Marcel Egger

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.

Calcium signalling in cardiac muscle: refractoriness revealed by coherentactivation

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 Ca2+-release sites was not followed by a refractoriness of Ca2+ 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.

Sarcoplasmic reticulum Ca2+ refilling controls recovery from Ca2+-induced Ca2+ release refractoriness in heart muscle

In cardiac muscle Ca2+-induced Ca2+ release (CICR) from the sarcoplasmic reticulum (SR) is initiated by Ca2+ influx via L-type Ca2+ channels. At present, the mechanisms underlying termination of SR Ca2+ release, which are required to ensure stable excitation-contraction coupling cycles, are not precisely known. However, the same mechanism leading to refractoriness of SR Ca2+ release could also be responsible for the termination of CICR. To examine the refractoriness of SR Ca2+ release, we analyzed Na+-Ca2+ exchange currents reflecting cytosolic Ca2+ signals induced by UV-laser flash-photolysis of caged Ca2+. Pairs of UV flashes were applied at various intervals to examine the time course of recovery from CICR refractoriness. In cardiomyocytes isolated from guinea-pigs and mice, &bgr;-adrenergic stimulation with isoproterenol-accelerated recovery from refractoriness by ≈2-fold. Application of cyclopiazonic acid at moderate concentrations (<10 &mgr;mol/L) slowed down recovery from refractoriness in a dose-dependent manner. Compared with cells from wild-type littermates, those from phospholamban knockout (PLB-KO) mice exhibited almost 5-fold accelerated recovery from refractoriness. Our results suggest that SR Ca2+ refilling mediated by the SR Ca2+-pump corresponds to the rate-limiting step for recovery from CICR refractoriness. Thus, the Ca2+ sensitivity of CICR appears to be regulated by SR Ca2+ content, possibly resulting from a change in the steady-state Ca2+ sensitivity and in the gating kinetics of the SR Ca2+ release channels (ryanodine receptors). During Ca2+ release, the concomitant reduction in Ca2+ sensitivity of the ryanodine receptors might also underlie Ca2+ spark termination by deactivation.

Regulatory Function of Na-Ca Exchange in the Heart: Milestones and Outlook

The Na/Ca exchange is a countertransport mechanism located in the cell membrane of almost every mammalian cell type. It can transport Ca 2+ across the membrane and against the electrochemical gradient for Ca 2+ by utilizing the electrochemical gradient for Na . Thirty years ago a Na/Ca exchange mechanism was identified in the squid giant axon and in heart muscle (Baker et al., 1968; Reuter & Seitz, 1968). By measuring Ca efflux evidence for a Na + and Ca countertransport system was found (Reuter & Seitz, 1968). Interestingly, an interdependence of Na + and Ca had been known for some time before these crucial experiments were carried out. Several studies had reported that contractility in the heart depended on the ratio of Ca : Na in the experimental solution. Before the exchange mechanism had been discovered, these observations were interpreted to mean some form of antagonism between Na + and Ca, possibly competition for a common receptor (Wilbrandt & Koller, 1948; Lüttgau & Niedergerke, 1958). Meanwhile, our knowledge about the function of the Na /Ca exchange in the heart and other tissues has dramatically increased, aided by the development and application of new methods and techniques to investigate this transporter. Several excellent books and reviews cover many aspects of Na /Ca exchange (Carafoli, 1985; Eisner & Lederer, 1985; Allen, Noble & Reuter, 1989; Blaustein, DiPolo & Reeves, 1991; Philipson, Nicoll & Li, 1993; Hilgemann, Philipson & Vassort, 1996; Khananshvili, 1998). The purpose of this review is to present an introductory overview for readers entering the field of Na /Ca exchange research. The emphasis is on the role of the Na/Ca exchanger in cardiac Ca 2+ signaling and its contribution to membrane current in cardiac myocytes. In addition, we point out several ongoing controversies as well as recent developments that promise to provide new approaches for carrying Na /Ca exchange research into the next millennium and, ultimately, from the “molecule to the bedside”. After the identification of the Na /Ca exchange mechanism research initially concentrated on the steadystate kinetics, the stoichiometry and electrogenicity of this transporter (e.g., Baker et al., 1969; Blaustein & Bantiago, 1977; Horackova & Vassort, 1979; Mullins, 1984). Important results were obtained with the voltageclamp technique in multicellular cardiac preparations but also with sarcolemmal vesicle preparations (Philipson, Behrson & Nishimoto, 1982; Reeves & Poronnik, 1987). Today, an electrogenic stoichiometry of 3 Na + : 1 Ca is generally accepted (Reeves & Hale, 1984; Eisner & Lederer, 1985). Several early studies were carried out to investigate the influence of Na /Ca exchange on cardiac contraction and relaxation. Slow and tonic contractions were found to be mediated by the Na /Ca exchange running in the Ca 2+ influx mode (for review see Eisner & Lederer, 1985) and the importance of this transporter for Ca removal and as a modulator for twitch tension was recognized (O’Neill, Valdeolmillos & Eisner, 1988; Bers & Bridge, 1989). With the advent of the patch-clamp technique, the current generated by the Na / Correspondence to: M. Egger

