Caffeine Pulses Research Articles

PDE1 inhibition and adenosine A2A receptor activation synergistically potentiates spontaneous calcium waves and transients in mouse ventricular myocytes

Abstract Background The phosphodiesterase-1C (PDE1C) and the adenosine A2A receptor (A2AR) are located in a macromolecular cluster and expected to modulate cAMP-dependent regulation of the intracellular calcium homeostasis in cardiac myocytes. In this contect, atrial fibrillation has been associated with a high incidence of spontanoeus arrhythmogenic calcium waves caused by excessive A2AR activation. On the other hand, inhibition of PDE1C and A2AR activation has also been reported to produce a synergistic and protective effect on cardiomyocyte survival, but little is known about the corresponding effects on the intracellular calcium homeostasis. Purpose This study, therfore, aimed to investigate how PDE1 inhibition and/or A2AR activation modulate sarcoplasmic reticulum (SR) calcium homeostasis in mouse ventricular myocytes. Methods Mouse ventricular myocytes (47 cells from 20 mice) were loaded with the calcium indicator Rhod-2 and spontaneous calcium release events at rest or caffeine induced calcium transients were visualized using resonance-scanning confocal microscopy at a frame rate of 150 Hz. The SR calcium content was estimated as the ratio of the peak fluorescence and the fluoresence the end of a 10 mM caffeine pulse (20s). Calcium leak was estimated as ratio of the flourescence emmision at baseline before and at the end of the caffeine pulse. Results The incidence of spontaneous calcium waves or transients increased from 0.7±0.3 events/min in control to 1.1±0.5 events/min and 3.4±1.0 events/min (p<0.01) after inhibition of PDE1 with the selective antagonist ITI-214 and the A2AR agonist CGS-21680 respectively. Concomitant PDE1 inhibition and A2AR activation synergistically increased the event frequency to 8.4±2.3 events/min (p<0.05). There were no significant differences in the corresponding SR calcium contents (4.4±0.3, 4.5±0.4, 4.7±0.4 and 4.1±0.2 for control, ITI-214, CGS-21680 and ITI-214+CGS-21680 respectively). However, there was a parallel increase in calcium leak from 8.7±4.2% in control to 21.8±9.8% with ITI-214, 17.6±7.2% with CGS-21680 and 37.5±11.1% when myocytes were exposed to both compounds (p<0.05), suggesting that the higher incidence of spontaneous calcium waves and transients observed upon PDE1 inhibition and/or A2AR activation is likely caused by a lowering of the threshold for spontaneous SR calcium release rather than by SR calcium overloading. Conclusion PDE1 inhibition exacerbates the incidence of spontaneous arrhythmogenic SR calcium release events in myocytes with high levels of A2AR activation, suggesting that PDE1 inhibition under these conditions increases the risk of arrhythmia. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Spanish Ministry of Science and Innovation

β-Adrenergic Pathway is Enhanced by Hormone-Induced Maturation of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes (iPS-CM)

iPS-CM are useful for studying patient-specific cardiac pathologies. However, they present an immature phenotype with incomplete excitation-contraction coupling, inefficient calcium-induced calcium release and lack of t-tubules. This compromises the study of calcium handling-related pathologies such as CPVT. To improve iPS-CM maturity, we treated the beating cardiomyocytes with triiodothyronine (T3) and dexamethasone (Dex), as recently described by Parikh and collaborators (2017). The aim of the study was to assess the changes in calcium handling in β-adrenergic stress conditions, and to determine the changes in gene expression associated to maturation. We generated iPS-CM using an heparin-based differentiation protocol followed by glucose starvation. T3 and Dex were applied from day 16 until day 30 of differentiation. We characterized calcium transients elicited by caffeine pulses in both basal and β-adrenergic stress conditions (100nM isoproterenol). Also, we measured the expression of a tubulogenesis marker (JPH2), calcium handling-associated genes (RYR2 and PLN) and the fetal and adult isoforms of SCN5A by qPCR. Our results obtained in the presence of isoproterenol showed an increase in caffeine sensitivity in hormone-treated vs. control cells (Kd of 2.85 ± 0.12mM and 1.74 ± 0.16mM, respectively). Also, we observed a decrease both in rise time (18.2%), and decay time (16.8%) of calcium transients in the hormone-treated vs. control cells. Our qPCR results showed an increase in the expression of the tubulogenesis marker JPH2 (2.20-fold) and calcium handling-related genes (RYR2 1.23-fold, PLN 1.61-fold). In addition, we found that hormone treatment promotes a switch in the relative expression of the adult respect to the neonatal isoform of SCN5A. Altogether, our results show that T3 and Dex enhance the β-adrenergic pathway response, and increase the expression of genes associated to a more mature functional and structural phenotype.

