Decreasing motion artifacts in calcium-dependent fluorescence transients from the perfused mouse heart using frequency filtering

A strategy has been developed for the removal of motion artifact and noise in calcium-dependent fluorescence transients from the perfused mouse heart using frequency filtering. An analytical model indicates that the spectral removal of motion artifacts is independent of the phase shift of the motion waveform in the frequency domain, and thus to the time shift (or delay) of motion in the time domain. This is based on the "shift theorem" of Fourier analysis, which avoids erroneous correction of motion artifact when using the motion signal obtained using reflectance from the heart. Several major steps are adopted to implement this model for elimination of motion as well as detection noise from the fluorescence transient signals from the calcium-sensitive probe Rhod-2. These include (1) extracting the fluorescence calcium transient signal from the raw data by using power spectrum density (PSD) in the frequency domain by subtracting the motion recorded using the reflectance of excitation light, (2) digitally filtering out the random noise using multiple bandpass filters centralized at harmonic frequencies of the transients, and (3) extracting high frequency noise with a Gaussian Kernel filter method. The processed signal of transients acquired with excessive motion artifact is comparable to transients acquired with minimal motion obtained by immobilizing the heart against the detection window, demonstrating the usefulness of this technique.     

Calibration of the calcium dissociation constant of Rhod(2)in the perfused mouse heart using manganese quenching

Both theoretical and experimental results are presented for in vivo calibration of the dissociation constant K(Ca)(d)of the calcium-sensitive fluorescent dye Rhod(2)in the perfused mouse heart, using manganese quenching of fluorescence transients. An analytical model is derived, based on the biochemical equilibrium of manganese competition with calcium for Rhod(2)binding. Expressing the differential of the changes between systole and diastole in fluorescence transient (delta Delta F(sys-dia)). delta DeltaF(sys-dia)in a beating heart as a function of the perfusate manganese concentration [Mn(2+)](p)allows correlation of the measured differential transient changes delta Delta F(sys-dia)with the calcium dissociation constant K(Ca)(d)of Rhod(2)and the calcium concentration in the heart. Numerical modeling indicates that the K(Ca)(d)predominantly affects the asymptotic slope of the delta Delta F(sys-dia)versus [Mn(2+)](p)curve at certain manganese concentrations, which suggests that the K(Ca)(d)can be inversely calculated by partially fitting the delta Delta F(sys-dia)distribution as a function of the perfusate manganese concentration. The feasibility of this approach is confirmed by quenching of calcium transients by manganese infusion into isolated perfused beating mouse hearts. The resulting calculated dissociation constant K(Ca)(d)of Rhod(2)is 720nM. Using the same approach, we are able to also estimate intracellular calcium concentrations of 700nM at peak systole and 300nM in diastole. This is in good agreement with values obtained by calibration of fluorescence values with a calcium saturation tetanization procedure in the same perfused mouse heart model.     

Estimation of the mitochondrial calcium pool in rat brain synaptosomes using Rhod-2 AM fluorescent dye

The literature data on the role of synaptic mitochondria in the regulation of the cytosolic calcium level are contradictory. In the present paper calcium storage by mitochondria in rat brain synaptosomes using the fluorescent dye Rhod-2 has been investigated. The addition of 60 mM KCl increases Rhod-2 fluorescence. This effect is completely abolished by replacing K⁺ with Na⁺ or withdrawing Ca²⁺ from the incubation medium. A proton ionophore, carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone, and a mixture of rotenone/oligomycin mitochondrial toxins cause a two-fold decrease in Rhod-2 fluorescence. Thapsigargin, an inhibitor of endoplasmic reticulum ATPase (1 μM), but not bafilomycin, an inhibitor of ATPase in synaptic vesicles (500 nM) also leads to a mitochondrial calcium influx. The addition of calcium to synaptosomes with the retained plasma membrane potential increased Rhod-2 fluorescence; however, this effect is insensitive to carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone. We have shown that mitochondria can serve as a calcium store in synaptosomes only in the case of a high cytosolic concentration of calcium.     

Quantitation of cytosolic [Ca2+] in whole perfused rat hearts using Indo-1 fluorometry.

