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.     

Ratiometric imaging of calcium during ischemiareperfusion injury in isolated mouse hearts using Fura-2

ABSTRACT: BACKGROUND: We present an easily implementable method for measuring Fura-2 fluorescence from isolated mouse hearts using a commercially available switching light source and CCD camera. After calibration, it provides a good estimate of intracellular [Ca2+] with both high spatial and temporal resolutions, permitting study of changes in dispersion of diastolic [Ca2+], Ca2+ transient dynamics, and conduction velocities in mouse hearts. In a proof-of-principle study, we imaged isolated Langendorff-perfused mouse hearts with reversible regional myocardial infarctions. METHODS: Isolated mouse hearts were perfused in the Landendorff-mode and loaded with Fura-2. Hearts were then paced rapidly and subjected to 15 minutes of regional ischemia by ligation of the left anterior descending coronary artery, following which the ligation was removed to allow reperfusion for 15 minutes. Fura-2 fluorescence was recorded at regular intervals using a high-speed CCD camera. The two wavelengths of excitation light were interleaved at a rate of 1 KHz with a computer controlled switching light source to illuminate the heart. RESULTS: Fura-2 produced consistent Ca2+ transients from different hearts. Ligating the coronary artery rapidly generated a well defined region with a dramatic rise in diastolic Ca2+ without a significant change in transient amplitude; Ca2+ handling normalized during reperfusion. Conduction velocity was reduced by around 50% during ischemia, and did not recover significantly when monitored for 15 minutes following reperfusion. CONCLUSIONS: Our method of imaging Fura-2 from isolated whole hearts is capable of detecting pathological changes in intracellular Ca2+ levels in cardiac tissue. The persistent change in the conduction velocities indicates that changes to tissue connectivity rather than altered intracellular Ca2+ handling may be underlying the electrical instabilities commonly seen in patients following a myocardial infarction.     

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 intramitochondrial pCa and pH by fura-2 and 2,7 biscarboxyethyl-5(6)-carboxyfluorescein (BCECF) fluorescence

Isolated heart mitochondria hydrolyze the acetoxymethyl esters of the Ca2+-sensitive fluorescent probe fura-2 and the fluorescent pH indicator biscarboxyethyl-5(6)-carboxyfluorescein (BCECF). The free acid forms of both probes are retained in the matrix and their fluorescence can be used to monitor the pCa and pH, respectively, of this compartment. When fura-2 loaded rat heart myocytes are lysed with digitonin, a portion of the dye is retained in the mitochondrial fraction and its fluorescence reports the uptake and release of Ca2+ by the mitochondria. It is concluded that fura-2 and BCECF may report mitochondrial as well as cytosol parameters when the probes are used in intact cells.     

19F nuclear magnetic resonance studies of free calcium in heart cells.

19F nuclear magnetic resonance is used in conjunction with 5,5'-difluoro-1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (5FBapta), a fluorinated calcium chelator, to report steady-state intracellular free calcium levels ([Ca2+]i) in populations of resting, quiescent, isolated adult heart cells. 31P nuclear magnetic resonance shows that 5FBapta-loaded cells maintain normal intracellular high-energy phosphates, pH, and free Mg2+. The intracellular free calcium concentration of well perfused, isolated heart cells is 61 +/- 5 nM, measured with 5FBapta, which has a dissociation constant (Kd) for calcium chelation of 500 nM. A similar value is obtained with Quin-MF, another fluorinated calcium chelator with Kd and maximum calcium sensitivity at 80 nM. We find that the steady-state level of intracellular free calcium is increased by decreased extra-cellular sodium concentration, omission of extracellular magnesium, decreased extracellular pH, hyperglycemia, and upon treatment with lead acetate. Further, extracellular ATP caused a large transient increase in [Ca2+]i. Thus, while heart cells maintain a very low level of intracellular free Ca2+, acute alterations in extracellular environment can cause derangement of calcium homeostasis, resulting in measurable increases in [Ca2+]i.     

Continuous measurement of the cytoplasmic pH in Lactococcus lactis with a fluorescent pH indicator

The cytoplasmic pH of Lactococcus lactis was studied with the fluorescent pH indicator 2',7'-bis-(2-carboxyethyl)-5 (and-6)-carboxyfluorescein (BCECF). A novel method was applied for loading bacterial cells with BCECF, which consists of briefly treating a dense cell suspension with acid in the presence of the probe. This results in a pH gradient, which drives accumulation of the probe in the cytoplasm. After neutralization the probe was well retained in cells stored on ice. BCECF-loaded cells were metabolically active, and were able to generate a pH gradient upon energization. The probe leaks out slowly at elevated temperatures. Efflux is stimulated upon energization of the cells, and is most likely catalyzed by an active transport system. It is a first-order process, and the rate constant could be deduced from the decrease of the fluorescence signal in periods of constant intracellular pH. This allowed a correction of the fluorescence signal for efflux of the probe. After calibration the cytoplasmic pH could be calculated from efflux-corrected fluorescence traces.     