Multiscale Determinants of Delayed Afterdepolarization Amplitude in Cardiac Tissue

Spontaneous calcium (Ca) waves in cardiac myocytes underlie delayed afterdepolarizations (DADs) that trigger cardiac arrhythmias. How these subcellular/cellular events overcome source-sink factors in cardiac tissue to generate DADs of sufficient amplitude to trigger action potentials is not fully understood. Here, we evaluate quantitatively how factors at the subcellular scale (number of Ca wave initiation sites), cellular scale (sarcoplasmic reticulum (SR) Ca load), and tissue scale (synchrony of Ca release in populations of myocytes) determine DAD features in cardiac tissue using a combined experimental and computational modeling approach. Isolated patch-clamped rabbit ventricular myocytes loaded with Fluo-4 to image intracellular Ca were rapidly paced during exposure to elevated extracellular Ca (2.7 mmol/L) and isoproterenol (0.25 μmol/L) to induce diastolic Ca waves and subthreshold DADs. As the number of paced beats increased from 1 to 5, SR Ca content (assessed with caffeine pulses) increased, the number of Ca wave initiation sites increased, integrated Ca transients and DADs became larger and shorter in duration, and the latency period to the onset of Ca waves shortened with reduced variance. In silico analysis using a computer model of ventricular tissue incorporating these experimental measurements revealed that whereas all of these factors promoted larger DADs with higher probability of generating triggered activity, the latency period variance and SR Ca load had the greatest influences. Therefore, incorporating quantitative experimental data into tissue level simulations reveals that increased intracellular Ca promotes DAD-mediated triggered activity in tissue predominantly by increasing both the synchrony (decreasing latency variance) of Ca waves in nearby myocytes and SR Ca load, whereas the number of Ca wave initiation sites per myocyte is less important.

Fluorescence Signal Kinetics of a New Ca2+ Probebound to Cardiac Ryanodine Receptors Point to Micro-Domain Detection of Ca2+

To probe the micro-domains of Ca2+-signaling, we constructed a probe, calstabi-cam, targeted to cardiac ryanodine receptors (RyR2) by FKBP12.6 (calstabin 2) incorporating calmodulin and EYFP as Ca2+-sensor. 48-72h after infection of cultured rat neonatal cardiomyocytes (rNCM), global measurements of calstabi-cam fluorescence were accompanied by simultaneous recordings of Ca2+ channel (ICa) and Na+/Ca2+-exchanger (INCX) and compared to measurements of fluo-4 Ca-transients. rNCMs continued spontaneous beating after dialysis with Cs+-based internal solutions (0.1-0.2mM EGTA at −50mV) as they generated calstabi-cam fluorescence signals that coincided with INCX oscillations, like those measured by fluo-4 that were blocked by 10µM ryanodine. Activation of ICa (∼5pA/pF) triggered slowly relaxing Ca-transients of calstabi-cam fluorescence (ΔF/F0 = −0.11; n=14). Similarly, short caffeine pulses also activated long lasting calstabi-cam signals (-ΔF/F0=0.15±0.012; n=28) accompanied by INCX (1.57±0.13pA/pF; n=28). Calstabi-cam signals activated by rapid Na+ withdrawal was significantly delayed (∼230ms) compared to those triggered by ICa (∼13ms) or caffeine (∼35ms). Similar results were found in feline adult cardiac myocytes where 1µM Isoproterenol increased ICa significantly without enhancing the associated calstabi-cam transients. These results suggest that the new probe responds more effectively to Ca2+ that is released into dyadic clefts by RyR2 than by sarcolemmal Ca2+ influx via Na/Ca exchanger. The extremely slow decay kinetics of Calstabi-cam signal is consistent with the notion that [Ca2+] in the dyadic cleft may rise to much higher values than those of cytosol.