Fluorometric determination of cytosolic calcium, [Ca2+]c, using Indo-1 in intact tissue, is limited by problems in obtaining calibration parameters for Indo-1 in vivo. Therefore, the goal of this study was to calibrate Indo-1 using in vitro constants, obtained from protein-containing reference solutions designed to produce similar Indo-1 spectral properties to those in vivo. Due to wavelength-dependent tissue light absorbance, the in vitro constants had to be absorbance-corrected using a novel method. The correction factor was calculated from the relationship between the Indo-1 fluorescence intensities at the two detection wavelengths. A mixture of proteins at approximately 28 mg/ml had a similar Indo-1 isosbestic wavelength (430 nm) to that found in vivo (427 nm), and a similar fluorescence ratio maximum with saturating Ca2+ to that found in vivo (after absorbance correction). Using calibration constants from this protein mixture, calculated [Ca2+]c in a Langendorf perfused rat heart was 187 nM during diastole, and 464 nM in systole. This new calibration method circumvented the considerable experimental problems of previous methods which required measurements with the cytosol fully depleted and fully saturated with Ca2+.     

Abnormalities in the regulation of blood platelet free cytosolic calcium in malignant hyperthermia. I. Human platelets.

The effect of halothane on the regulation of blood platelet free cytosolic calcium was investigated in Quin-2-loaded cells from patients susceptible to Malignant Hyperthermia (MH) and healthy controls, respectively. The resting level of free cytosolic calcium was slightly, but statistically significantly, enhanced in platelets from patients (90 +/- 10 nM vs 110 +/- 35 nM). Halothane induced a dose-dependent, rapid Ca2+ release from intracellular stores both in normal and in MH derived cells, but the resulting increase in cytosolic calcium was significantly higher in the latter (2 mM halothane: [Ca2+]i = 117 +/- 12 nM vs 218 +/- 117 nM; 4 mM halothane: 225 +/- 35 nM vs. 417 +/- 201 nM). Whereas in platelets from healthy donors a complete reversibility of the halothane effect could be observed within 30-45 min, the cytosolic Ca2+ transients in platelets from patients were different from those in normals either in a higher initial peak or in a diminished decline velocity or in both. The basal Ca2+ permeability of the platelet plasma membrane was very low. Generally, halothane caused a dose-dependent increase in Ca2+ permeability. However, the influx of external calcium was significantly higher in platelets from patients than in controls (2 mM halothane: delta [Ca2+]i = 69 +/- 12 nM vs 135 +/- 63 nM; 4 mM halothane: 127 +/- 33 nM vs. 258 +/- 111 nM). Combining the results, the suggestion can be made that susceptibility to MH is characterized by a generalized membrane defect.(ABSTRACT TRUNCATED AT 250 WORDS)     

LY294002, but not wortmannin,increases intracellular calcium and inhibits calcium transients in bovine and human airway smooth muscle cells

To characterize the effect that a phosphatidylinositol 3-kinase (PI3-kinase) inhibitor, LY294002, has on cytosolic calcium concentrations ([Ca2+]i), bovine airway smooth muscle cells (BASMC) and cultured human bronchial smooth muscle cells (HBSMC) were loaded with fura 2-AM, imaged as single cells and [Ca2+]i measured ratiometrically. LY294002 (50 microM) increased [Ca2+]i by 294+/-76 nM (P<0.01, n=13) and 230+/-31 nM (P<0.001, n=10) in BASMC and HBSMC, respectively, and increases occurred in the absence of extracellular calcium. In contrast, after pre-treatment with thapsigargin, LY294002 no longer increased [Ca2+]i. This calcium mobilization by LY294002 was associated with a significant functional effect since LY294002 also inhibited calcium transients to carbachol (45+/-23 nM), caffeine (45+/-32 nM), and histamine (20+/-22 nM), with controls of 969+/-190, 946+/-156, and 490+/-28 nM, respectively. Wortmannin, a different PI3-kinase inhibitor, neither increased [Ca2+]i nor inhibited transients. Also, LY294002 increased [Ca2+]i in the presence of wortmannin, U-73122, and xestospongin C. We concluded that LY294002 increased [Ca2+]i, at least in part, by mobilizing intracellular calcium stores and inhibited calcium transients. The effects of LY294002 on [Ca2+]i were not dependent on wortmannin-sensitive PI3-kinases, phospholipase C, or inositol trisphosphate receptors (IP3R). For BASMC and HBSMC, LY294002 has effects on calcium regulation that could be important to recognize when studying PI3-kinases.     

