Skip Navigation

Cardiovascular Research 2003 59(3):695-704; doi:10.1016/S0008-6363(03)00467-X
© 2003 by European Society of Cardiology
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Stamm, C.
Right arrow Articles by del Nido, P. J
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Stamm, C.
Right arrow Articles by del Nido, P. J
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Copyright © 2003, European Society of Cardiology

Cytosolic calcium in the ischemic rabbit heart: assessment by pH- and temperature-adjusted rhod-2 spectrofluorometry

Christof Stamma,*, Ingeborg Friehsb, Yeong-Hoon Choib, David Zurakowskic, Francis X McGowand and Pedro J del Nidob

aUniversität Rostock, Department of Cardiac Surgery, Schillingallee 35, 18057 Rostock, Germany
bChildren’s Hospital Boston, Department of Cardiac Surgery, Harvard Medical School, 300 Longwood Avenue, Boston, MA 02115, USA
cDepartment of Biostatistics, Harvard Medical School, 300 Longwood Avenue, Boston, MA 02115, USA
dDepartment of Anesthesiology, Harvard Medical School, 300 Longwood Avenue, Boston, MA 02115, USA

christof.stamm{at}med.uni-rostock.de

* Corresponding author. Tel.: +49-381-494-6101; fax: +49-381-494-6102.

Received 2 April 2003; revised 22 May 2003; accepted 28 May 2003


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusions
 References
 
Objectives: Cytosolic calcium ([Ca2+]i) mediates ischemia–reperfusion (I/R) injury, but magnitude and time course of I/R-induced [Ca2+]i overload remain unclear. Fluorescent indicators are commonly used to measure [Ca2+]i, and have a dissociation constant (Kd) that depends on pH and temperature. We hypothesized that changes of Kd during I/R lead to misleading interpretations of [Ca2+]i recordings. Methods: (1) In isolated rabbit hearts (n=4 each), intracellular pH was measured during I/R at 37°C, 20°C, and 4°C with and without cardioplegic arrest by 31P-NMR-spectroscopy. (2) Kd for rhod-2 and calcium was determined at varying pH and temperature in in vitro experiments. (3) Isolated rabbit hearts were subjected to I/R, and [Ca2+]i was recorded by surface rhod-2 spectrofluorometry. Finally, [Ca2+]i was computed using either the conventional Kd, or the pH- and temperature-adjusted Kd. Results: Kd(Ca2+Rhod-2) remained stable between pH 7.1 and 6.8, but increased exponentially with lower pH and/or temperature. Calculations using a static Kd indicated that [Ca2+]i rose only mildly during warm ischemia and did not rise during cardioplegic arrest, while a large Ca2+ influx appeared to occur during early reperfusion. When the pH and temperature-adjusted Kd was used for calculation, [Ca2+]i rose significantly during ischemia (431±37% during 20 min 37°C ischemia, and 78±19% during 20 min cardioplegic arrest at 20°C). During early reperfusion, [Ca2+]i decreased rapidly, without significant further [Ca2+]i elevation. Conclusions: In contrast to previous reports, [Ca2+]i accumulation occurs during unprotected ischemia as well as hypothermic ischemia with cardioplegic arrest, without further net Ca2+ influx on reperfusion. This finding has important implications for timing of protective strategies during myocardial ischemia.

KEYWORDS Calcium (cellular); Ischemia; Cardioplegia; Cardiovascular surgery


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 5. Conclusions
 References
 
It is generally acknowledged that myocardial ischemia–reperfusion injury provokes cytosolic free calcium ([Ca2+]i) accumulation [1,2]. During ischemia, several mechanisms result in failure of intracellular Ca2+ compartmentalization and extrusion from the cell against high concentration gradients. At the onset of reperfusion, intracellular Ca2+ overload is thought to be further exacerbated by uncontrolled influx from the extracellular space, but controversy remains about extent and time course of ischemia–reperfusion-induced changes in calcium homeostasis. The pathophysiologic consequences include energy-depleting futile calcium cycling, impaired relaxation, and systolic contractile dysfunction. Fluorescence indicators are commonly used to assess intracellular Ca2+ handling, and the respective techniques are straightforward in single cells or tissue preparations such as ventricular trabeculae or atrial muscle strips. However, true myocardial ischemia and reperfusion can be experimentally performed only in the intact perfused organ, in which intracellular Ca2+ is much more difficult to measure. The complex methodology problems include light absorbance by chromatic molecules (i.e. myoglobin, hemoglobin), tissue autofluorescence (i.e. NAD+), motion artefacts of the beating heart, and the impact of temperature and pH on Ca2+-induced signal emission. Signal quality greatly depends on the fluorescence indicator used, and each indicator has particular advantages and disadvantages. We have recently introduced the long-wavelength Ca2+ indicator rhod-2 for recording of beat-to-beat Ca2+ transients, because rhod-2 possesses several advantages over other fluorescence indicators that are of particular importance in the heart. However, the signal of any cation-sensitive indicator is affected by changes in intracellular pH as well as temperature, both of which can vary greatly in clinically relevant models of myocardial ischemia and reperfusion [3]. The extent of pH- or temperature-induced signal changes depends on the physical properties and binding affinity for Ca2+ and protons, and is unknown for most of the indicators used today.