Functionally Isolated Sarcoplasmic Reticulum Model: Intrinsic Regulation of SR Ca2+ Release and Tetracaine Effect

The fraction of the sarcoplasmic reticulum (SR) Ca2+ content released at a twitch (fractional release, FR) in cardiomyocytes is regulated by the L-type Ca2+ current (ICa,L) and the SR Ca2+ content ([Ca2+]SR). In the experimental model of the functionally isolated SR (FISRM), cardiomyocytes are perfused with Na+, Ca2+-free solution, which makes the cells unexcitable and thermodynamically inhibits the sarcolemmal Na+/Ca2+ exchanger. Intracellular free Ca2+ concentration ([Ca2+]i) was measured with fluo-3 and Ca2+ transients due to SR release were evoked by 100 ms-long caffeine pulses. FISRM was used to study the intrinsic relationship between FR and [Ca2+]SR in the absence of ICa,L, the physiological trigger for SR Ca2+ release. After SR depletion by prolonged caffeine application, 1-20 electrical stimuli were applied to attain different [Ca2+]SR (from 55.3±10.0 to 123.0±10.3 μM). The variation in total [Ca2+] (Δ[Ca2+]T) during transients was estimated from [Ca2+]i and Ca2+ buffering parameters. FR (Δ[Ca2+]T/[Ca2+]SR) showed a non-linear relationship with [Ca2+]SR (from 5.4±5.4 to 82.8±7.2%, p< 0.05), as previously observed when SR Ca2+ release was triggered by ICa,L. This behavior was reproduced with a mathematical model of FISRM, after inclusion of intra-SR and cytosolic Ca2+ buffers, which resulted in more realistic simulations. In addition, FISRM was used to study the inhibitory effect of tetracaine on FR. The tetracaine concentration that caused 50% inhibition of FR (IC50) was 58.9±5.7 μM. The FISRM may be a useful experimental model to investigate in intact cells the selective effects on SR Ca2+ release of drugs that also affect sarcolemmal currents, such as tetracaine. Support: CNPq.

An adaptive wavelength interval selection by modified particle swarm optimization algorithm: simultaneous spectral or differential pulse voltammetric determination of multiple components with overlapping peaks

The elimination of wavelengths which contain useless or irrelevant information for calibration model is becoming one of the key steps in multicomponent spectral analysis even in situations where the partial least-squares (PLS) regression is applied. Because of the continuity of most kinds of spectral responses, the adaptive wavelength interval selection by modified particle swarm optimization (PSO) could be proposed in the present study. The proposed method was used for simultaneous spectrophotometric determination of caffeine, aspirin, and phenacetin and simultaneous differential pulse voltammetric determination of nitrophenol isomers. The method was eventually applied to commercial drugs. For comparison, a conventional full-spectrum PSO analysis was also performed. Experimental results demonstrate that the adaptive wavelength interval selection by modified PSO is capable of improving the future predictive ability of the model.

Transient Ca2+ depletion of the sarcoplasmic reticulum at the onset of reperfusion

Myocardial stunning is a contractile dysfunction that occurs after a brief ischaemic insult. Substantial evidence supports that this dysfunction is triggered by Ca2+ overload during reperfusion. The aim of the present manuscript is to define the origin of this Ca2+ increase in the intact heart. To address this issue, Langendorff-perfused mouse hearts positioned on a pulsed local field fluorescence microscope and loaded with fluorescent dyes Rhod-2, Mag-fluo-4, and Di-8-ANEPPS, to assess cytosolic Ca2+, sarcoplasmic reticulum (SR) Ca2+, and transmembrane action potentials (AP), respectively, in the epicardial layer of the hearts, were submitted to 12 min of global ischaemia followed by reperfusion. Ischaemia increased cytosolic Ca2+ in association with a decrease in intracellular Ca2+ transients and a depression of Ca2+ transient kinetics, i.e. the rise time and decay time constant of Ca2+ transients were significantly prolonged. Reperfusion produced a transient increase in cytosolic Ca2+ (Ca2+ bump), which was temporally associated with a decrease in SR-Ca2+ content, as a mirror-like image. Caffeine pulses (20 mM) confirmed that SR-Ca2+ content was greatly diminished at the onset of reflow. The SR-Ca2+ decrease was associated with a decrease in Ca2+ transient amplitude and a shortening of AP duration mainly due to a decrease in phase 2. To the best of our knowledge, this is the first study in which SR-Ca2+ transients are recorded in the intact heart, revealing a previously unknown participation of SR on cytosolic Ca2+ overload upon reperfusion in the intact beating heart. Additionally, the associated shortening of phase 2 of the AP may provide a clue to explain early reperfusion arrhythmias.