Relationship between cytosolic calcium and oxygen consumption in isolated rat hearts.

Maximum oxygen consumption was attained in isolated perfused rat hearts using high perfusate calcium and/or isoproterenol, or phenylephrine. The amplitude of calcium transients was directly related to oxygen consumption until oxygen consumed per beat reached maximum. At saturating oxygen consumption the amplitude of [Ca2+]i transients continued to increase, indicative of a calcium overload. In all cases +dP/dt correlated proportionately with +dCa2+/dt. Augmented developed pressure, related to isoproterenol-induced increase in cytosolic cAMP, cannot be attributed totally to elevated levels of [Ca2+]i transients. Adenosine (10(-5) M) added to the medium containing isoproterenol (10(-6) M) negated the isoproterenol-induced increase in cAMP and returned cardiac performance, oxygen consumption, and amplitude of [Ca2+]i transients to control state.     

Optical Mapping of Action Potentials and Calcium Transients in the Mouse Heart

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. Cardiovascular physiological phenotyping of the mouse heart can be easily done using fluorescence imaging employing various probes for transmembrane potential (V_m), calcium transients (CaT), and other parameters. Excitation-contraction coupling is characterized by action potential and intracellular calcium dynamics; therefore, it is critically important to map both V_m and CaT simultaneously from the same location on the heart^1-4. Simultaneous optical mapping from Langendorff perfused mouse hearts has the potential to elucidate mechanisms underlying heart failure, arrhythmias, metabolic disease, and other heart diseases. Visualization of activation, conduction velocity, action potential duration, and other parameters at a myriad of sites cannot be achieved from cellular level investigation but is well solved by optical mapping^1,5,6. In this paper we present the instrumentation setup and experimental conditions for simultaneous optical mapping of V_m and CaT in mouse hearts with high spatio-temporal resolution using state-of-the-art CMOS imaging technology. Consistent optical recordings obtained with this method illustrate that simultaneous optical mapping of Langendorff perfused mouse hearts is both feasible and reliable.     

Increases in cytosolic calcium ion concentration can be dissociated from the killing of cultured hepatocytes by tert-butyl hydroperoxide

Digital imaging fluorescence microscopy was used to study the effect of tert-butyl hydroperoxide (TBHP) on the cytosolic free calcium concentration ([Ca2+]i) of single rat hepatocytes in primary culture. Within minutes of the addition of TBHP, individual hepatocytes displayed one or more peaks of increased [Ca2+]i that promptly returned to the prestimulation level. This was followed by a slower increase of [Ca2+]i that reached a plateau of 696 +/- 260 nM (basal 194 +/- nM) after 20 min. Another rise in [Ca2+]i, abrupt and much larger, preceded the death of the cells after about 45 min. Pretreatment of the hepatocytes with deferoxamine, a ferric iron chelator, or the addition of the antioxidants N,N'-diphenyl-p-phenylenediamine or catechol prevented the loss of viability. Neither the number of hepatocytes displaying the initial [Ca2+]i transients nor the magnitude of these oscillations was affected by deferoxamine, N,N'-diphenyl-p-phenyl-enediamine, or catechol. However, both the plateau phase and the abrupt rise in [Ca2+]i were prevented. Treatment of the hepatocytes with TBHP in a low calcium buffer (less than 2 microM Ca2+) reduced or abolished the initial [Ca2+]i transients and eliminated both the plateau phase and abrupt rise in [Ca2+]i. The onset of cell death was delayed by 10 min in the low calcium medium. Addition of 3.5 mM EGTA to the cultures lowered the basal calcium concentration, prevented both the initial [Ca2+]i spikes and the delayed changes, and further prolonged the onset of cell death. These data indicate that the killing of the cultured hepatocytes by TBHP can be dissociated from changes in intracellular calcium homeostasis. An influx of extracellular Ca2+ ions may aggravate somewhat the mechanisms of cell injury by an oxidative stress and accelerate the time of onset of cell death.     