In order to achieve a better understanding of intracellular calcium during ischemia in the intact heart, we measured the change in intracellular pH during several ischemia/reperfusion protocols that mimic clinically and experimentally relevant situations, determined the dissociation constant Kd for rhod-2 and Ca2+ at the respective pH and temperature, and determined cytosolic calcium by spectrofluorometry in whole hearts, using the appropriate Kd.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 5. Conclusions
 References
 
Our approach to obtain pH and temperature-corrected measurements of intracellular calcium using rhod-2 included the following steps:

1. Measurement of intracellular pH in isolated rabbit hearts subjected to ischemia at 37°C, 20°C, and 4°C without cardioplegic arrest, and at 20°C with high-potassium cardioplegic arrest, using 31P nuclear magnetic resonance.
2. Determination of the specific Kd for rhod-2 and Ca2+ in series of in vitro experiments at pH 7.1–6.2 in increments of 0.1, each at the above described temperatures.
3. Recording of intracellular Ca2+ signals in intact perfused rabbit hearts using rhod-2 spectrofluorometry, during ischemia at 37°C without cardioplegic arrest and at 20°C with cardioplegic arrest: two commonly used experimental protocols that are of particular clinical relevance.
4. Computation of the intracellular free Ca2+ concentration using the specific pH and temperature-corrected Kd for a given protocol and time point.

2.1 Isolated heart perfusion
The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). New Zealand white rabbits (2–2.5 kg) were euthanized by intravenous injection of ketamine (100 mg/kg), and heparin (500 U/kg). Hearts were rapidly excised and placed in 4°C cold buffer solution. After cannulation of the aorta, hearts were perfused in the Langendorff mode at 80 mmHg constant perfusion pressure with modified Krebs–Henseleit (KH) buffer (115 mmol/l NaCl, 26 mmol/l NaHCO3, 11 mmol/l glucose, 1.8 mmol/l MgSO4, 1.8 mmol/l KH2PO4, 4.7 mmol/l KCl, 1.25 mmol/l CaCl2, and 10 U/l insulin), that had been equilibrated with a 95% O2/5% CO2 gas mixture and passed through a 0.2 micrometer nylon filter. The final buffer pH was 7.35–7.45, PO2 was 550–600 mmHg, and PCO2 was 30–40 mmHg. A fluid-filled latex balloon connected to a micromanometry catheter (Millar Instruments, Houston, TX) was placed in the left ventricle via the left atrium. After 30 min stabilization, the hearts were placed in the respective perfusion chamber for 31P NMR spectroscopy or rhod-2 spectrofluorometry. Global ischemia was induced by occlusion of the aortic cannula. Cardiac temperature was monitored throughout the experiment, including the period of ischemia, using a temperature probe inserted in the right ventricle. During stabilization and reperfusion myocardial temperature was adjusted to 37°C. During the period of ischemia, temperature was maintained at 37°C or lowered to 20°C by adjusting the ambient temperature in the perfusion chamber.

2.2 31P NMR spectroscopy
Hearts were perfused with phosphate-free modified KH buffer using a customized perfusion system. The isolated heart in its perfusion chamber was positioned within a 20-mm solenoid radiofrequency coil. NMR spectra were acquired in an 8.45 Tesla vertical bore Bruker spectrometer (Bruker Instruments, Billerica, MA). Spectra were obtained by signal averaging 120 scans with a 2-s delay, resulting in a time resolution of 4.5 min. Intracellular pH was calculated from the shift of the inorganic phosphate (Pi) peak using the following equation [4]:

Formula
where x is the position of the Pi peak with respect to the PCr peak in p.p.m.

Following a stabilization period of 20–30 min, hearts were subjected to 45 min ischemia at 37°C (n=4), at 20°C (n=4), and at 4°C (n=4). In another group of hearts, cardioplegic arrest was induced by injection of perfusion buffer containing 20 mM K+ (n=4).