Variable luminal sarcoplasmic reticulum Ca 2+ buffer capacity in smooth muscle cells

Sarcoplasmic reticulum contains the internal Ca 2+ store in smooth muscle cells and its lumen appears to be a continuum that lacks diffusion barriers. Accordingly, the free luminal Ca 2+ level is the same all throughout the SR; however, whether the Ca 2+ buffer capacity is the same in all the SR is unknown. We have estimated indirectly the luminal Ca 2+ buffer capacity of the SR by comparing the reduction in SR Ca 2+ levels with the corresponding increase in [Ca 2+] i during activation of either IP 3Rs with carbachol or RyRs with caffeine, in smooth muscle cells from guinea pig urinary bladder. We have determined that carbachol-sensitive SR has a 2.4 times larger Ca 2+ buffer capacity than caffeine-sensitive SR. Rapid inhibition of SERCA pumps with thapsigargin revealed that this pump activity accounts for 80% and 60% of the Ca 2+ buffer capacities of carbachol- and caffeine-sensitive SR, respectively. Moreover, the Ca 2+ buffer capacity of carbachol-sensitive SR was similar to caffeine-sensitive SR when SERCA pumps were inhibited. Similar rates of Ca 2+ replenishments suggest similar levels of SERCA pump activities for either carbachol- or caffeine-sensitive SR. Paired pulses of caffeine, in conditions of low Ca 2+ influx, indicate the relevance of luminal SR Ca 2+ buffer capacity in the [Ca 2+] i response. To further study the importance of luminal SR Ca 2+ buffer capacity in the release process we used low levels of heparin to partially inhibit IP 3Rs. This condition revealed carbachol-induced transient increase of luminal SR Ca 2+ levels provided that SERCA pumps were active. It thus appears that SERCA pump activity keeps the luminal SR Ca 2+-binding proteins in the high-capacity, low-affinity conformation, particularly for IP 3R-mediated Ca 2+ release.

Reperfusion after Ichemia Causes Cytosolic Calcium Overload Due to Rapid Calcium Release from the Sarcoplasmic Reticulum

After a brief ischemic insult, a sustained contractile dysfunction occurs manifested as a sluggish recovery of pump function (myocardial stunning). Substantial evidence supports that myocardial dysfunction is triggered by Ca2+ overload during reperfusion (R). Previous results from different laboratories including our own, describe a cascade of events triggered by R that involves the activation of Na+/H+ and Na+/Ca2+ (NCX) exchangers, with enhanced Ca2+ influx. Whether this Ca2+ influx directly produces the increase in cytosolic Ca2+ or this increase occurs as a consequence of sarcoplasmic reticulum (SR) Ca2+ release triggered in turn by the Ca2+ influx, is not known. To address this issue, we performed 12 min of global no-flow ischemia followed by R in the isovolumic Langendorff perfused mouse heart positioned on a Pulsed Local Field Fluorescence microscope and loaded with fluorescent dyes (Rhod-2 or Mag-Fluo-4 to assess cytosolic or SR Ca2+, respectively). The results indicated an initial increase in diastolic Ca2+ during early R that gradually returned to pre-ischemic levels. This increase was associated with a decrease in SR Ca2+ content that recovered within 10 min, as a mirror image of the diastolic Ca2+ profile. Additional experiments in which caffeine pulses (20 mM) were applied, confirmed that SR Ca2+ content was greatly diminished at the onset of R and gradually recovered within 10 min of R. The present findings indicate that the increase in diastolic Ca2+ that occurs upon R is due to a SR Ca2+ release and not just because of the Ca2+ entry through the reverse NCX mode, as has been previously thought.

Ouabain augments and maintains the catecholamine release responses evoked by repetitive pulses of potassium, caffeine or histamine in perifused bovine chromaffin cells

Cardiac glycosides such as ouabain augment the release of neurotransmitters and hormones from various organs, tissues and cell systems. Here we have investigated novel aspects of the ouabain effects on fast-perifused bovine adrenal chromaffin cells subjected to repetitive stimulation during long-time periods with secretagogues that enhance Ca 2+ entry (i.e. 100 mM K +solution) or causing the release into the cytosol of the Ca 2+ stored in the endoplasmic reticulum (i.e. 20 mM caffeine or 100 μM histamine). After 1 h of intermittent stimulation, the amperometrically measured catecholamine release responses decayed to 50% with K + pulses, and to 10–15% with caffeine or histamine pulses. Ouabain (10 μM) augmented 2-fold the K + secretory responses and kept them high, along an hour. When given after the responses had decayed upon repetitive caffeine or histamine pulsing, ouabain gradually restored such responses to their initial control values. On the basis of these results, we raise the hypothesis that ouabain may facilitate the handling by the cell of the endoplasmic reticulum Ca 2+ fluxes that are known to be involved in secretory vesicle transport and the regulation of exocytosis. This may have physiological and pathological interest in the light of an endogenous ouabain steroid found in the adrenal cortex.