Influence of calcium antagonists on thrombin-induced calcium mobilization and platelet-vessel wall interactions.

Elevation of cytosolic ionized calcium plays a critical role in human platelet activation. We have evaluated three well-characterized calcium antagonists for their ability to prevent thrombin-induced calcium mobilization in Fura 2 AM-loaded platelets and also their ability to inhibit platelet-vessel wall interactions. Thrombin (0.2 U/ml) caused significant elevation of cytosolic calcium (basal 84 +/- 18, activated 546 +/- 76 nM; n = 3). Verapamil, diltiazem, and nifedipine (100 microM) did not exert any inhibitory effect on thrombin-mediated calcium elevation. Untreated platelets perfused through a Baumgartner chamber containing a rabbit aorta preparation reacted with exposed and denuded subendothelium. The percentage of the total area covered by control platelet thrombi was 39.6 +/- 3.4. Diltiazem and Nifedipine significantly reduced the percentage of area covered by platelet thrombi, but the drugs were not as effective as aspirin (8.2 +/- 1.4). Calcium antagonists studied did not inhibit thrombin-stimulated elevation of cytosolic calcium in blood platelets. Although these drugs have been shown to prevent in vitro platelet aggregation and offer some protection against risks for atherosclerosis and thrombosis, they failed to significantly inhibit platelet-vessel wall interactions leading to formation of spread platelets and aggregates.     

A method for determining the individual optical characteristics of posttranslational fluorescent forms of fluorescent proteins with the use of nonlinear laser fluorimetry

A method for determining the individual optical characteristics (fluorescence quantum yield, the rate constant and quantum yield of singlet-triplet conversion, excitation of fluorescence cross-section, extinction coefficient) and concentration correlations between the fluorescent forms of fluorescent proteins arising in the reaction of posttranslational chromophore formation has been developed, which is based on combined application of absorption spectroscopy and classical and nonlinear laser fluorimretry. The method allows one to determine the share of fluorescent forms in the mixture of chromoproteins. The individual optical characteristics of the red form of the fluorescent protein mRFP1 has been determined: the fluorescence quantum yield eta = 0.24 +/- 0.03; the extinction coefficient in the maximum of absorbance band (584 nm) epsilon = 213 +/- 40 mM(-1) cm(-1) (the cross-section of absorbance sigma = (8.2 +/- 1.5).10(-16) cm2); the constant of singlet-triplet conversion rate K32 = (0 +/- 0.6)-10970 s(-1). The part of the red form in the mixture of chromoproteins is 26 +/- 6%.     

The role of calcium in follicle-stimulating hormone signal transduction in Sertoli cells.

Sertoli cells are hormonally regulated by follicle-stimulating hormone (FSH) acting upon a G-protein-linked cell surface FSH receptor. FSH increases intracellular cyclic AMP but the involvement of other signal transduction mechanisms including intracellular calcium in FSH action are not proven. Using freshly isolated rat Sertoli cells we measured cytosolic free ionized calcium levels by dual-wavelength fluorescence spectrophotometry using the calcium-sensitive fluorescent dye Fura2-AM. The cytosolic calcium concentration in unstimulated Sertoli cells was 89 +/- 2 nM (n = 151 experiments) and was markedly increased by either calcium channel ionophores (ionomycin, Bay K8644) or plasma membrane depolarization consistent with the presence of voltage-sensitive and -independent calcium channel in Sertoli cell membranes. Ovine FSH stimulated a specific, sensitive (ED50, 5.0 ng of S-16/ml), and dose-dependent (maximal at 20 ng/ml) rise in cytosolic calcium commencing within 60 s to reach levels of 192 +/- 31 nM after 180 s and lasting for at least 10 min. The effect of FSH was replicated by forskolin, cholera toxin, and dibutyryl cyclic AMP, suggesting that cyclic AMP may mediate the FSH-induced rise in cytosolic calcium. The FSH-induced rise in cytosolic calcium required extracellular calcium and was abolished by calcium channel blockers specific for dihydropyridine (verapamil, nicardipine), nonvoltage-gated (ruthenium red) or all calcium channels (cobalt). Thus FSH action on Sertoli cells involves a specific, rapid, and sustained increase in cytosolic calcium which requires extracellular calcium and involves both dihydropyridine-sensitive, voltage-gated calcium channels and voltage-independent, receptor-gated calcium channels in the plasma membranes of rat Sertoli cells. The replication by cyclic AMP of the effects of FSH suggests that calcium may be a signal-amplification or -modulating mechanism rather than an alternate primary signal transduction system for FSH in Sertoli cells.     