2.3 Dissociation constants for rhod-2 and Ca2+
To determine the Kd for rhod-2 and Ca2+ at changing pH and temperature, fluorescence measurements were made in 11 samples of varying Ca2+ solutions containing 10 µM rhod-2, which were prepared for each combination of pH and temperature. The solution contained 100 mM KCl, 1 mM Mg2+, and 0.5 mM myoglobin in order to mimic ionic strength, autofluorescence, and absorption properties of the cardiomyocyte cytosol. The various Ca2+ concentrations were prepared from two stock solutions of known Ca2+ concentration (calcium calibration buffer kit #1 with Mg2+, Molecular Probes) using the method by Tsien and Pozzan [8]. This method is based on the principle that when the concentrations of Ca2+ and EGTA are very close to each other, the only free Ca2+ available is that which is in equilibrium with EGTA. Thus, [Ca2+]free is a function of the dissociation constant of Ca2+EGTA, Kd(Ca2+EGTA), which by itself is dependent on ionic strength, pH, and temperature. Therefore, the effective free Ca2+ concentration of each calibration solution has to be determined based on the specific dissociation constants of EGTA for Ca2+ for each given pH and temperature. Both Kd(Ca2+EGTA) and the free Ca2+ concentration in the undiluted Ca2+EGTA buffer solution at a given temperature and pH were derived using the web-based computer program Webmaxclite v1.00 (Stanford University, http://www.stanford.edu/~cpatton/maxclite.htm). The free Ca2+ concentration in the Ca2+ dilution series obtained by mixing Ca2+EGTA and K2EGTA was then computed according to the equation:

Formula

Once the actual free Ca2+ concentration in each sample was known, the Kd for rhod-2 and Ca2+ was determined. Samples were prepared in a 3 ml quartz cuvette, which was placed in a water-jacketed temperature controlled cuvette holder in the SLM 8100 spectrofluorometer, and all solutions were constantly stirred during fluorescence measurements. Excitation light of 524 nm was used, and emission was recorded between 540 and 620 nm in 1 nm increments with an integration time of 0.5 s (Fig. 1). Fluorescence intensity at 589 nm was then used to construct the rhod-2 calibration curves. The Kd for rhod-2 and Ca2+ was computed based on the actual free Ca2+ concentration in each sample and the resulting fluorescence using automated software supplied by the vendor (Dissociation Constant Calculator, http://www.probes.com/), based on the equation:

Formula


Figure 1
View larger version (18K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1 Intracellular pH measured by 31P NMR spectroscopy in whole hearts subjected to a 45 min period of ischemia at different temperatures with or without high-potassium cardioplegic arrest. Indicated are the mean values of four experiments each and the respective standard deviation.

 
Principally, data are plotted as the log of the [Ca2+]free (x-axis) versus the log [(F–Fmin)/(Fmax–F)] (y-axis). This double-log plot gives an x-intercept that is the log of the Kd for rhod-2 under the given conditions.

2.4 Rhod-2 spectrofluorometry
Measurement of beat-to-beat intracellular Ca2+ transients was performed as we have previously described and validated in detail [5]. During the 30 min stabilization period, hearts were loaded with the Ca2+-sensitive dye rhod-2 AM (Molecular probes, Eugene, OR) by perfusion with the cell-permeable acetoxymethylester (Rhod-2 AM; 0.5 mg/0.25 ml DMSO infused over 2 min without recirculation). Dye-loading was followed by a 15 min washout to remove any extracellular dye. A modified spectrofluorometer (SLM-Aminco, Springfield, IL) was set to provide excitation light at 524 nm and recorded emission light at 589 nm. Recordings were performed with a time-resolution of 2 ms for analysis of single Ca2+ transients (recordings over 5 s) and 40 ms for observance of changes in Ca2+ levels over longer time periods. Since rhod-2 has no spectral shift after Ca2+ binding, it is necessary to account for differences in dye loading or changes in tissue dye concentration over time (leakage or photobleaching). Therefore, tissue absorbance was quantified using the ratio of scattered excitation light at 524 nm (peak rhod-2 absorbance in myocardial tissue) and 589 nm (isosbestic point for rhod-2 in myocardium). The change in absorbance over time was then used to normalize emission light intensity by calculating fluorescence/absorbance (F/A) for each time point. In order to quantify [Ca2+]i, the maximum Ca2+-induced rhod-2 fluorescence (Fmax) must be determined at the end of each experiment because the fluorescence intensity is proportional to the amount of dye loaded. Therefore, Fmax was determined at the end of the experiment as follows: To induce Ca2+ release from the sarcoplasmic reticulum, 2,2'-dithiodipyridine (100 µM) was infused over a period of 2 min, immediately followed by calcium ionophore A23187 [GenBank] (calcimycin) in 10 ml 10% Ca2+ solution to maximize Ca2+ entry from the extracellular space. Fluorescence was recorded with a time resolution of 40 ms during the infusion, and maximum fluorescence was determined to calculate systolic and diastolic [Ca2+]i using the following equation:

Formula
where [Ca2+]i is the free intracellular Ca2+ concentration, Kd is the dissociation constant for rhod-2 with Ca2+, Ft is fluorescence at a specific time point, Fo is background fluorescence measured before dye loading, At and Amax are tissue light absorbance at the specific time point or at the end of the experiment, respectively.