Effects of peroxide on the fluorescence of the Ca2+ probe Fluo 3 and the pH probe BCECF

Fluorescence probes are invaluable tools in monitoring intracellular ion concentrations. They have also been used for studying how reactive oxygen species alter these concentrations and yet there are no studies indicating how reactive oxygen species directly affect the characteristics of the probes. Our concern was that if reactive oxygen were to affect characteristics of these probes, these measurements would be inconsequential. Therefore, we examined the effects of peroxide on the Ca²⁺-sensitive dye Fluo 3 and the pH sensitive dye BCECF. Peroxide concentrations below 10 mM did not alter fluorescence or binding characteristics of either dye. Since the concentrations of peroxide used in most pathophysiological experiments are in the micromolar range, we conclude that these probes are appropriate for monitoring the effects of peroxide on intracellular ion concentrations.     

Estimation of matrix pH in isolated heart mitochondria using a fluorescent probe

Isolated heart mitochondria hydrolyze the acetoxymethyl esters of the Ca2+-sensitive fluorescent probe fura-2 and the pH-sensitive 2',7'-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF). The resulting charged forms of the probes are retained in the mitochondrial matrix and appear well-suited for the estimation of pCa and pH in this compartment. The mitochondria esterase activity is stimulated by Ca2+, inhibited by butacaine and quinine, and shows an alkaline pH optimum. The esterase has a similar affinity for the two probes (Km about 1.5 microM) and a somewhat higher Vmax for BCECF. Intramitochondrial pH can be determined by recording the ratio of the fluorescence of matrix BCECF at its excitation maximum of 509 nm to that at 450 nm, an excitation wavelength that is unresponsive to pH. A calibration plot relating the fluorescence ratio to pH is constructed using detergent-lysed mitochondria and the excitation maximum of 500 nm for BCECF in aqueous solution. Estimates of matrix pH by BCECF fluorescence in its useful range (pH 6 to 8) agree well with values obtained using the distribution of 5,5-dimethyl-2,4-oxazolidenedione. In protocols in which the fluorescence with excitation at 450 nm does not vary, a direct recording of BCECF fluorescence with excitation at 509 nm can be used to follow the kinetics of matrix pH changes.     

Determination of Fura-2 dissociation constants following adjustment of the apparent Ca-EGTA association constant for temperature and ionic strength

The accurate calibration of Fura-2 fluorescence in living cells is dependent upon the apparent dissociation constant (Kd) of Fura-2 for Ca2+. If Ca-EGTA calibration buffers are used to construct an in vitro calibration curve, then the calculated value of the apparent Ca-EGTA association constant (K'CaEGTA) will have an important influence on the Kd of Fura-2 and thus the calculated free [Ca2+] in cells. In order to simulate experimental conditions, the individual proton and Ca2+ association constants for EGTA in these experiments were adjusted for both ionic strength and temperature using a semi-empirical form of the Debye-Huckel limiting law and the Van't Hoff isochore, respectively, as described by Harrison and Bers. The modified individual binding constants were then employed in the calculation of K'CaEGTA using the SPECS computer program of Fabatio. At pH = 7.05, ionic strength = 0.15 M, temp = 20 degrees C, K'CaEGTA = 3.232 x 10(6) M-1; at pH = 6.84, temp = 36 degrees C, K'CaEGTA = 1.652 x 10(6) M-1. These values differed substantially from those obtained with unadjusted individual association constants. Calibration buffers of varying [Ca2+] were prepared using the corrected values of K'CaEGTA, and Fura-2 fluorescence ratios were measured during superfusion of these buffers in the experimental chamber at both 20 degrees C and 37 degrees C. The Kd of Fura-2 for Ca2+ was determined to be 236 nM at 20 degrees C and 285 nM at 37 degrees C, utilizing the value of K'CaEGTA adjusted by the method of Harrison and Bers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ionic selectivity of low-affinity ratiometric calcium indicators: mag-Fura-2, Fura-2FF and BTC

