The cardiac sarcoplasmic reticulum: filled with Ca2+ and surprises.
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Animals↗
Biomedical subjects
Ernst Niggli
Publications and source records attributed to Ernst Niggli.
Agonist specific L-type Ca(2+)-current stimulation in ventricular myocytes by a novel steroid-like compound.
In cardiac muscle the amplitude of Ca(2+) transients can be increased by enhancing Ca(2+) influx. Among the processes leading to increased Ca(2+) influx, agonists of the L-type Ca(2+)-channel can play an important role. Known pharmacological Ca(2+)-channel agonists act on different binding sites on the channel protein, which may lead not only to enhanced peak currents, but also to distinct changes in other biophysical characteristics of the current. In this study, membrane currents were recorded with the patch-clamp technique in the whole-cell configuration in guinea pig isolated ventricular myocytes in combination with confocal fluorescence Ca(2+) imaging techniques and a variety of pharmacological tools. Testing a new positive inotropic steroid-like compound, we found that it increased the L-type Ca(2+)-current by 2.5-fold by shifting the voltage-dependence of activation by 20.2 mV towards negative potentials. The dose-response relationship revealed two vastly different affinities (EC(50(high-affinity))=4.5+/-1.7 nM, EC(50(low-affinity))=8.0+/-1.1 microM) exhibiting differential pharmacological interactions with three classes of Ca(2+)-current antagonists, suggesting more than one binding site on the channel protein. Therefore, we identified and characterized a novel positive inotropic compound (F90927) as a member of a new class of Ca(2+)-channel agonists exhibiting unique features, which set it apart from other presently known L-type Ca(2+)-channel agonists.
Animals↗
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 P(4(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.
Cells, Cultured↗
The early years of cellular cardiac electrophysiology and Silvio Weidmann (1921-2005).
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Action Potentials↗
The nitrodibenzofuran chromophore: a new caging group for ultra-efficient photolysis in living cells.
Photochemical uncaging of bio-active molecules was introduced in 1977, but since then, there has been no substantial improvement in the properties of generic caging chromophores. We have developed a new chromophore, nitrodibenzofuran (NDBF) for ultra-efficient uncaging of second messengers inside cells. Photolysis of a NDBF derivative of EGTA (caged calcium) is about 16-160 times more efficient than photolysis of the most widely used caged compounds (the quantum yield of photolysis is 0.7 and the extinction coefficient is 18,400 M(-1) cm(-1)). Ultraviolet (UV)-laser photolysis of NDBF-EGTA:Ca(2+) rapidly released Ca(2+) (rate of 20,000 s(-1)) and initiated contraction of skinned guinea pig cardiac muscle. NDBF-EGTA has a two-photon cross-section of approximately 0.6 GM and two-photon photolysis induced localized Ca(2+)-induced Ca(2+) release from the sarcoplasmic recticulum of intact cardiac myocytes. Thus, the NDBF chromophore has great promise as a generic and photochemically efficient protecting group for both one- and two-photon uncaging in living cells.
Animals↗
A novel steroid-like compound F90927 exerting positive-inotropic effects in cardiac muscle.
Here we report a novel steroid-like compound F90363, exhibiting positive inotropy in vivo and in vitro in various cardiac muscle preparations. F90363 is a racemic mixture composed of the stereoisomers (-)-F90926 and (+)-F90927. Only F90927 exerted positive inotropy, while F90926 induced a weak negative inotropy, but only at concentrations 10(3) times higher than F90927 and most likely resulting from an unspecific interaction. The rapid time course of the action of F90927 suggested a direct interaction with a cellular target rather than a genomic alteration. We could identify the L-type Ca2+ current I(Ca(L)) as a main target of F90927, while excluding other components of cardiac Ca2+ signalling as potential contributors. In addition, several other signaling pathways known to lead to positive inotropy (e.g. alpha- and beta-adrenergic stimulation, cAMP pathways) could be excluded as targets of F90927. However, vessel contraction and stiffening of the cardiac muscle at high doses (>30 microM, 0.36 mg kg(-1), respectively) prevent the use of F90927 as a candidate for drug development. Since the compound may still find valuable applications in research, the aim of the present study was to identify the cellular target and the mechanism of inotropy of F90927.