[Ca2+]i was recorded during the following ischemia/reperfusion protocols: 20 min unprotected ischemia at 37°C without cardioplegic arrest followed by 10 min reperfusion (n=6); 20 min ischemia at 20°C with cardioplegic arrest (20 mM K+) followed by 10 min reperfusion. The fluorescence signal was then used to calculate [Ca2+]i in 5 min intervals, using the Kd(Ca2+Rhod-2) of 710 nM for every time point. This is the Kd(Ca2+Rhod-2) in a simulated intracellular myocardial environment that was determined by del Nido et al. when they first described rhod-2 spectrofluorometry for measurement of [Ca2+]i in isolated rabbit hearts [5]. Then, the calculation was repeated, but now the adjusted Kd for a given pH (derived from the NMR experiments) and temperature (according to the experimental protocol) was used for each time point.

2.5 Statistics
Linear and nonlinear regression analysis was utilized to determine the best fitting models for describing the relationship between pH and Kd at a given temperature. The Levenberg–Marquardt method was applied to derive nonlinear models. The coefficient of determination (R2) was used to assess the proportion variability in Kd accounted for by pH in each regression model with goodness-of-fit evaluated by the F-test. SPSS statistical package (version 11.0) was used.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 5. Conclusions
 References
 
3.1 Intracellular pH
As depicted in Fig. 1, pH decreased during ischemia. The extent of intracellular acidification depended largely on the temperature, reaching pH 6.05 after 45 min ischemia at 37°C, pH 6.4 at 20°C, and pH 6.6 at 4°C. Ischemia at 20°C with cardioplegic arrest resulted in mild acidification, reaching pH 6.8 after 45 min. Irrespective of temperature or cardioplegic arrest, pH increased rapidly during early reperfusion, reaching near-normal levels between pH 7.0 and pH 7.1 at 5 min reperfusion in all groups.

3.2 Kd for rhod-2 and Ca2+
Fig. 2 depicts a typical series of in vitro rhod-2 fluorescence emission scans, recorded at increasing free Ca2+ concentrations (here: pH 7.1, 20°C). As expected, increasing Ca2+ in the solution results in an increase in fluorescence intensity. The impact of temperature on Ca2+-induced rhod-2 fluorescence is demonstrated in Fig. 3A. Lowering temperature from 37°C to 20°C resulted in a marked rightward shift of the Ca2+-fluorescence relationship, indicating that less rhod-2 fluorescence is induced at a given Ca2+ concentration. Further reducing temperature to 4°C increased the rightward shift moderately. Hence, it should be noted that lowering temperature from 37°C to room temperature, as easily happens in experimental settings studying ischemic and thus non-perfused hearts, has a significant effect on intensity of the fluorescence signal. The relationship between pH and Ca2+-induced rhod-2 fluorescence at 37°C is shown in Fig. 3B. It is evident that lowering pH from pH 7.1 to pH 6.8 had no significant effect on Ca2+-induced rhod-2 fluorescence, both curves are virtually congruent. At pH 6.5, however, a marked rightward shift was apparent, again indicating a reduction in Ca2+-induced rhod-2 fluorescence, and this rightward shift was exacerbated by further acidification of the solution to pH 6.2. It can thus be assumed that mild intracellular acidification, as occurs i.e. during a brief period of ischemia with cardioplegic arrest, has little impact on the rhod-2 signal intensity.


Figure 2
View larger version (24K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2 Rhod-2 fluorescence emission spectra measured at increasing calcium concentration in a simulated intracellular environment at a given pH and temperature (here: pH 7.1, 20°C. Fluorescence intensity at 589 nm (interrupted line) was then used to determine the dissociation constant for rhod-2 and calcium.

 

Figure 3
View larger version (18K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3 Fluorescence intensity measured at increasing calcium concentrations in a simulated intracellular environment at varying temperature (A) and at different pH (B). The y-axis depicts fluorescence emission at 589 nm during excitation at 524 nm. pH or temperature-corrected free calcium concentrations of the EGTA-buffered calibration solution were used on the x-axis. Note that a lowering temperature from 37°C to 20°C results in a significant rightward shift of the curve, indicating a decrease in fluorescence emitted at a given calcium concentration (A). The emission signal curves at pH 7.1 and pH 6.8 are virtually congruent, whereas a rightward shift is apparent at pH 6.5 and pH 6.2 (B). The depicted data were obtained in single preparation. To ensure reproducibility, the Kd measurements were repeated twice and gave virtually identical values.

 
Once the actual Kd for rhod-2 and Ca2+ was determined for each pH increment at a given temperature, we plotted the relationship between Kd(Ca2+Rhod-2) and pH (Fig. 4). At 37°C and pH 7.1, Kd was calculated as 480 nM, and remained more or less stable up to pH 6.8 (Kd=484 nM). With further lowered pH, Kd rose steeply, was determined to equal 2459 nM at pH 6.2. A quadratic model had the highest R2 among the regression models tested (R2=0.996) and represented the most accurate description of the relationship between pH and Kd (F-test=961.5, P<0.0001). It is described by the following equation:

Formula


Figure 4
View larger version (10K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4 Relationship between the dissociation constant for rhod-2 and calcium and intracellular pH in whole hearts at 37°C (A) and at 20°C (B). Depicted are measured values (open circles) and the quadratic regression (see Results section for model characteristics). Note that Kd remains relatively stable with acidifying pH up to pH 6.6. When intracellular pH drops further, Kd rises, indicating a decreasing affinity of rhod-2 for calcium. The depicted data were obtained in single preparation. To ensure reproducibility, the Kd measurements were repeated twice and were virtually identical.

