Valeriy Lukyanenko

Regulation of calcium release by calcium inside the sarcoplasmic reticulum in ventricular myocytes

To study the effects of changes in sarcoplasmic reticulum (SR) intraluminal Ca2+ on the Ca2+ release mechanism, we correlated the activity of single cardiac ryanodine receptor (RyR) channels, monitored in planar bilayers, with the properties of spontaneous elementary Ca2+ release events (sparks) in intact ventricular myocytes, monitored by scanning confocal microfluorimetry. Under both normal conditions and Ca2+ overload, induced by elevation of extracellular [Ca2+], Ca2+ sparks represented single populations of events. During Ca2+ overload, the frequency of sparks increased from 0.8 to 3.1 events per second per 100 μm line scanned, and their amplitude increased from 100 nM to 400 nM. The duration of the Ca2+ sparks, however, was not altered. Changes in the properties of Ca2+ sparks were accompanied by only an ≈ 30% increase in the SR Ca2+ content, as determined by emptying the intracellular Ca2+ stores using caffeine. When single Ca2+ release channels were incorporated into lipid bilayers and activated by cytoplasmic Ca2+ (≈ 100 nM) and ATP (3 mM), elevation of Ca2+ on the luminal side from 20 μM to 0.2–20 mM resulted in a 1.2-fold to 7-fold increase, respectively, in open probability (Po). This potentiation of Po was due to an increase in mean open time and frequency of events. The relative effect of luminal Ca2+ was greater at low levels of cytoplasmic [Ca2+] than at high levels of cytoplasmic [Ca2+], and no effect of luminal Ca2+ was observed to occur in channels activated by 0.5–50 μM cytoplasmic Ca2+ in the absence of ATP. Our results suggest that SR Ca2+ release channels are modulated by SR intraluminal Ca2+. These alterations in properties of release channels may account for, or contribute to, the mechanism of spontaneous Ca2+ release in cardiac myocytes

Ca2+ sparks and Ca2+ waves in saponin-permeabilized rat ventricular myocytes

1 We carried out confocal Ca2+ imaging in myocytes permeabilized with saponin in ‘internal’ solutions containing: MgATP, EGTA and fluo‐3 potassium salt. 2 Permeabilized myocytes exhibited spontaneous Ca2+ sparks and waves similar to those observed in intact myocytes loaded with fluo‐3 AM. 3 In the presence of ‘low’[EGTA] (0·05 mm), Ca2+ waves arose regularly, even at relatively low [Ca2+] (50–100 nm, free). Increasing [EGTA] resulted in decreased frequency and propagation velocity of Ca2+ waves. Propagating waves were completely abolished at [EGTA] > 0·3 mm. 4 The frequency of sparks increased as a function of [Ca2+] (50–400 nm range) with no sign of a high affinity Ca2+‐dependent inactivation process. 5 The rate of occurrence of Ca2+ sparks was increased by calmodulin and cyclic adenosine diphosphate‐ribose (cADPR).

Dynamic Regulation of Sarcoplasmic Reticulum Ca2+ Content and Release by Luminal Ca2+-Sensitive Leak in Rat Ventricular Myocytes

In cardiac muscle, excitation-contraction (E-C) coupling is determined by the ability of the sarcoplasmic reticulum (SR) to store and release Ca(2+). It has been hypothesized that the Ca(2+) sequestration and release mechanisms might be functionally linked to optimize the E-C coupling process. To explore the relationships between the loading status of the SR and functional state of the Ca(2+) release mechanism, we examined the effects of changes in SR Ca(2+) content on spontaneous Ca(2+) sparks in saponin-permeabilized and patch-clamped rat ventricular myocytes. SR Ca(2+) content was manipulated by pharmacologically altering the capacities of either Ca(2+) uptake or leak. Ca(2+) sparks were recorded using a confocal microscope and Fluo-3 and were quantified considering missed events. SR Ca(2+) content was assessed by application of caffeine. Exposure of permeabilized cells to anti-phospholamban antibodies elevated the SR Ca(2+) content and increased the frequency of sparks. Suppression of the SR Ca(2+) pump by thapsigargin lowered [Ca(2+)](SR) and reduced the frequency of sparks. The ryanodine receptor (RyR) blockers tetracaine and Mg(2+) transiently suppressed the frequency of sparks. Upon washout of the drugs, sparking activity transiently overshot control levels. Low doses of caffeine transiently potentiated sparking activity upon application and transiently depressed the sparks upon removal. In patch-clamped cardiac myocytes, exposure to caffeine produced only a transient increase in the probability of sparks induced by depolarization. We interpret these results in terms of a novel dynamic control scheme for SR Ca(2+) cycling. A central element of this scheme is a luminal Ca(2+) sensor that links the functional activity of RyRs to the loading state of the SR, allowing cells to auto-regulate the size and functional state of their SR Ca(2+) pool. These results are important for understanding the regulation of intracellular Ca(2+) release and contractility in cardiac muscle.