Animals↗
Founder of cardiac cellular electrophysiology: honouring Silvio Weidmann, 7 April 1921- 11 July 2005.
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Electrophysiology↗
Paradoxical SR Ca2+ release in guinea-pig cardiac myocytes after beta-adrenergic stimulation revealed by two-photon photolysis of caged Ca2+.
In heart muscle the amplification and shaping of Ca(2+) signals governing contraction are orchestrated by recruiting a variable number of Ca(2+) sparks. Sparks reflect Ca(2+) release from the sarcoplasmic reticulum (SR) via Ca(2+) release channels (ryanodine receptors, RyRs). RyRs are activated by Ca(2+) influx via L-type Ca(2+) channels with a specific probability that may depend on regulatory mechanisms (e.g. beta-adrenergic stimulation) or diseased states (e.g. heart failure). Changes of RyR phosphorylation may be critical for both regulation and impaired function in disease. Using UV flash photolysis of caged Ca(2+) and short applications of caffeine in guinea-pig ventricular myocytes, we found that Ca(2+) release signals on the cellular level were largely governed by global SR content. During beta-adrenergic stimulation resting myocytes exhibited smaller SR Ca(2+) release signals when activated by photolysis (62.3% of control), resulting from reduced SR Ca(2+) content under these conditions (58.6% of control). In contrast, local signals triggered with diffraction limited two-photon photolysis displayed the opposite behaviour, exhibiting a larger Ca(2+) release (164% of control) despite reduced global and local SR Ca(2+) content. This apparent paradox implies changes of RyR open probabilities after beta-adrenergic stimulation, enhancing local regenerativity and reliability of Ca(2+) signalling. Thus, our results underscore the importance of phosphorylation of RyRs (or of a related protein), as a regulatory physiological mechanism that may also provide new therapeutic avenues to recover impaired Ca(2+) signalling during cardiac disease.
Adrenergic beta-Agonists↗
Rapid turnover of the "functional" Na(+)-Ca2+ exchanger in cardiac myocytes revealed by an antisense oligodeoxynucleotide approach.
Antisense oligodeoxynucleotides (AS-ODNs) were used in combination with transient functional expression of the cardiac Na(+)-Ca2+ exchanger (NCX1) to correlate suppression of the Na(+)-Ca2+ exchange function with down-regulation of NCX1 protein expression. In a de-novo expression system (Sf9 cells), a decrease in both, NCX1 mRNA and protein after AS-ODN application was paralleled by diminished NCX1 activity, a typical hallmark of a true "antisense effect". Although AS-ODN uptake was also efficient in rat neonatal cardiac myocytes, in whole-cell extracts of these cells treated with AS-ODNs, the amount of NCX1 protein determined in a quantitative binding assay remained almost unchanged, despite a prompt loss of NCX1 function. Immunocytochemical staining of myocytes revealed that most of the immunoreactivity was not localized in the plasma membrane, but in intracellular compartments and was barely affected by AS-ODN treatment. These results indicate that the "functional half-life" of the NCX1 protein in the plasma membrane of neonatal cardiac myocytes is surprisingly short, much shorter than reported half-lifes of about 30 h for other membrane proteins.
Animals↗
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, beta-adrenergic stimulation with isoproterenol-accelerated recovery from refractoriness by approximately 2-fold. Application of cyclopiazonic acid at moderate concentrations (<10 micromol/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.
Acetates↗
Applications of multi-photon microscopy in cell physiology.
Owing to its many optical and physical advantages for fluorescence excitation, multi-photon microscopy has found a wide range of uses in biology, both in structural and functional studies. In this review we highlight various applications of this technique in different fields of cell physiology and biophysical research. This includes studies on second messenger and ionic signals, on cellular metabolism as well as on genetically engineered probes and indicators. In addition, this techniques has been successfully applied for diffraction-limited photolysis of caged compounds. We also point out some of the problems that were encountered along the still rapidly evolving path of this technique, and draw attention to some of the ongoing developments that will further extend and improve the usefulness of multi-photon excitation, such as fluoresce life-time imaging (FLIM), fluorescence resonance energy transfer (FRET) and entangled photon microscopy approaches.
