca2+ release from the SR. In freshly isolated ventricular myocytes Ca2+ influx was homogeneous while in short-term cultured cells pronounced Ca2+ gradients could be found during Ca2+ influx. Spatial non-uniformities in the amplitude of local Ca2+ entry transients were likely to cause subcellularly restricted Ca2+ release. 4. The changes in the spatial properties of depolarization-induced Cai2+ signals during short-term culture were paralleled by a decrease (to 65%) in the total cell capacitance. In addition, staining the sarcolemma with the membrane-selective dye Di-8-ANEPPS revealed that, in cultured myocytes, t-tubular membrane connected functionally to the surface membrane was reduced or absent. 5. These results demonstrate that the short-term culture of adult ventricular myocytes results in the concomitant loss of functionally connected t-tubular membrane. The lack of the t-tubular system subsequently caused spatially non-uniform SR Ca2+ release. Evidence is presented to show that in ventricular myocytes lacking t-tubules non-uniform SR Ca2+ release was, most probably, the result of inhomogeneous Ca2+ entry during ICa. These findings directly demonstrate the functional importance of the t-tubular network for uniform ventricular Ca2+ signalling.

Subcellular properties of triggered Ca2+ waves in isolated citrate-loaded guinea-pig atrial myocytes characterized by ratiometric confocal microscopy.

1. Spatiotemporal aspects of subcellular Ca2+ signalling were studied in cultured adult guinea-pig atrial myocytes. A mixture of the Ca2+ indicators fluo-3 and Fura Red in combination with laser-scanning confocal microscopy was used for [Ca2+]i measurements while membrane currents were recorded simultaneously. 2. In citrate-loaded atrial myocytes not every Ca2+ current (ICa) could trigger Ca2+ release from the sarcoplasmic reticulum (SR). Two types of Ca2+ signals could be observed: Ca2+ transients resulting from (i) Ca2+ influx alone and (ii) additional Ca2+ release. 3. Ca2+ release elicited by voltage steps of 100-150 ms duration was either apparently homogeneous or propagated as Ca2+ waves through the entire cell. With brief ICa (50-75 ms), Ca2+ waves with limited subcellular propagation were observed frequently. These waves always originated from either end of the myocyte. 4. The time course of changes in Na(+)-Ca2+ exchange current (INaCa) depended on the subcellular properties of the underlying Ca2+ transient and on the particular cell geometry. Apparently homogeneous Ca2+ release was accompanied by an inward change of INaCa the onset phase of which was fused with ICa. Changes in INaCa caused by a Ca2+ wave propagating through the entire cell showed a W shape, which could be attributed to differences of the fractional surface-to-volume ratio in different cell segments during propagation of the Ca2+ wavefront. Those waves with limited spreading only activated a small component of INaCa. 5. The different subcellular patterns of Ca2+ release signals can be explained by spatial inhomogeneities in the positive feedback of the SR. This depends on the local SR Ca2+ loading state under the control of the local Ca2+ influx during activation of ICa. Due to the higher surface-to-volume ratio at the two ends of the myocyte, SR loading and therefore the positive feedback in Ca(2+)-induced Ca2+ release may be higher at the ends, locations where Ca2+ waves are preferentially triggered. 6. We conclude that the individual cell geometry may be an important determinant of subcellular Ca2+ signalling not only in cardiac muscle cells but presumably also in other types of cells that depend on Ca2+ signalling. In addition, the cell geometry in combination with varying subcellular Ca2+ release patterns can greatly affect the time course of Ca(2+)-activated membrane currents.