 
At 20°C and pH 7.1, Kd was 935 nM, and again remained stable up to pH 6.8 (Kd=877 nM). Then, the curve rose parallel to that at 37°C, reaching 4.8 µM at pH 6.2. Again, a quadratic model best described the relationship between pH and Kd (R2=0.995, F-test=711.9, P<0.0001):

Formula

At 4°C and pH 7.1, however, Kd was calculated as 10-fold higher than at 37°C (4354 nM), remained at that level up to pH 6.8, and then increased even more. Because the high Kd does not permit accurate estimation of [Ca2+]i at 4°C the data were not further analyzed.

3.3 [Ca2+]i measurements
Fig. 5A depicts the rhod-2 fluorescence recorded from the LV surface of an intact perfused rabbit heart at 589 nm during 20 min ischemia at 37°C, followed by 10 min reperfusion. Signal intensity rises by approximately 100% during the ischemic period. On reperfusion, there is a rapid and pronounced increase in fluorescence, followed by a rather rapid decrease over the next 5 min. When [Ca2+]i is calculated after determining Fmax, using a constant Kd (here: 710 nM) (Fig. 5B), mean [Ca2+]i appears to increase only mildly during ischemia, followed by an immediate increase on early reperfusion. However, when the pH-adjusted Kd is used to quantify [Ca2+]i at each time point (Fig. 5C), it becomes evident that [Ca2+]i rises by almost 400% throughout the period of ischemia (431±37% in four experiments), followed by a rapid decrease during early reperfusion, without reaching pre-ischemic levels during the first 10 min of reperfusion. Beat-to-beat Ca2+ transients during 15 min unprotected ischemia are shown in Fig. 6. As mean [Ca2+]i increases, the amplitude of the Ca2+ transient decreases. After 20 min ischemia, no rhythmic Ca2+ transients were recordable. In order to mimic the cardiac surgical situation of cardioplegic arrest at lowered temperature, we recorded cytosolic Ca2+ in rabbit hearts subjected to 20 min ischemia at room temperature with high-potassium cardiac arrest. As shown in Fig. 7A, injection of the cardioplegic solution effectively abolished cytosolic Ca2+ transients. Over the following period of ischemia at 20°C, the rhod-2 fluorescence signal increased only by approximately 10%. On reperfusion, there was again a rapid increase in signal intensity, followed by a decline over the next 5 min. When [Ca2+]i was then calculated using a fixed Kd of 710 nM, the plot depicting mean [Ca2+]i (Fig. 7B) follows the raw fluorescence signal in parallel. However, when the pH-adjusted Kd is used in to determine the actual cytosolic Ca2+ concentration, it becomes clear that [Ca2+]i increases by approximately 80% during ischemia (78±19% in four experiments), and decreases immediately on reperfusion (Fig. 7C).


Figure 5
View larger version (17K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 5 (A) Rhod-2 fluorescence at 589 nm recorded from the left ventricular surface of a Langendorff-perfused rabbit heart subjected to 20 min ischemia at 37°C (interrupted lines). (B) Cytosolic calcium concentration was calculated using the conventional dissociation constant for rhod-2 and calcium in an intracellular environment, 710 nM. Note that the maximum rise in Ca2+i appears to occur during early reperfusion. (C) Here, cytosolic calcium concentration was computed using the pH-adjusted dissociation constant for rhod-2 and calcium at 37°C. Note that here, cytosolic calcium increases markedly during the period of ischemia, and decreases during early reperfusion.

 

Figure 6
View larger version (17K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 6 Calcium transients recorded from the LV surface of a heart subjected to 15 min normothermic ischemia. (A) The cytosolic calcium concentration was calculated at each time point using the pH-adjusted dissociation constant (Kd) for rhod-2 and calcium. (B) [Ca2+]i as calculated using a constant Kd (here: 710 nM).