Mechanism of P2X receptor‐mediated Increase in Cardiac Myocyte Contractility: Importance of Augmentation of SR Ca2+ Loading

Activation of cardiac P2X receptor can stimulate an increase in myocyte contractility. However, the underlying mechanism is not known. The objective here is to investigate the positive inotropic mechanism of cardiac P2X receptor using mice with cardiac-specific overexpression of the human P2X4 receptor (Tg). The P2X agonist 2-meSATP can evoke a greater inward current in Tg than in wild type (WT) cells. We tested the hypothesis that an increased P2X receptor-mediated inward current is associated with an increased sarcoplasmic reticulum (SR) Ca2+ loading and that this increased SR content is a mechanism for the enhanced myocyte contractility mediated via P2X receptors. The SR content was determined by the Na+/Ca++ exchanger-mediated inward current during a rapid caffeine pulse. Activation of the overexpressed P2X4 receptor by 2-meSATP caused a significant increase in the caffeine-mediated inward current, showing an augmented SR Ca2+ loading by the agonist in Tg cells. To determine whether P2X receptor activation alters myofilament Ca2+ sensitivity, simultaneous measurement of Ca2+ transients by fura-2 fluorescence ratio and cell shortening (CS) were determined in Tg cells at 0.2 to 2Hz stimulus frequencies. The plot of CS against fura-2 fluorescence ratio at different Hz was linear and similar before and after 2-meSATP, suggesting that P2X receptor activation does not change myofilament Ca2+sensitivity. 2-meSATP did not change L-type Ca current in Tg or WT cells and did not stimulate cyclic AMP accumulation. We conclude that activation of cardiac P2X receptor can augment SR Ca2+ content independent of cyclic AMP and that this increased content was associated with and likely contributed to the P2X receptor-mediated stimulation of myocyte contractility.

Effect of the nitrogen source on caffeine degradation by Aspergillus tamarii.

To evaluate caffeine degradation and nitrogen requirements during Aspergillus tamarii growth in submerged culture. Aspergillus tamarii spores produced on a coffee infusion agar medium added with sucrose were used. Several caffeine and ammonium sulphate concentrations (0-1 and 0-1.36 g l-1, respectively) were tested simultaneously on fungal biomass production and caffeine degradation. An additional caffeine pulse (4 g l-1) was added for all experiments after 48 h of fermentation. Results revealed that when using 0.90 g l-1 of caffeine and 0.14 g l-1 of ammonium sulphate, biomass production and caffeine degradation were enhanced. Highest biomass production (Xmax = 9.87 g l-1) with a specific growth rate (micro) of 0.073 h-1 and caffeine degradation rate of 0.033 g l-1 h-1, was observed under these conditions. Caffeine degradation as well as biomass production were characterized. These studies set the stage for future characterization studies of intracellular enzymes involved in caffeine degradation. Moreover, results observed may help in the biotreatment of residues from the coffee agroindustry.

Novel stimulatory actions of the phytoestrogen genistein: effects on the gain of cardiac excitation-contraction coupling.