Accurate measurement of elevated intracellular calcium levels requires indicators with low calcium affinity and high selectivity. We examined fluorescence spectral properties and ionic specificity of three low-affinity, ratiometric indicators structurally related to Fura-2: mag-Fura-2 (furaptra), Fura-2FF, and BTC. The indicators differed in respect to their excitation wavelengths, affinity for Ca2+ (Kd approximately 20 microM, 6 microM and 12 microM respectively) and selectivity over Mg2+ (Kd approximately 2 mM for mag-Fura-2, > 10 mM for Fura-2FF and BTC). Among the tested indicators, BTC was limited by a modest dynamic range upon Ca2+ binding, susceptibility to photodamage, and sensitivity to alterations in pH. All three indicators bound other metal ions including Zn2+, Cd2+ and Gd3+. Interestingly, only in the case of BTC were spectral differences apparent between Ca2+ and other metal ions. For example, the presence of Zn2+ increased BTC fluorescence 6-fold at the Ca2+ isosbestic point, suggesting that this dye may be used as a fluorescent Zn2+ indicator. Fura-2FF has high specificity, wide dynamic range, and low pH sensitivity, and is an optimal low-affinity Ca2+ indicator for most imaging applications. BTC may be useful if experimental conditions require visible wavelength excitation or sensitivity to other metal ions including Zn2+.     

The slowing of Ca2+ signals by Ca2+ indicators in cardiac muscle.

The use of high-affinity fluorescent probes for monitoring intracellular free Ca2+ in cardiac muscle is now widespread. We have investigated the consequences of introducing intracellular buffers with the properties of Fura-2 or Indo-1 on the action potential, Ca2+ transient and contractile activity of the myocardium. Our theoretical results suggest that, at the high intracellular concentrations of these fluorescent probes used on occasion to improve the signal-to-noise ratio of the emitted fluorescence, modulation of action potential profile and attenuation of the amplitudes of the Ca2+ transient and contraction can occur, together with subtle changes in the kinetics of these events.     

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.     

Measurement of the concentration of free cytoplasmic calcium in the process of platelet aggregation using a fluorescent method

A multiwavelength method for measuring free cytosolic calcium concentration is proposed. It is based on the registration of the fluorescent spectrum of calcium--sensitive probe indo-1 and deconvolution of the spectrum into components corresponding to free and bound forms of the probe. Calcium concentration is calculated as a product of calcium-probe dissociation constant by calcium-bound to free form concentration ratio. The obtained values are independent of variations in light-scattering properties of the medium and total dye concentration in the optical channel. It is shown that during ADP-induced platelet aggregation calcium concentration rises without measurable delay after the addition of the inducer and significantly decreases by the time the aggregation begins.     

Free calcium concentrations in bullfrog rods determined in the presence of multiple forms of Fura-2.

We employed the fluorescent calcium indicator Fura-2, loaded into intact retinas of the bullfrog Rana catesbeiana, to measure free calcium concentrations in the rod outer-segment cytosol. We determined that traditional methods of calculation yielded erroneous values of calcium. This error results from the presence of at least two distinct pools of Fura-2 in rod outer segments. Application of manganese quenches each pool, but quenching occurs at different rates. Using this fact, we show that the pools can be isolated by brief exposure to manganese and examined separately. One of these pools has the same fluorescent properties as the free salt of Fura-2 we use in our in vitro calibrations. The other source of fluorescence has more unusual properties. Although insensitive to calcium concentrations in the physiological range, it contributes significant anomalous fluorescence when cytosolic free calcium concentrations are elevated by application of IBMX. Nevertheless, the experimentally isolated, classic pool of Fura-2 is well behaved and allows us to calculate calcium concentrations relative to the Kd of Fura-2 by the usual ratio method. We show that when rods are exposed to saturating light, the free calcium concentration in their outer segments falls to a level not significantly different from zero within 20-30 s.     

Alpha-stat calibration of indo-1 fluorescence and measurement of intracellular free calcium in rat ventricular cells at different temperatures.

To explore how to manage pH when calibrating Ca2+ probes at different temperatures, the dissociation constant (Kd) of indo-1 was determined both in pH-stat (pH is fixed despite the temperature) and in alpha-stat (pH changes with temperature as in cells). The results showed that the Kd was much more sensitive to temperature in pH-stat than in alpha-stat, demonstrating that alpha-stat calibration should be preferred when using a Ca2+ probe to measure intracellular free calcium ([Ca2+]i) at different temperatures. Based on the calibration in situ and in alpha-stat, we showed a striking increase of [Ca2+]i from 141+/-8 nM at 30 degrees C to 218+/-22 nM at 10 degrees C in indo-1 loaded rat ventricular cells, which supports that intracellular calcium overload takes place in cardiac myocytes of non-hibernating mammals during hypothermia.     