Animals↗
Increased exchange current but normal Ca2+ transport via Na+-Ca2+ exchange during cardiac hypertrophy after myocardial infarction.
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 (I(NaCa)), whereas [Ca2+]i changes were simultaneously recorded with a confocal microscope. I(NaCa) 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 I(NaCa) 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.
Animals↗
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 (I(Na))- and L-type Ca2+ current (I(Ca,L))-induced Ca2+ transients in voltage-clamped ventricular myocytes isolated from guinea-pigs. Both currents, I(Na) and I(Ca,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 microM verapamil to partially block L-type Ca2+ current reduced the corresponding Ca2+ transients down to individual Ca2+ sparks. In contrast, I(Na)-induced Ca2+ responses were still spatially homogeneous and lacked Ca2+ sparks even for small current amplitudes. By using high resistance patch pipettes (> 4 MOmega) to exaggerate the loss of voltage control during I(Na), Ca2+ sparks appeared superimposed on a homogeneous Ca2+ release component and were exclusively triggered during the flow of I(Na). In the presence of 10 microM ryanodine both I(Ca,L) and I(Na) elicited small, residual Ca2+ transients that were spatially homogeneous but displayed distinctively different temporal profiles. We conclude that I(Na) is indeed able to cause Ca2+ release in guinea-pig ventricular myocytes. In contrast to I(Ca,L)-induced Ca2+ transients, which are built up from the recruitment of individual Ca2+ sparks, the I(Na)-evoked cellular responses were always homogeneous, indicating that their underlying elementary Ca2+ release event is distinct from the Ca2+ spark. Thus, I(Na)-induced Ca2+ transients are composed of smaller Ca2+ signals, most likely Ca2+ quarks.
Animals↗
Calcium quarks.
Elementary subcellular Ca2+ signals arising from the opening of single ion channels may offer the possibility to examine the stochastic behavior and the microscopic chemical reaction rates of these channel proteins in their natural environment. Such an analysis can yield detailed information about the molecular function that cannot be derived from recordings obtained from an ensemble of channels. In this review, we summarize experimental evidence suggesting that Ca2+ sparks, elementary Ca2+ signaling events of cardiac and skeletal muscle excitation contraction coupling, may be comprised of a number of smaller Ca2+ signaling events, the Ca2+ quarks.
Animals↗
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+).
Adenosine Triphosphate↗
Involvement of calcium in rhythmic activity induced by disinhibition in cultured spinal cord networks.
Disinhibition of rat spinal networks induces a spontaneous rhythmic bursting activity. The major mechanisms involved in the generation of such a bursting are intrinsic neuronal firing of a subpopulation of interneurons, recruitment of the network by recurrent excitation, and autoregulation of neuronal excitability. We have combined whole cell recording with calcium imaging and flash photolysis of caged-calcium to investigate the contribution of [Ca(2+)](i) to rhythmogenesis. We found that calcium mainly enters the neurons through voltage-activated calcium channels and N-methyl-D-aspartate (NMDA) channels as a consequence of the depolarization during the bursts. However, [Ca(2+)](i) could neither predict the start nor the termination of bursts and is therefore not critically involved in rhythmogenesis. Also calcium-induced calcium release is not involved as a primary mechanism in bursting activity. From these findings, we conclude that in the rhythmic activity induced by disinhibition of spinal cord networks, the loading of the cells with calcium is a consequence of bursting and does not functionally contribute to rhythm generation.
Animals↗
Ca(2+) Sparks in Cardiac Muscle: Is There Life Without Them?
The discovery of elementary Ca(2+) signaling events, the Ca(2+) sparks, has profoundly changed our view of cardiac Ca(2+) signaling and, in particular, excitationcontraction coupling. In addition, a partial disruption of cardiac Ca(2+) signaling may be important in clinical cardiac conditions such as congestive heart failure. Understanding cardiac diseases on the cellular and molecular levels may provide a key to new pharmacological strategies in the near future.
Journal Article↗