 

Figure 7
View larger version (15K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 7 (A) Rhod-2 fluorescence at 589 nm recorded from the left ventricular surface of a Langendorff-perfused rabbit heart subjected to 20 min ischemia with high-potassium cardioplegic arrest at 20°C (interrupted lines). (B) Cytosolic calcium concentration was calculated using the conventional dissociation constant for rhod-2 and calcium in an intracellular environment, 710 nM. Note that [Ca2+]i does not seem to rise during ischemia, but a large increase in [Ca2+]i appears to occur during early reperfusion. (C) Here, the cytosolic calcium concentration was computed using the pH-adjusted dissociation constant for rhod-2 and calcium at 37°C. Note that cytosolic calcium increases mildly but steadily throughout the period of ischemia, and decreases during early reperfusion.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
5. Conclusions
 References
 
As expected, based on the physicochemical properties of a Ca2+ indicator dye such as rhod-2, we found the dissociation constant Kd for rhod-2 and Ca2+ to be dependent on pH and temperature. At lower pH, accumulating protons compete with Ca2+ ions at the binding sites resulting in a net decrease of Ca2+ affinity of the dye, while lower temperature decreases the quantum efficiency of the dye, i.e. blunting the fluorescence intensity emitted when a Ca2+ ion binds to rhod-2. Both mechanisms inevitably increase the effective Kd for rhod-2 and Ca2+. Hence, if the fluorescence signal appears not to change while intracellular pH and/or temperature drop, although [Ca2+]i does in fact rise. Or, in other words, the actual increase in [Ca2+]i during ischemia is systemically underestimated if Kd is not adjusted according to the changes in pH or temperature. The pathophysiologic relevance is that, in contrast to previous reports based on Ca2+ recordings quantified using a static Kd, [Ca2+]i mainly rises during the period of ischemia and not during early reperfusion. Based on our findings, the increase in signal intensity on reperfusion is caused by the rapidly normalizing intracellular pH (and thus decreasing Kd). The clinical implication is that strategies aiming at inhibiting the rise in [Ca2+]i during ischemia may be more effective than those that focus on the early reperfusion period.

Various techniques to assess the [Ca2+]i have been developed. NMR measurements using BAPTA-based Ca2+-sensitive indicators first allowed for observation of changes in [Ca2+]i over time [6], and beat-to-beat recordings of Ca2+ transients became possible using fluorescent or luminescent indicators. Measurements of intracellular Ca2+ handling during whole-organ ischemia and reperfusion have been reported since the late 1980s using fluorescent indicators such as Indo-1, Fura-2, Fluo-3, or the visible light-emitting protein aequorin. Although we focused on rhod-2, it is important to acknowledge that the principles of pH- and temperature depending Kd apply to all ion-sensitive indicator molecules. Following Lattanzio and Pressman’s recordings of intracellular Ca2+ transients using the first-generation indicator Quin-2 [7,8], Lee et al. established the measurement of [Ca2+]i in intact rabbit hearts using Indo-1 [9]. They described a rapid increase of systolic and diastolic [Ca2+]i during the first 30 s of global normothermic ischemia that reached a plateau after 90 s. Subsequently, the same group described the changes in [Ca2+]i during 2–3 min ischemia in greater detail [10]. Again, they observed a rapid increase of the Indo-1 fluorescence ratio that appeared to reach a plateau in less than 60 s. However, they did not attempt to quantify [Ca2+]i using the dissociation constant, assuming that the raw fluorescence signal correlates closely with [Ca2+]i. Since all BAPTA-based indicators are to some extent pH sensitive, the investigators performed a series of experiments lowering pH in a mixture of Ca2+ and Indo-1, and no change in fluorescence intensity was obvious. In response, Lattanzio systematically studied the pH dependency of the Kd for Indo-1 and Ca2+ in the setting of myocardial acidosis [11,12]. By loading isolated rat hearts with Indo-1 and the pH sensitive indicator BCECF, he demonstrated that a rise of mean [Ca2+]i from 170 nM to 390 nM (230%) during 8 min global ischemia was not reflected by the Indo-1 fluorescence ratio, because the simultaneous drop in pH decreased the affinity of Indo-1 for Ca2+. Jimenez et al. described the redistribution of Ca2+ between the membranous and cytosolic compartment during 60 min global ischemia using fura-2, and did not observe a significant rise of [Ca2+]i at 20 min [13]. However, these measurements were not corrected for pH-induced changes in Kd either, and hence the actual increase in [Ca2+]i was probably underestimated. More recently, Morgan and coworkers studied the intracellular Ca2+ homeostasis during ischemia and reperfusion using the bioluminescent protein aequorin, which is injected subepicardially and emits photons as visible light upon binding to Ca2+. Using this technique, Kihara et al. recorded [Ca2+]i in isolated ferret hearts during 3 min global normothermic ischemia, and observed only a mild increase in [Ca2+]i [14]. Later, the technique was adapted to rat hearts, and they observed an initial increase in Ca2+ transient amplitude during 10 min global ischemia, followed by a very mild increase in diastolic [Ca2+]i [15]. During reperfusion, however, a rapid increase in [Ca2+]i was described and interpreted as Ca2+ influx from the extracellular space. Subsequently, the aequorin technique was also used in isolated mice hearts [16]. Again, only a mild increase in [Ca2+]i during the ischemic period of 15 min was described, followed by a significant increase in signal intensity on early reperfusion. The physicochemical properties of the protein aequorin are of course not comparable with those of the BAPTA-based fluorescence indicators, and a systematic investigation of pH and temperature dependency of the dissociation constant for aequorin and Ca2+ has not been reported. However, it can be assumed that the affinity of aequorin for Ca2+ is also pH-sensitive, and that its quantum efficiency is a function of the ambient temperature. If this is indeed the case, one can infer that the true increase of [Ca2+]i in these studies was also systematically underestimated, while the apparent rapid [Ca2+]i increase on reperfusion is in fact a function of the normalizing intracellular pH.