Direct measurement of cytosolic calcium and pH in living Chlamydomonas reinhardtii cells

Intracellular free Ca2+ and H+ were quantified in Chlamydomonas reinhardtii, using the fluorescent ion indicators Fura-2 and BCECF. We demonstrate that both indicators can be loaded into living cells as acetoxymethylesters. The esters were hydrolyzed intracellularly to genuine Fura-2 and BCECF capable of indicating changes in Ca2+i and H+i. Fura-2 accumulated in the cytoplasm to a concentration of 50 microM, whereas BCECF reached a concentration of 200 microM. The average Ca2+i was estimated to be 180 +/- 40 nM and the average pHi was 7.4 +/- 0.1. To document the applicability of the ion indicators in Chlamydomonas, we tested their responses to several stimuli. We observed increases in cytoplasmic Ca2+ in response to elevated external Ca2+ on membrane-permeable acids, which are known to induce flagellar excision in Chlamydomonas. The membrane-permeable acids caused a decrease in cytoplasmic pH. Pulses of photosynthetically active light lead to transient pHi changes. Finally, concomitant measurements of rhodopsin-triggered and voltage-sensitive photocurrents indicated that Ca2+ influx is accompanied by a transient depolarisation of the plasmalemma. These experiments document that Fura-2 and BCECF are versatile dyes for studying various ionic processes in Chlamydomonas.     

Determination of intracellular pH of BALB/c-3T3 cells using the fluorescence of pyranine

In terms of accuracy and sensitivity, intracellularly trapped, pH-dependent fluorescent probes are appropriate to accurately measure intracellular pH. These probes are commonly introduced into living cells in esterified form, wherein the free acid is produced through enzymatic hydrolysis. The fluorescence characteristics of the ester and the free acid can differ markedly and spectral uncertainty can occur. We describe here the measurement of intracellular pH using 8-hydroxypyrene-1,3,6-trisulfonic acid (pyranine) that has been scrape-loaded into BALB/c-3T3 mouse cells. The excitation spectrum of pyranine is pH sensitive, with an isosbestic point at 415 nm and peaks at 405 and 465 nm which decrease and increase with pH, respectively. The 465/405 ratio can be used to monitor the pH, while the fluorescence at 415 nm indicates the total dye-dependent signal remaining. The scrape-loaded dye persists in cells for periods up to 6 h. We have calibrated this dye in situ using nigericin/high K+, and have found that the pKa of the dye in situ is 7.82, as compared to 7.68 in vitro. We have observed that the cells can slowly equilibrate their intracellular pH to near control levels when presented with either an acute alkaline or acid load.     

应用AR—CM—MIC阳离子测定系统检测单个神经元内游离钙

运用Ca~(2 )指示剂Fura-2作为细胞内钙离子的荧光探针,采用精密的AR-CM-MIC阳离子测定系统,检测了分离的单个神经细胞内游离钙离子浓度的动态变化,同时观察了钙离子载体、钙螯合剂等多种药物对细胞内钙浓度的影响,并追踪刺激前后的瞬间变化,探讨此项技术应用于检测细胞内游离钙的灵敏度及适用范围,取得了良好的效果。     

On experimental artifacts in the use of metal ion chelators for the determination of the cation binding constants of alpha-lactalbumin. A reply

The binding constant of Ca2+ to the strong cation site of bovine alpha-lactalbumin has been measured directly by monitoring the free calcium concentration by Quin 2 fluorescence. A dissociation constant of 1-4 nM was calculated, which confirms the strong calcium binding properties of this protein. In order to examine whether the metal ion chelators EDTA or EGTA affect the cation binding equilbria by binding to bovine alpha-lactalbumin, calcium binding equilibria were carefully measured under highly stabilized pH and temperature conditions. Within the concentration ranges required for competitive binding by these ligands (EDTA or EGTA) (less than 1-3 mM) these chelators produced no artifacts, in contradiction to the data of Kronman and Bratcher (Kronman, M. J., and Bratcher, S. C. (1983) J. Biol. Chem. 258, 5707-5709).     

On the noninvasive measurement of intracellular free magnesium by 31P NMR spectroscopy

We previously introduced a noninvasive measurement of the concentration of free Mg2+ in intact cells and tissues using 31P NMR. To resolve a controversy in the literature concerning the affinity of Mg2+ for ATP used in our procedure, the apparent dissociation constant of MgATP under simulated intracellular conditions has been determined by three independent magnetic resonance methods, including a newly developed combination procedure for determining this value at intracellular ATP levels. The new combination method, which utilizes 31P NMR to determine the degree of Mg2+ chelation of ATP and the dye antipyrylazo III for optical determination of free Mg2+, yielded a value of (50 +/- 10) microM for this apparent dissociation constant at pH 7.2 in the presence of 0.15 M K+ and 25 degrees C. We further show that hydroxyquinolines are not satisfactory indicators for optical determination of the Mg2+-nucleotide dissociation constant. From our determinations a low value of free Mg2+ (less than 1 mM) is established for all of the tissues studied, including perfused heart muscle, contrary to a recent report in the literature. Saturating human erythrocytes with Mg2+ results in an alpha- and beta-phosphorus resonance separation for intracellular ATP that is indistinguishable from that observed in a noncellular MgATP control under similar conditions, showing that MgATP resonances in this cell are unaffected by the cellular environment.