Increased Exchange Current but Normal Ca2+ Transport via Na+-Ca2+ Exchange During Cardiac Hypertrophy After Myocardial Infarction

Abstract— Hypertrophied and failing cardiac myocytes generally show alterations in intracellular Ca2+ handling associated with changes in the contractile function and arrhythmogenicity. The cardiac Na+-Ca2+ exchange (NCX) is an important mechanism for Ca2+ extrusion and cell relaxation. Its possible involvement in changes of excitation-contraction coupling (EC-coupling) with disease remains uncertain. We analyzed the NCX function in rat ventricular myocytes 5 to 6 months after experimental myocardial infarction (PMI) produced by left coronary artery ligation and from sham-operated (SO) hearts. Caged Ca2+ was dialyzed into the cytoplasm via a patch-clamp pipette and Ca2+ was released by flash photolysis to activate NCX and measure the associated currents (INaCa), whereas [Ca2+]i changes were simultaneously recorded with a confocal microscope. INaCa density normalized to the [Ca2+]i jumps was 2.6-fold higher in myocytes from PMI rats. The level of total NCX protein expression in PMI myocytes was also increased. Interestingly, although the INaCa density in PMI cells was larger, PMI and SO myocytes presented virtually identical Ca2+ transport via the NCX. This discrepancy was explained by a reduced surface/volume ratio (34.8%) observed in PMI cells. We conclude that the increase in NCX density may be a mechanism to maintain the required Ca2+ extrusion from a larger cell to allow adequate relaxation.

Spatiotemporal features of Ca2+ buffering and diffusion in atrial cardiac myocytes with inhibited sarcoplasmic reticulum.

Ca(2+) signaling in cells is largely governed by Ca(2+) diffusion and Ca(2+) binding to mobile and stationary Ca(2+) buffers, including organelles. To examine Ca(2+) signaling in cardiac atrial myocytes, a mathematical model of Ca(2+) diffusion was developed which represents several subcellular compartments, including a subsarcolemmal space with restricted diffusion, a myofilament space, and the cytosol. The model was used to quantitatively simulate experimental Ca(2+) signals in terms of amplitude, time course, and spatial features. For experimental reference data, L-type Ca(2+) currents were recorded from atrial cells with the whole-cell voltage-clamp technique. Ca(2+) signals were simultaneously imaged with the fluorescent Ca(2+) indicator Fluo-3 and a laser-scanning confocal microscope. The simulations indicate that in atrial myocytes lacking T-tubules, Ca(2+) movement from the cell membrane to the center of the cells relies strongly on the presence of mobile Ca(2+) buffers, particularly when the sarcoplasmic reticulum is inhibited pharmacologically. Furthermore, during the influx of Ca(2+) large and steep concentration gradients are predicted between the cytosol and the submicroscopically narrow subsarcolemmal space. In addition, the computations revealed that, despite its low Ca(2+) affinity, ATP acts as a significant buffer and carrier for Ca(2+), even at the modest elevations of [Ca(2+)](i) reached during influx of Ca(2+).

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≈ 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.

Synthesis and Two-photon Photolysis of 6-(ortho-Nitroveratryl)-Caged IP3 in Living Cells

The synthesis of a photolabile derivative of inositol‐1,4,5‐trisphosphate (IP3) is described. This new caged second messenger (6‐ortho‐nitroveratryl)‐IP3 (6‐NV‐IP3) has an extinction coefficient of 5000 M−1 cm−1 at 350 nm, and a quantum yield of photolysis of 0.12. Therefore, 6‐NV‐IP3 is photolyzed with UV light about three times more efficiently than the widely used P4(5)‐1‐(2‐nitrophenyl)ethyl‐caged IP3 (NPE‐IP3). 6‐NV‐IP3 has a two‐photon cross‐section of about 0.035 GM at 730 nm. This absorbance is sufficiently large for effective two‐photon excitation in living cells at modest power levels. Using near‐IR light (5 mW, 710 nm, 80 MHz, pulse‐width 70 fs), we produced focal bursts of IP3 in HeLa cells, as revealed by laser‐scanning confocal imaging of intracellular Ca2+ concentrations. Therefore, 6‐NV‐IP3 can be used for efficient, subcellular photorelease of IP3, not only in cultured cells but also, potentially, in vivo. It is in the latter situation that two‐photon photolysis should reveal its true forte.