Genistein, a phytoestrogen found abundantly in soy products, is thought to be cardioprotective, partly through its ability to act as a natural Ca2+ channel antagonist. However, the precise nature and significance of such direct cardiac actions remain obscure. We investigated the hypothesis that genistein exerts important additional actions on cardiac excitation-contraction coupling (ECC). Genistein acutely increased cell shortening and the Ca2+ transient in field stimulated guinea-pig ventricular myocytes despite potently inhibiting the L-type Ca2+ current, I(Ca,L). The specific phosphotyrosine phosphatase inhibitor, bpV(phen), diminished the stimulatory effects of genistein on myocyte contractility, suggesting that the mechanism partly involved tyrosine kinase inhibition. Genistein increased sarcoplasmic reticulum (SR) Ca2+ load as measured with a caffeine pulse in Na+-free/ Ca2+-free solution. Furthermore, in the continued presence of caffeine, genistein increased the time constant of decline of the caffeine-induced Ca2+ transient, implying impaired sarcolemmal Na+/Ca2+ exchanger function. Tetanization studies in intact myocytes revealed that 43% of cells exhibited increased myofilament Ca2+ sensitivity in the presence of genistein. These findings demonstrate novel cardiac actions of genistein on the SR Ca2+ load, Na+/Ca2+ exchanger, and myofilament Ca2+ sensitivity, which result in an overall increase in myocyte contractility and consequently the gain of ECC.

Comparison of sarcoplasmic reticulum Ca 2+-ATPase function in human, dog, rabbit, and mouse ventricular myocytes

It has been reported that sarcoplasmic reticulum (SR) Ca 2+ uptake is more rapid in rat than rabbit ventricular myocytes, but little information is available on the relative SR Ca 2+ uptake activity in others species, including humans. We induced Ca 2+ transients with a short caffeine pulse protocol (rapid solution switcher, 10 mM caffeine, 100 ms) in single ventricular myocytes voltage clamped (-80 mV) with pipettes containing 100 μM fluo-3 and nominal 0 Ca 2+, in 0 Na + o/0 Ca 2+ o solution to inhibit Na/Ca exchange. SR in non-paced human, dog, rabbit, and mouse ventricular myocytes could be readily loaded with Ca 2+ under our experimental conditions with a pipette [Ca 2+] = 100 nM. Resting [Ca 2+] i was similar in four types of ventricular myocytes. Activation of the Ca 2+-release channel with a 100-ms caffeine pulse produced a rise in [caffeine] i to slightly above 2 mM, the threshold for caffeine activation of Ca 2+ release. This caused a similar initial rate of rise and peak [Ca 2+] i in the four types of ventricular myocytes. However, there were significant differences in the duration of the plateau (top 10%) [Ca 2+] i transients and the time constant of the [Ca 2+] i decline (reflecting activity of the SR Ca 2+-ATPase), with values for human > dog > rabbit > mouse. In paced myocytes under physiologic conditions, SR Ca 2+ content was greater in mouse than in rabbit myocytes, while peak I Ca,L was smaller in mouse. These findings confirm substantial species difference in SR Ca 2+-ATPase activity, and suggest that the smaller the animal and the more rapid the heart rate, greater the activity of the SR Ca 2+-ATPase. In addition, it appears that substantial species differences exist in the degree of SR Ca 2+ loading and I Ca,L under physiologic conditions.

Functional coupling between glycolysis and excitation-contraction coupling underlies alternans in cat heart cells.

Electromechanical alternans was characterized in single cat atrial and ventricular myocytes by simultaneous measurements of action potentials, membrane current, cell shortening and changes in intracellular Ca2+ concentration ([Ca2+]i). Using laser scanning confocal fluorescence microscopy, alternans of electrically evoked [Ca2+]i transients revealed marked differences between atrial and ventricular myocytes. In ventricular myocytes, electrically evoked [Ca2+]i transients during alternans were spatially homogeneous. In atrial cells Ca2+ release started at subsarcolemmal peripheral regions and subsequently spread toward the centre of the myocyte. In contrast to ventricular myocytes, in atrial cells propagation of Ca2+ release from the sarcoplasmic reticulum (SR) during the small-amplitude [Ca2+]i transient was incomplete, leading to failures of excitation-contraction (EC) coupling in central regions of the cell. The mechanism underlying alternans was explored by evaluating the trigger signal for SR Ca2+ release (voltage-gated L-type Ca2+ current, ICa,L) and SR Ca2+ load during alternans. Voltage-clamp experiments revealed that peak ICa,L was not affected during alternans when measured simultaneously with changes of cell shortening. The SR Ca2+ content, evaluated by application of caffeine pulses, was identical following the small-amplitude and the large-amplitude [Ca2+]i transient. These results suggest that the primary mechanism responsible for cardiac alternans does not reside in the trigger signal for Ca2+ release and SR Ca2+ load. beta-Adrenergic stimulation with isoproterenol (isoprenaline) reversed electromechanical alternans, suggesting that under conditions of positive cardiac inotropy and enhanced efficiency of EC coupling alternans is less likely to occur. The occurrence of electromechanical alternans could be elicited by impairment of glycolysis. Inhibition of glycolytic flux by application of pyruvate, iodoacetate or beta-hydroxybutyrate induced electromechanical and [Ca2+]i transient alternans in both atrial and ventricular myocytes. The data support the conclusion that in cardiac myocytes alternans is the result of periodic alterations in the gain of EC coupling, i. e. the efficacy of a given trigger signal to release Ca2+ from the SR. It is suggested that the efficiency of EC coupling is locally controlled in the microenvironment of the SR Ca2+ release sites by mechanisms utilizing ATP, produced by glycolytic enzymes closely associated with the release channel.