Effect of thyroid state on cytosolic free calcium in resting and electrically stimulated cardiac myocytes

The effects of the thyroid state on the cytosolic free Ca2+ concentration, [Ca2+]i, of resting and K+-depolarized cardiomyocytes were studied using the fluorescent Ca2+ indicator fura2. The mean resting [Ca2+]i in euthyroid myocytes (89 +/- 8 nM) was not significantly different from that in hyperthyroid myocytes (100 +/- 14 nM). The resting O2-consumption rate was identical for both groups when expressed per mg protein, but a 35% higher value was observed in the hyperthyroid group when expressed per cell on account of the cellular hypertrophy induced by thyroid hormone. Potassium induced depolarization (50 mM [K+]0) raised the level of [Ca2+]i by 50% in both groups. When ATP-coupled respiration was blocked with oligomycin, the 50 mM K+-induced rise in [Ca2+]i was accompanied in both groups by a 40% rise in glycolytic activity as inferred from measurement of lactate production. Ca2+-fluorescence transients were recorded from electrically stimulated myocytes of euthyroid, hyperthyroid and hypothyroid rats. The time taken to reach peak fluorescence (TPL) and that to 50% decay of peak fluorescence (RL0.5) decreased in the direction hypothyroid----hyperthyroid, indicating an increase in Ca2+ fluxes in the same direction. Isoproterenol (1 microM) enhanced the peak Ca2+ fluorescence in electrically stimulated hypothyroid and euthyroid myocytes but not in hyperthyroid myocytes. Both the TPL and RL0.5 were decreased by isoproterenol in euthyroid, but more so in hypothyroid myocytes. None of these parameters were influenced by isoproterenol in the hyperthyroid group. We conclude that (1) thyroid hormone increases neither the O2-consumption rate nor the level of [Ca2+]i of resting cardiomyocytes and (2) the effects of the beta-receptor-agonist isoproterenol on Ca2+ transients of electrically stimulated myocytes, are inversely related to the documented changes in beta-receptor density in heart tissue occurring with alterations in the thyroid state.     

Mapping action potentials and calcium transients simultaneously from the intact heart

Intracellular calcium handling plays an important role in cardiac electrophysiology. Using two fluorescent indicators, we developed an optical mapping system that is capable of measuring calcium transients and action potentials at 256 recording sites simultaneously from the intact guinea pig heart. On the basis of in vitro measurements of dye excitation and emission spectra, excitation and emission filters at 515 +/- 5 and >695 nm, respectively, were used to measure action potentials with di-4-ANEPPS, and excitation and emission filters at 365 +/- 25 and 485 +/- 5 nm, respectively, were used to measure calcium transients with indo 1. The percent error due to spectral overlap was small when action potentials were measured (1.7 +/- 1.0%, n = 3) and negligible when calcium transients were measured (0%, n = 3). Recordings of calcium transients, action potentials, and isochrone maps of depolarization time and the time of calcium transient onset indicated negligible error due to fluorescence emission overlap. These data demonstrate that the error due to spectral overlap of indo 1 and di-4-ANEPPS is sufficiently small, such that optical mapping techniques can be used to measure calcium transients and action potentials simultaneously in the intact heart.     

Simultaneous recording of calcium transients in skeletal muscle using high- and low-affinity calcium indicators.