Dual effects of tetracaine on spontaneous calcium release in rat ventricular myocytes.

1. Confocal microfluorometry was used to study the effects of tetracaine on spontaneous Ca2+ release from the sarcoplasmic reticulum (SR) in isolated rat ventricular myocytes. 2. At low concentrations (0.25‐1.25 mM), tetracaine caused an initial inhibition of spontaneous release events (Ca2+ sparks) and Ca2+ waves, which was followed by a gradual increase in Ca2+ release activity. The frequency and magnitude of sparks were first decreased and then increased with respect to control levels. At high concentrations (> 1.25 mM), tetracaine abolished all forms of spontaneous release. 3. Exposure of the myocytes to tetracaine resulted in a gradual increase in the SR Ca2+ load as indexed by changes in the magnitude of caffeine‐induced Ca2+ transients. 4. In cardiac SR Ca(2+)‐release channels incorporated into lipid bilayers, tetracaine (> 0.25 mM) induced a steady inhibition of channel activity. Addition of millimolar Ca2+ to the luminal side of the channel caused an increase in channel open probability under control conditions as well as in the presence of various concentrations of tetracaine. 5. We conclude that the primary effect of tetracaine on SR Ca(2+)‐release channels is inhibition of channel activity both in vitro and in situ. The ability of tetracaine to reduce spark magnitude suggests that these events are not due to activation of single channels or non‐reducible clusters of channels and, therefore, supports the multichannel origin of sparks. We propose that the paradoxical late potentiation of release by submaximal concentrations of tetracaine is caused by a gradual increase in SR Ca2+ load and subsequent activation of the Ca(2+)‐release channels by Ca2+ inside the SR.

The role of luminal Ca2+ in the generation of Ca2+ waves in rat ventricular myocytes

1 We used confocal Ca2+ imaging and fluo‐3 to investigate the transition of localized Ca2+ releases induced by focal caffeine stimulation into propagating Ca2+ waves in isolated rat ventricular myocytes. 2 Self‐sustaining Ca2+ waves could be initiated when the cellular Ca2+ load was increased by elevating the extracellular [Ca2+] ([Ca2+]o) and they could also be initiated at normal Ca2+ loads when the sensitivity of the release sites to cytosolic Ca2+ was enhanced by low doses of caffeine. When we prevented the accumulation of extra Ca2+ in the luminal compartment of the sarcoplasmic reticulum (SR) with thapsigargin, focal caffeine pulses failed to trigger self‐sustaining Ca2+ waves on elevation of [Ca2+]o. Inhibition of SR Ca2+ uptake by thapsigargin in cells already preloaded with Ca2+ above normal levels did not prevent local Ca2+ elevations from triggering propagating waves. Moreover, wave velocity increased by 20 %. Tetracaine (0·75 mM) caused transient complete inhibition of both local and propagating Ca2+ signals, followed by full recovery of the responses due to increased SR Ca2+ accumulation. 3 Computer simulations using a numerical model with spatially distinct Ca2+ release sites suggested that increased amounts of releasable Ca2+ might not be sufficient to generate self‐sustaining Ca2+ waves under conditions of Ca2+ overload unless the threshold of release site Ca2+ activation was set at relatively low levels (< 1·5 μM). 4 We conclude that the potentiation of SR Ca2+ release channels by luminal Ca2+ is an important factor in Ca2+ wave generation. Wave propagation does not require the translocation of Ca2+ from the spreading wave front into the SR. Instead, it relies on luminal Ca2+ sensitizing Ca2+ release channels to cytosolic Ca2+.