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 microM). 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+.

Caffeine-induced contractions in developing rabbit heart.

Mature myocardium utilizes calcium released by the sarcoplasmic reticulum (SR) for cell contraction. Transient exposure of mature myocytes to caffeine is known to directly trigger Ca2+ release from the SR. In contrast, neonatal rabbit heart cells rely on transsarcolemmal Ca2+ influx for tension generation. SR function is decreased in immature heart and appears to play a minimal role as a calcium source. Accordingly, we hypothesized that neonatal rabbit myocytes would not respond to a caffeine pulse. Isolated neonatal and adult myocytes were paced to load the SR with calcium and then exposed to a 1-s pulse of 10 mM caffeine. As previously described, adult myocytes exhibited a brisk contraction in response to caffeine. Unexpectedly, neonatal myocytes also exhibited a similar, brisk response. These caffeine-induced contractions were not dependent on extracellular Ca2+ but were dependent upon the loading of SR Ca2+ stores. When SR Ca2+ stores were depleted by exposure to caffeine, mature myocytes exhibited only small, slow contractions in response to electrical field stimulation. Replenishing the SR Ca2+ stores resulted in normal, brisk contractions. In contrast, electrically stimulated contractions in immature myocytes were largely unaffected by caffeine-induced SR depletion. Thus, although neonatal myocytes are capable of loading and releasing calcium from the SR, such SR calcium release is not normally required for contraction in the developing heart. The minor role of SR Ca2+ release in immature rabbit heart may not result from immaturity of the SR, but rather from an inadequate mechanism to trigger SR calcium release.

Release and sequestration of calcium by ryanodine-sensitive stores in rat hippocampal neurones.

1. The properties of ryanodine-sensitive Ca2+ stores in CA1 pyramidal cells were investigated in rat hippocampal slices by using whole-cell patch-clamp recordings combined with fura-2-based fluorometric digital imaging of cytoplasmic Ca2+ concentration ([Ca2+]i). 2. Brief pressure applications of caffeine onto the somata of pyramidal cells caused large transient increases in [Ca2+]i (Ca2+ transients) of 50-600 nM above baseline. 3. The Ca2+ transients evoked by caffeine at -60 mV were not associated with an inward current, persisted after blocking voltage-activated Ca2+ currents and were completely blocked by bath-applied ryanodine. Similar transients were also evoked at +60 mV. Thus, these transients reflect Ca2+ release from intracellular ryanodine-sensitive Ca2+ stores. 4. The Ca2+ transients evoked by closely spaced caffeine pulses rapidly decreased in amplitude, indicating progressive depletion of the Ca2+ stores. The amplitude of the Ca2+ transients recovered spontaneously with an exponential time constant of 59 s. Recovery was accelerated by depolarization-induced elevations in [Ca2+]i and blocked by cyclopiazonic acid (CPA) and thapsigargin, indicating that store refilling is mediated by endoplasmic reticulum Ca(2+)-ATPases. 5. Even without prior store depletion the caffeine-induced Ca2+ transients disappeared after 6 min exposure to CPA, suggesting that ryanodine-sensitive Ca2+ stores are maintained at rest by continuous Ca2+ sequestration. 6. Caffeine-depleted Ca2+ stores did not refill in Ca(2+)-free saline, suggesting that the refilling of the stores depends upon Ca2+ influx through a 'capacitative-like' transmembrane influx pathway operating at resting membrane potential. The refilling of the stores was also blocked by Ni2+ and gallopamil (D600). 7. Elevations of basal [Ca2+]i produced by bath-applied KCl markedly potentiated (up to 6-fold) the caffeine-induced Ca2+ transients. The degree of potentiation was positively related to the increase in basal [Ca2+]i. The Ca2+ transients remained potentiated up to 9 min after reversing the KCl-induced [Ca2+]i increase. Thus, the ryanodine-sensitive Ca2+ stores can 'overcharge' when challenged with an increase in [Ca2+]i and slowly discharge excess Ca2+ after basal [Ca2+]i returns to its resting level. 8. Pressure applications of caffeine onto pyramidal cell dendrites evoked local Ca2+ transients similar to those separately evoked in the respective somata. Thus, dendritic ryanodine-sensitive Ca2+ stores are also loaded at rest and can function as independent compartments. 9. In conclusion, the ryanodine-sensitive Ca2+ stores in hippocampal pyramidal neurones contain a releasable pool of Ca2+ that is maintained by a Ca2+ entry pathway active at subthreshold membrane potentials. Ca2+ entry through voltage-gated Ca2+ channels transiently overcharges the stores. Thus, by acting as powerful buffers at rest and as regulated sources during activity, Ca2+ stores may control the waveform of physiological Ca2+ signals in CA1 hippocampal pyramidal neurones.