To monitor cytosolic [Ca2+] over a wide range of concentrations in functioning skeletal muscle cells, we have used simultaneously the rapid but relatively low affinity calcium indicator antipyrylazo III (AP III) and the slower but higher affinity indicator fura-2 in single frog twitch fibers cut at both ends and voltage clamped with a double vaseline gap system. When both dyes were added to the end pool solution the cytosolic fura-2 concentration reached a steady level equal to the end pool concentration within approximately 2.5 h, a time when the AP III concentration was still increasing. For depolarizing pulses of increasing amplitude, the fura-2 fluorescence signal approached saturation when the simultaneously recorded AP III absorbance change was far from saturation. Comparison of simultaneously recorded fura-2 and AP III signals indicated that the mean values of the on and off rate constants for calcium binding to fura-2 in 18 muscle fibers were 1.49 x 10(8) M-1 s-1 and 11.9 s-1, respectively (mean KD = 89 nM), if all AP III in the fiber is assumed to behave as in calibrating solution and to be in instantaneous equilibrium with [Ca2+]. [Ca2+] transients calculated from the fura-2 signals using these rate constants were consistent with the [Ca2+] transients calculated from the AP III signals. Resting [Ca2+] or small changes in [Ca2+] which could not be reliably monitored with AP III could be monitored with fura-2 with little or no interference from changes in [Mg2+] or from intrinsic signals. The fura-2 signal was also less sensitive to movement artifacts than the AP III signal. After a [Ca2+] transient the fura-2 signal demonstrated a relatively small elevation of [Ca2+] that was maintained for many seconds.     

Impairment of myocardial calcium homeostasis by antibodies against the adenine nucleotide translocator.

The adenine nucleotide translocator (ANT) is an autoantigen in myocarditis and dilated cardiomyopathy. Carrier-specific antibodies impair myocardial energy metabolism and heart function. They cross-react with a myolemmal calcium channel and alter calcium fluxes in isolated myocytes. To test whether antibodies against the ANT can alter calcium homeostasis in intact hearts, guinea pigs were immunized with the carrier protein and their isolated hearts loaded with the intracellular calcium indicator INDO-1. The diastolic and systolic ratios of fluorescence signals at 410 nm and 510 nm (emission wavelengths of the calcium-bound and calcium-free indicator), 'd-s410/510', were measured by excitation at 364 nm. This index of the transient calcium concentration associated with the contraction cycle correlated with the external heart work (EHW) in non-immunized controls. EHW of immunized animals was lower (76 +/- 62 vs 153 +/- 47 mJ/g/min in controls, p < 0.005) and the amplitude of d-s410/510 was elevated (27.6 +/- 4.1% of the average ratio of the whole heart cycle vs 21.7 +/- 1.2% in controls, p < 0.005) and essentially independent of EHW. Isoproterenol stimulation increased EHW in all hearts but d-s410/510 was hightened in control hearts, only. Thus, a disorder between cytosolic calcium transients and work was recorded in hearts from guinea pigs immunized with the ANT. It may contribute to an immunopathic mechanism of heart failure subsequent to myocarditis.     

Erythropoietin increases cytosolic free calcium concentration and thrombin induced changes in cytosolic free calcium in platelets from spontaneously hypertensive rats

Using fura-2 cytosolic free calcium concentrations were measured in intact washed platelets from 9 spontaneously hypertensive rats (SHR) and from 9 age-matched normotensive Wistar-Kyoto rats (WKY). In resting platelets cytosolic free calcium concentration was significantly higher in SHR than in WKY (171.8 +/- 64.4 nM vs 93.1 +/- 59.0 nM, p less than 0.05). After preincubation with erythropoietin cytosolic free calcium concentration was significantly higher in SHR than in WKY (197.5 +/- 83.2 vs 93.0 +/- 60.1, p less than 0.01). Using platelets from SHR erythropoietin increased mean resting cytosolic free calcium concentration by 14.9% (p less than 0.05) and mean thrombin induced changes of cytosolic free calcium by 58.3% (p less than 0.01). In contrast, erythropoietin caused no significant increase in the resting calcium concentration or in thrombin induced changes of cytosolic free calcium in platelets from WKY. It is concluded that erythropoietin is involved in the pathogenesis of hypertension by elevating cytosolic free calcium concentration.     

