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.

Voltage-independent calcium release in heart muscle.

The Ca2+ that activates contraction in heart muscle is regulated as in skeletal muscle by processes that depend on voltage and intracellular Ca2+ and involve a positive feedback system. How the initial electrical signal is amplified in heart muscle has remained controversial, however. Analogous protein structures from skeletal muscle and heart muscle have been identified physiologically and sequenced; these include the Ca2+ channel of the sarcolemma and the Ca2+ release channel of the sarcoplasmic reticulum. Although the parallels found in cardiac and skeletal muscles have provoked valuable experiments in both tissues, separation of the effects of voltage and intracellular Ca2+ on sarcoplasmic reticulum Ca2+ release in heart muscle has been imperfect. With the use of caged Ca2+ and flash photolysis in voltage-clamped heart myocytes, effects of membrane potential in heart muscle cells on Ca2+ release from intracellular stores have been studied. Unlike the response in skeletal muscle, voltage across the sarcolemma of heart muscle does not affect the release of Ca2+ from the sarcoplasmic reticulum, suggesting that other regulatory processes are needed to control Ca2(+)-induced Ca2+ release.

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.

Sodium/calcium exchanger in heart muscle: molecular biology, cellular function, and its special role in excitation-contraction coupling.

The Na/Ca exchanger has been examined with respect to its molecular biology, its cellular function, and its role in excitation-contraction coupling. The Na/Ca exchanger plays a central part in excitation-contraction coupling, setting the level of sarcoplasmic reticular calcium and contributing to the triggering of sarcoplasmic reticular calcium release. Functional biophysical studies with isolated single cells and caged calcium provide evidence that the Na/Ca exchanger works as a two step sequential transporter. In the heart there are about 250 exchangers.mu-2, operating at a turnover rate of up to about 2500.s-1, with the exchanger carrying -2.56 charges under normal conditions. The Na/Ca exchanger has been recently cloned from diverse mammalian species and several tissues and is largely conserved. It is clear, however, that the function of the Na/Ca exchanger is different in the different tissues. Thus work is in progress in several laboratories, including ours, to determine how the Na/Ca exchanger achieves its tissue specific function. Several modulatory motifs have been seen in studies of the exchanger that may explain some of the tissue specific differences. Interestingly the modulation of the Na/Ca exchanger (for example, by protons, sodium, calcium, ATP, calmodulin) seems to arise from interactions with the intracellular loop.

Ratiometric confocal Ca(2+)-measurements with visible wavelength indicators in isolated cardiac myocytes.

We present a new method for ratiometric Ca2+ measurements using indicators with excitation spectra in the visible range of wavelengths. Laser-scanning confocal microscopy was used to record intracellular Ca(2+)-signals with high temporal and spatial resolution in single cardiac myocytes. The patch-clamp technique was applied to load the cells with the fluorescent Ca(2+)-indicators and to follow the membrane currents with the fluorescence signals simultaneously. Intracellular free Ca(2+)-concentration ([Ca2+]i) was estimated with a ratiometric method. An in vitro calibration procedure was used to convert the fluorescence ratio obtained with two different Ca(2+)-indicators (Fluo-3 and Fura-Red) into Ca(2+)-concentrations. Fluo-3 showed an increase in fluorescence upon a rise in intracellular Ca(2+)-concentration, while the Fura-Red fluorescence decreased. Since the fluorescence of Fluo-3 was around 2-fold brighter than the Fura-Red signal the cells were loaded with a 1:2 mixture of the two indicators. The large increase of the fluorescence ratio during a rise in [Ca2+]i (up to 4-fold) allowed us to record time-resolved signals with this mixture even when monitored in a very small subcellular volume (around 1 micron3). Long lasting continuous recordings of the fluorescence were possible because the dye-mixture exhibited no detectable bleaching with illumination periods of up to 30 s. The use of the Fluo-3/Fura-Red ratio method should significantly facilitate and improve quantitative measurements of [Ca2+]i with high temporal and spatial resolution. Moreover, this approach is especially valuable when used with confocal microscopes which are usually equipped with lasers in the visible light range. Furthermore, it may be possible to use the same approach with mixtures of other indicators to estimate the concentration of other biologically important ions/compounds with a ratiometric calibration.

Photolysis of caged compounds characterized by ratiometric confocal microscopy: a new approach to homogeneously control and measure the calcium concentration in cardiac myocytes.