Initial contractile response of isolated rat heart cells to halothane, enflurane, and isoflurane.

In several beating cardiac muscle preparations, a short-lived increase in twitch tension or amplitude has been observed when they were exposed abruptly to solutions containing halothane or enflurane. As exposure to the anesthetics was continued, the expected negative inotropic effect became evident after the short-lived increase in twitch. No such increase in twitch has been reported during exposure to isoflurane. It has been hypothesized that this short-lived increase in twitch is caused by an enhancement of calcium release from the sarcoplasmic reticulum, but other mechanisms have not been excluded. Freshly isolated, single rat ventricular cells were stimulated to beat at room temperature and abruptly exposed to solutions containing halothane (0.25-0.64 mM), enflurane (0.69-1 mM), or isoflurane (0.31-0.54 mM). During these exposures, twitch amplitude was measured and intracellular calcium concentration was followed using the calcium-sensitive dye indo-1. In some experiments, the whole-cell patch-clamp technique was used to measure membrane current. In addition, in several cells the sarcoplasmic reticulum calcium content was assessed through the response to brief pulses of caffeine. Both the twitch amplitude and the intracellular calcium transient were increased temporarily in cells abruptly exposed to halothane or enflurane. No such behavior was found with isoflurane. After continued exposure to all three agents, both the twitch amplitude and the calcium transient were less than control. During the beats exhibiting an increase in twitch, no alteration in the relation between cell length (twitch amplitude) and the intracellular calcium transient was found compared with control conditions. In addition, the temporary increase in twitch amplitude occurred in cells contracting under voltage-clamp control when halothane was introduced, and it was not associated with any increase in the calcium current. The sarcoplasmic reticulum calcium content at the time of the halothane-induced increase in twitch also was not increased. The short-lived increase in twitch after abrupt exposure to halothane or enflurane is related to increased intracellular calcium during the beat and not to any changes in myofilament sensitivity to calcium. Because these results eliminate most alternative explanations for this phenomenon, the authors conclude that halothane, and probably also enflurane, increases the fraction of calcium released from the sarcoplasmic reticulum with each heart beat. Isoflurane appears to lack this action.

Free radicals enhance Na+/Ca2+ exchange in ventricular myocytes

Oxygen-derived free radicals (OFR) have been implicated in the pathogenesis of intracellular Ca2+ overload and the arrhythmias that characterize cardiac reperfusion. These arrhythmias may in large part be due to activation of the pathological transient inward current (ITI). However, the identity of the ITI generated by OFR is uncertain. We previously found that H2O2, an OFR-generating compound, markedly stimulated the ITI elicited by brief caffeine pulses in patch-clamped guinea pig ventricular myocytes. In the present study, using patch-clamped rabbit ventricular myocytes loaded with the Ca(2+)-sensitive indicator fura 2, we have further characterized this ITI and have identified its major component to be Na+/Ca2+ exchange based on its dependence on extracellular Na+ and sarcoplasmic reticulum Ca2+ release, its sensitivity to Ni2+, and the effects of its inhibition on relaxation. The effect on ITI was not unique to H2O2, because another free radical-generating system, xanthine + xanthine oxidase, produced a similar response. We hypothesize that enhancement of Na+/Ca2+ exchange by OFR during reperfusion, when intracellular Na+ is elevated, may promote intracellular Ca2+ overload and triggered arrhythmias.