In the majority of the studies investigating post-ischemic Ca2+ homeostasis, unprotected normothermic ischemia is utilized to simulate clinical myocardial ischemia. However, various cardioprotective measures are routinely used during cardiac surgical procedures, but little is known about the effects of cardioplegic arrest and hypothermic ischemia on intracellular Ca2+ handling. When we measured [Ca2+]i during cardioplegic arrest at 20°C, only a very mild increase was noted when a constant Kd was used for calculation. By using the appropriate pH and temperature corrected Kd at a given time point, it became evident that, in fact, there was an 80% increase in [Ca2+]i. This indicates that even routinely used methods of cardioprotection are associated with a significant intracellular Ca2+ overload. Determination of [Ca2+]i at low temperature, however, is not possible with current methods. The exceedingly high Kd for rhod-2 and Ca2+ is probably due to the low quantum efficiency of the dye at low temperatures, but perhaps also due to altered binding kinetics for Ca2+ at the binding site in the rhod-2 molecule that decreases the affinity of the dye for Ca2+. Whatever the mechanism, the effective Kd is too high to allow for an assessment of [Ca2+]i with reasonable accuracy.

4.1 Rhod-2 for measurement of [Ca2+]i
Rhod-2, which was first introduced in 1989, is a typical modern long-wavelength indicator that is valuable for experiments in cells and tissues with high levels of autofluorescence [17]. Rhod-2 fluorescence intensity increases >100-fold upon binding Ca2+, and a good signal-to-noise ratio can be achieved with very low intracellular rhod-2 concentrations. The estimated intracellular rhod-2 concentration in our model in less than 2 µM, compared with millimolar concentrations of older indicators such as Quin-2, or typically 20–30 µM with fura-2 [18]. The superior signal quality of rhod-2 allows for real-time recordings with extremely high spatial and/or temporal resolution in tissue [19,20]. For example, Qian et al. recorded high-quality Ca2+ transients with an imaging frequency of 300 frames/s in blood-perfused rabbit hearts and were able to show marked spatial heterogeneity of the Ca2+ transient during the early phase of ischemia [21]. A fluorescent Ca2+ indicator should have an appropriate Ca2+ affinity, ideally somewhat higher than the basal [Ca2+]i. To high an affinity (i.e. low Kd) will lead to significant Ca2+ buffering and hence disturb intracellular Ca2+ homeostasis, and too low an affinity (i.e. high Kd) will necessitate excessively high indicator concentrations to achieve a good enough signal. The rhod-2 Kd of approximately 500–700 nM under baseline conditions is ideal for measurements in cardiomyocytes, and gives a very good signal-to-noise ratio that allows for recording of single transients with time resolutions up to 2 ms and their analysis without the need for signal averaging techniques

4.2 Limitations of the study
There are several limitations to our study that are important to acknowledge. Intracellular pH and Ca2+ were not measured simultaneously but in separate sets of experiments using different methods (31P NMR spectroscopy and rhod-2 spectrofluorometry). However, the change in intracellular pH is usually very uniform in a given ischemia–reperfusion protocol, and the inference made should at least allow for proof-of-principle. The cytosolic concentration of other intracellular cations such as Mg2+ or Na+ also change significantly during myocardial ischemia. While a changing Na+ concentration in a biological system is unlikely to influence the affinity of indicator for Ca2+, the bivalent cations Mg2+ probably competes significantly with Ca2+ at the binding site. The cytosolic Mg2+ concentration is known to increase markedly during ischemia, and we did not adjust for this change in our experiments. Furthermore, oxygen free radicals, which are produced in abundance during ischemia and reperfusion, may also interfere with the binding affinity of a fluorescent indicator, but the magnitude of this effect remains unclear.


   5. Conclusions
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusions
References
 
Observation of [Ca2+]i during myocardial ischemia and reperfusion using fluorescent indicators such as rhod-2 is feasible, but care must be taken to account for changes in the Kd for rhod-2 and Ca2+ that are induced by changing pH and temperature. If Kd is not adjusted accordingly, [Ca2+]i is systematically underestimated, and this should apply to other indicators as well. Since experimental settings vary greatly, the Kd values presented here should not be considered absolute, but Kd should be re-evaluated whenever a new experimental protocol requires determination of [Ca2+]i.