Dual probe fluorescence monitoring of intracellular free calcium during ischemia in mouse heart by using continuous compensation for pH dependence of the dissociation constant of Fura-2, and the interference of myoglobin

Mitochondrial damage is the main source of cellular injury upon ischemia-reperfusion, and calcium loading has been implicated in this phenomenon. The use of optical probes for calcium monitoring of the intact heart is hampered by internal filter effects of intracellular hemoproteins, endogenous fluorescence, and their sensitivity to pH. We describe here a method for measurement of intracellular free calcium in isolated myoglobin-deficient perfused mouse hearts under conditions of large intracellular pH fluctuations by simultaneous fluorescence monitoring of the calcium-probe Fura-2 and the pH probe BCECF through dual wavelength excitation of both probes. In myoglobin-containing mouse heart endogenous chromophores interfere with Fura-2 fluorometry. It is shown that a paradoxical decrease in Fura-2 fluorescence occurs during ischemia in isolated mouse hearts. Simultaneous recording of BCECF fluorescence (calibrated against pH measurement with phosphorus NMR) and data reduction based on continual recalculation of the apparent dissociation constant of the calcium-probe complex revealed that a marked increase in intracellular free calcium occurs, and that the Fura-2 fluorescence decrease was caused by an increase in dissociation constant due to intracellular acidification. Intracellular free calcium rose almost linearly during a 20-min period of ischemia and returned to basal values rapidly upon the commencement of perfusion.     

Dual probe fluorescence monitoring of intracellular free calcium during ischemia in mouse heart by using continuous compensation for pH dependence of the dissociation constant of Fura-2, and the interference of myoglobin

Mitochondrial damage is the main source of cellular injury upon ischemia–reperfusion, and calcium loading has been implicated in this phenomenon. The use of optical probes for calcium monitoring of the intact heart is hampered by internal filter effects of intracellular hemoproteins, endogenous fluorescence, and their sensitivity to pH.We describe here a method for measurement of intracellular free calcium in isolated myoglobin-deficient perfused mouse hearts under conditions of large intracellular pH fluctuations by simultaneous fluorescence monitoring of the calcium-probe Fura-2 and the pH probe BCECF through dual wavelength excitation of both probes. In myoglobin-containing mouse heart endogenous chromophores interfere with Fura-2 fluorometry.It is shown that a paradoxical decrease in Fura-2 fluorescence occurs during ischemia in isolated mouse hearts. Simultaneous recording of BCECF fluorescence (calibrated against pH measurement with phosphorus NMR) and data reduction based on continual recalculation of the apparent dissociation constant of the calcium-probe complex revealed that a marked increase in intracellular free calcium occurs, and that the Fura-2 fluorescence decrease was caused by an increase in dissociation constant due to intracellular acidification. Intracellular free calcium rose almost linearly during a 20-min period of ischemia and returned to basal values rapidly upon the commencement of perfusion.     

Role of mitochondria in Ca(2+) homeostasis of mouse pancreatic acinar cells

The effects of mitochondrial Ca(2+) uptake on cytosolic Ca(2+) concentration ([Ca(2+)](c)) were investigated in mouse pancreatic acinar cells using cytosolic and/or mitochondrial Ca(2+) indicators. When calcium stores of the endoplasmic reticulum (ER) were emptied by prolonged incubation with thapsigargin (Tg) and acetylcholine (ACh), small amounts of calcium could be released into the cytosol (Delta[Ca(2+)](c)=46 +/- 6 nM, n=13) by applying mitochondrial inhibitors (combination of rotenone (R) and oligomycin (O)). However, applications of R/O, soon after the peak of Tg/Ach-induced Ca(2+) transient, produced a larger cytosolic calcium elevation (Delta[Ca(2+)](c)=84 +/- 6 nM, n=9), this corresponds to an increase in the total mitochondrial calcium concentration ([Ca(2+)](m)) by approximately 0.4 mM. In cells pre-treated with R/O or Ru360 (a specific blocker of mitochondrial Ca(2+) uniporter), the decay time-constant of the Tg/ACh-induced Ca(2+) response was prolonged by approximately 40 and 80%, respectively. Tests with the mitochondrial Ca(2+) indicator rhod-2 revealed large increases in [Ca(2+)](m) in response to Tg/ACh applications; this mitochondrial uptake was blocked by Ru360. In cells pre-treated with Ru360, 10nM ACh elicited large global increases in [Ca(2+)](c), compared to control cells in which ACh-induced Ca(2+) signals were localised in the apical region. We conclude that mitochondria are active elements of cellular Ca(2+) homeostasis in pancreatic acinar cells and directly modulate both local and global calcium signals induced by agonists.