Spatial characteristics of sarcoplasmic reticulum Ca2+ release events triggered by L-type Ca2+ current and Na+ current in guinea-pig cardiac myocytes.

Ca2+ signals in cardiac muscle cells are composed of spatially limited elementary events termed Ca2+ sparks. Several studies have also indicated that Ca2+ signals smaller than Ca2+ sparks can be elicited. These signals have been termed Ca2+ quarks and were proposed to result from the opening of a single Ca2+ release channel of the sarcoplasmic reticulum. We used laser‐scanning confocal microscopy to examine the subcellular properties of Na+ current (INa)‐ and L‐type Ca2+ current (ICa,L)‐induced Ca2+ transients in voltage‐clamped ventricular myocytes isolated from guinea‐pigs. Both currents, INa and ICa,L, evoked substantial, global Ca2+ transients. To examine the spatiotemporal properties of such Ca2+ signals, we performed power spectral analysis of these Ca2+ transients and found that both lacked spatial frequency components characteristic for Ca2+ sparks. The application of 10 μm verapamil to partially block L‐type Ca2+ current reduced the corresponding Ca2+ transients down to individual Ca2+ sparks. In contrast, INa‐induced Ca2+ responses were still spatially homogeneous and lacked Ca2+ sparks even for small current amplitudes. By using high resistance patch pipettes (> 4 MΩ) to exaggerate the loss of voltage control during INa, Ca2+ sparks appeared superimposed on a homogeneous Ca2+ release component and were exclusively triggered during the flow of INa. In the presence of 10 μm ryanodine both ICa,L and INa elicited small, residual Ca2+ transients that were spatially homogeneous but displayed distinctively different temporal profiles. We conclude that INa is indeed able to cause Ca2+ release in guinea‐pig ventricular myocytes. In contrast to ICa,L‐induced Ca2+ transients, which are built up from the recruitment of individual Ca2+ sparks, the INa‐evoked cellular responses were always homogeneous, indicating that their underlying elementary Ca2+ release event is distinct from the Ca2+ spark. Thus, INa‐induced Ca2+ transients are composed of smaller Ca2+ signals, most likely Ca2+ quarks.

Angiotensin II–Mediated Adaptive and Maladaptive Remodeling of Cardiomyocyte Excitation–Contraction Coupling

Cardiac hypertrophy is associated with alterations in cardiomyocyte excitation–contraction coupling (ECC) and Ca2+ handling. Chronic elevation of plasma angiotensin II (Ang II) is a major determinant in the pathogenesis of cardiac hypertrophy and congestive heart failure. However, the molecular mechanisms by which the direct actions of Ang II on cardiomyocytes contribute to ECC remodeling are not precisely known. This question was addressed using cardiac myocytes isolated from transgenic (TG1306/1R [TG]) mice exhibiting cardiac specific overexpression of angiotensinogen, which develop Ang II–mediated cardiac hypertrophy in the absence of hemodynamic overload. Electrophysiological techniques, photolysis of caged Ca2+ and confocal Ca2+ imaging were used to examine ECC remodeling at early (≈20 weeks of age) and late (≈60 weeks of age) time points during the development of cardiac dysfunction. In young TG mice, increased cardiac Ang II levels induced a hypertrophic response in cardiomyocyte, which was accompanied by an adaptive change of Ca2+ signaling, specifically an upregulation of the Na+/Ca2+ exchanger–mediated Ca2+ transport. In contrast, maladaptation was evident in older TG mice, as suggested by reduced sarcoplasmic reticulum Ca2+ content resulting from a shift in the ratio of plasmalemmal Ca2+ removal and sarcoplasmic reticulum Ca2+ uptake. This was associated with a conserved ECC gain, consistent with a state of hypersensitivity in Ca2+-induced Ca2+ release. Together, our data suggest that chronic elevation of cardiac Ang II levels significantly alters cardiomyocyte ECC in the long term, and thereby contractility, independently of hemodynamic overload and arterial hypertension.