Time for primary review 27 days.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusions
 References
 

  1. Meldrum D.R., Cleveland J.C., Sheridan B.C., et al. Cardiac surgical implications of calcium dyshomeostasis in the heart. Ann Thorac Surg (1996) 61:1273–1280.[Abstract/Free Full Text]
  2. Halow J.M., Figueredo V.M., Shames D.M., Camacho S.A., Baker A.J. Role of slowed Ca2+ transient decline in slowed relaxation during myocardial ischemia. J Mol Cell Cardiol (1999) 31:1739–1748.[CrossRef][Web of Science][Medline]
  3. Martinez-Zaguilan R., Martinez G.M., Latanzio F., Gillies R.J. Simultaneous measurement of intracellular pH and Ca2+ using the fluorescence of SNARF-1 and fura-2. Am J Physiol (1991) 260:C297–C307.[Web of Science][Medline]
  4. Moon R.B., Richards J.H. Determination of intracellular pH by 31P magnetic resonance. J Biol Chem (1973) 248:7276–7278.[Abstract/Free Full Text]
  5. del Nido P.J., Glynn P., Buenaventura P., Salama G., Koretsky A.P. Fluorescence measurement of calcium transients in perfused rabbit heart using rhod 2. Am J Physiol (1998) 274:H728–H741.[Web of Science][Medline]
  6. Yue D.T., Wier W.G. Estimation of intracellular [Ca2+] by nonlinear indicators. Biophys J (1985) 48:533–537.[Web of Science][Medline]
  7. Lattanzio F.A., Pressman B.C. Alterations in intracellular calcium activity and contractility of isolated perfused rabbit hearts by ionophores and adrenergic agents. Biochem Biophys Res Commun (1986) 139:816–821.[CrossRef][Web of Science][Medline]
  8. Tsien R., Pozzan T. Measurement of cytosolic free Ca2+ with Quin 2. Methods Enzymol (1989) 172:230–262.[Web of Science][Medline]
  9. Lee H.C., Smith N., Mohabir R., Clusin W.T. Cytosolic calcium transients from the beating mammalian heart. Proc Natl Acad Sci USA (1987) 84:7793–7797.[Abstract/Free Full Text]
  10. Lee H.C., Mohabir R., Smith N.B., Franz M.R., Clusin W.T. Effect of ischemia on calcium-dependant fluorescence transients in rabbit hearts containing Indo-1. Circulation (1988) 78:1047–1059.[Abstract/Free Full Text]
  11. Lattanzio F.A. The effects of pH and temperature on fluorescent calcium indicators as determined with chelex-100 and EDTA buffer systems. Biochem Biophys Res Commun (1990) 171:102–108.[CrossRef][Web of Science][Medline]
  12. Lattanzio F.A., Bartschat D.K. The effect of pH on rate constant, ion selectivity and thermodynamic properties of fluorescent calcium and magnesium indicators. Biochem Biophys Res Commun (1991) 177:184–191.[CrossRef][Web of Science][Medline]
  13. Jimenez E., del Nido P.J., Feinberg H., Levitsky S. Redistribution of myocardial calcium during ischemia. Relationship to onset of contracture. J Thorac Cardiovasc Surg (1993) 105:988–994.[Abstract]
  14. Kihara Y., Grossman W., Morgan J.P. Direct measurement of changes in intracellular calcium transients during hypoxia, ischemia, and reperfusion of the intact mammalian heart. Circ Res (1989) 65:1029–1044.[Abstract/Free Full Text]
  15. Meissner A., Morgan J.P. Contractile dysfunction and abnormal Ca2+ modulation during postischemic reperfusion in rat heart. Am J Physiol (1995) 268:H100–H111.[Web of Science][Medline]
  16. Hampton T.G., Amende I., Travers K.E., Morgan J.P. Intracellular calcium dynamics in mouse model of myocardial stunning. Am J Physiol (1998) 274:H1821–H1827.[Web of Science][Medline]
  17. Minta A., Kao J.P., Tsien R.Y. Fluorescent indicators for cytosolic calcium based on rhodamine and fluorescein chromophores. J Biol Chem (1989) 264:8171–8178.[Abstract/Free Full Text]
  18. Rink T.J. Measurement of cytosolic calcium: fluorescent calcium indicators. Mineral Electrolyte Metab (1988) 14:7–14.
  19. Du C., MacGowan G.A., Farkas D.L., Koretsky A.P. Quantitative measurement of calcium transients in perfused mouse hearts using rhod-2 fluorometry. Proc SPIE (1999) 3602:214–219.[CrossRef]
  20. Choi B.R., Salama G. Simultaneous maps of optical action potentials and calcium transients in guinea pig hearts: mechanisms underlying concordant alternans. J Physiol (2000) 529:171–188.[Abstract/Free Full Text]
  21. Qian Y.W., Clusin W.T., Lin S.F., Han J., Sung R.J. Spatial heterogeneity of calcium transient alternans during the early phase of myocardial ischemia in the blood-perfused rabbit heart. Circulation (2001) 104:2082–2087.[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?



This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Stamm, C.
Right arrow Articles by del Nido, P. J
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Stamm, C.
Right arrow Articles by del Nido, P. J
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?