Termination of Ca2+ release during Ca2+ sparks in rat ventricular myocytes

1 Confocal Ca2+ imaging was used to measure spontaneous release events (Ca2+ sparks) in fluo‐3‐loaded isolated rat ventricular myocytes. 2 The microscopic Ca2+ release flux underlying Ca2+ sparks was derived by adapting the methods used previously to describe macroscopic Ca2+ release from cell‐averaged Ca2+ transients. 3 The magnitude of the local release fluxes varied from 2 to 5 μM ms−1, depending on SR Ca2+ loading conditions. Following spontaneous activation, the release flux rapidly decayed (τ= 6–12 ms). The rate of termination of release flux was found to be directly related to the magnitude of the flux (r2= 0.88). 4 The rate of termination of local release flux was slowed in the presence of FK506, a compound that is known to reduce inactivation of SR Ca2+ channels in vitro. 5 These results suggest that termination of release flux during sparks is not due to a spontaneous stochastic decay process or local depletion of Ca2+ from the SR, but rather involves an active extinguishing mechanism such as Ca2+‐dependent inactivation or adaptation.

Inhibition of Ca(2+) sparks by ruthenium red in permeabilized rat ventricular myocytes.

We have compared the effects of the sarcoplasmic reticulum (SR) Ca(2+) release inhibitor, ruthenium red (RR), on single ryanodine receptor (RyR) channels in lipid bilayers, and on Ca(2+) sparks in permeabilized rat ventricular myocytes. Ruthenium red at 5 microM inhibited the open probability (P(o)) of RyRs approximately 20-50-fold, without significantly affecting the conductance or mean open time of the channel. At the same concentration, RR inhibited the frequency of Ca(2+) sparks in permeabilized myocytes by approximately 10-fold, and reduced the amplitude of large amplitude events (with most probable localization on the line scan) by approximately 3-fold. According to our theoretical simulations, performed with a numerical model of Ca(2+) spark formation, this reduction in Ca(2+) spark amplitude corresponds to an approximately 4-fold decrease in Ca(2+) release flux underlying Ca(2+) sparks. Ruthenium red (5 microM) increased the SR Ca(2+) content by approximately 2-fold (from 151 to 312 micromol/l cytosol). Considering the degree of inhibition of local Ca(2+) release events, the increase in SR Ca(2+) load by RR, and the lack of effects of RR on single RyR open time and conductance, we have estimated that Ca(2+) sparks under normal conditions are generated by openings of at least 10 single RyRs.

Underlying mechanisms of symmetric calcium wave propagation in rat ventricular myocytes.

Calcium waves in heart cells are mediated by diffusion-coupled calcium-induced calcium release. The waves propagate in circular fashion. This is counterintuitive in view of the accepted ultrastructure of the cardiac myocyte. The density of calcium release sites in the transverse direction is four times higher than in the longitudinal direction. Simulations with release sites localized along Z-lines and isotropic diffusion yielded highly elliptical, nonphysiological waves. We hypothesized that subcellular organelles counteracted the higher release site density along the Z-lines by acting as transverse diffusion barriers and sites of active calcium uptake. We quantified the reduction of transverse diffusion by microinjecting cells with the nonreactive dye fluorescein. The ratio of the radial diffusion coefficient to the longitudinal coefficient was 0.39. Inhibition of mitochondrial uptake by rotenone accelerated the wave in the transverse direction. Simulations with release sites clustered at the Z-lines and a transverse diffusion coefficient 50% of the longitudinal coefficient generated waves of ellipticity 2/1 (major axis along the Z-line). Introducing additional release sites between the Z-lines at a density 20% of that on the Z-lines produced circular waves. The experiments and simulations support the presence of transverse diffusion barriers, additional uptake sites, and possibly intermediate release sites as well.