Here we describe the subcellularly uniform control of the intracellular Ca2+ concentration ([Ca2+]i) by flash photolysis of caged Ca2+ or a caged Ca2+ buffer. A mixture of the two Ca2+ indicators Fluo-3 and Fura-red was used together with a laser-scanning confocal microscope to reveal spatial aspects of intracellular Ca2+ signals. The patch clamp technique in the whole-cell variant was applied to load the cells with the indicator mixture together with either DM-nitrophen or diazo-2 and to measure changes in the membrane current. An in vivo calibration was performed to convert the Fluo-3/Fura-red fluorescence ratios to [Ca2+] values. The resulting calibration curve suggested an apparent KD of 1.6 microM, Rmax of 2.15, Rmin of 0.08 and a Hill-coefficient of 0.75 for the indicator mixture. Controlled rupture of the cell membrane revealed a large fraction of immobile intracellular Fura-red fluorescence that may account for the reduced in vivo Rmax value when compared to the in vitro value of 3.1. In cardiac myocytes, flash photolytic release of Ca2+ from DM-nitrophen generated inwardly directed Na+/Ca2+ exchange currents and Ca2+ signals that were graded with the discharged flash-energy. Rapid line-scans revealed subcellularly homogeneous [Ca2+] jumps regardless of the discharged flash energy. Ca2+ signals evoked by L-type Ca2+ currents (ICa) could be terminated rapidly in a spatially homogeneous manner by UV flash photolysis of diazo-2. No side-effects of the photolytic products of DM-nitrophen or diazo-2 with the mixture of Fluo-3/Fura-red were detectable in our experiments. The combination of UV flash photolysis and laser scanning confocal microscopy enabled us to control [Ca2+]i homogeneously on the subcellular level. This approach may improve our understanding of the subcellular properties of cardiac Ca2+ signalling. The technique can also be applied in other cell types and with other signalling systems for which caged compounds are available.

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.

Real-time confocal microscopy and calcium measurements in heart muscle cells: towards the development of a fluorescence microscope with high temporal and spatial resolution.

Preliminary experiments and characterization of a modified confocal fluorescence microscope have been carried out and are presented in this article. We have made use of commercially available hardware and software (having modified and extended the system) and are continuing the process of making modifications to this system to enable us to carry out investigations in living and mobile cells. We report on identified problems in measuring intracellular calcium in myocardial cells, important lessons that have been learned and new findings regarding myocardial cells. Specifically, we have found that myocardial cellular organelles (e.g. nuclei and mitochondria) are sharply defined unlike images obtained with standard epifluorescence microscopes using intracellular indicators. Furthermore, we show that the organelles can accumulate or largely exclude certain indicators relative to the concentration in the cytosol. Additionally, we have examined the use of the new calcium indicator fluo-3 in contracting heart cells and have more clearly defined certain limitations in its use in this preparation. We are working on modifications of the present equipment to enable the system to work with an ultraviolet laser as a light source. The confocal microscope offers the prospect of extraordinarily good spatial and temporal resolution under specific conditions in heart cells.

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+.

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.

Specific inhibition of Na-Ca exchange function by antisense oligodeoxynucleotides.

The Na-Ca exchanger is essential for the Ca2+ homeostasis in many cell types. This transporter has been difficult to investigate because no specific inhibitor is available. We have synthesized an antisense oligodeoxynucleotide directed against the rat cardiac Na-Ca exchanger mRNA. To estimate the activity of the Na-Ca exchange in single cultured myocytes, the exchange current (INaCa) was measured with the voltage-clamp technique while the intracellular Ca2+ concentration ([Ca2+]i) was simultaneously recorded. Most cells exposed to antisense oligodeoxynucleotide showed neither an INaCa nor an increase of [Ca2+]i upon extracellular Na+ removal. Liberation of Ca2+ by flashphotolysis of caged Ca2+ was not followed by a decay of [Ca2+]i in cells exposed to the antisense oligonucleotide, whereas in control cells resting [Ca2+]i was reached 6 s after the flash. Control experiments with non-sense and mismatched oligonucleotides were performed to exclude unspecific inhibitory effects. These results demonstrate that the Na-Ca exchange was specifically and completely suppressed and that antisense oligodeoxynucleotides represent a useful tool to investigate the cellular and molecular properties of the Na-Ca exchanger.

[Automatic over-all and detail photography of autopsies]. / Automatische Ubersichts- und Detailfotografie bei Autopsien.

Due to lack of time or skill of the staff involved important autopsy findings are often not photographically documented. In order to overcome this deficiency an automatic photographic device was constructed consisting of one horizontally movable camera on the ceiling with a normal range objective (MICRO-NIKKOR 55 mm/f 2,8) and one fixed camera with a teleobjective (NIKKOR 200 mm/f 4). Each camera is coupled with a winder and two powerful electronic flash lights. The shot is electrically triggered by a remote control push button. No focussing, no finding of picture sector and no diaphragm setting are necessary. A detailed description of the set up, the type of equipment used (including prices) and the settings applied are given.