Am J Physiol Heart Circ Physiol 307: H1687–H1690, 2014; doi:10.1152/ajpheart.00468.2014.
Perspectives
Detecting calcium in cardiac muscle: fluorescence to dye for Peter Lipp and Lars Kaestner Institute for Molecular Cell Biology and Research Centre for Molecular Imaging and Screening, Medical Faculty, Saarland University, Homburg, Germany Submitted 3 July 2014; accepted in final form 22 September 2014
Ca2⫹ as a second messenger in general and as a mediator between electrical excitation and cellular contraction in the heart is well accepted. Accordingly, numerous Ca2⫹-sensitive fluorescent dyes are available nowadays, rendering the choice of the appropriate dye for a particular application a challenge. It appears to be important to provide calibrated Ca2⫹ signals rather than fluorescence transients, especially when comparing data between studies. In case calibrated signals cannot be provided, then the time course of fluorescence transients should be interpreted with caution and such signals should be referred to as fluorescence transients rather than Ca2⫹ transients. Additionally, we discuss how major properties of the fluorescence dyes should be considered with a special emphasis on their Ca2⫹ dissociation properties. Furthermore, we highlight putative risks of tripping, especially in the context of multicellular recordings, and how to circumvent them. As an ubiquitous intracellular messenger, Ca2⫹ ions play an important role in almost all living cells (4). A particularly dynamic role has been assigned to Ca2⫹ in muscle cells because here they not only control gene expression and other slower signaling processes but serve as a direct transducer of the electrical excitation of the plasma membrane into the activation of the physiological response of the cell, the contraction, by activation of the contractile machinery (5). Almost 50 years ago, Jöbsis and O’Connor (10) reported the measurements of intracellular Ca2⫹ transients in skeletal muscle using the Ca2⫹-sensitive dye murexide, which changes its absorbance upon binding of Ca2⫹ ions. Later, these absorbance dyes were improved and an important member of this family, arsenazo III, was widely used to study intracellular Ca2⫹ in skeletal muscle [see Miledi et al. (17)]. In parallel approaches, studies employing injections of luminescent photoproteins, especially aequorin, led to seminal findings for Ca2⫹ signaling in many cell types, but particularly in cardiac muscle (1,2) (for transduction of genetically encoded sensors, see below). The field of intracellular Ca2⫹ research only took off after Ca2⫹ sensors were synthesized that displayed substantial fluorescence changes upon Ca2⫹ binding (9). When using Ca2⫹ fluorophores, it appears to be important to provide calibrated Ca2⫹ signals rather than fluorescence transients, especially when comparing data between studies. In case calibrated signals cannot be provided, then the time course of fluorescence transients should be interpreted with caution and such signals should be referred to as fluorescence transients rather than Ca2⫹ transients. Nowadays, researchers face a choice of more than 50 different Ca2⫹-sensitive small molecule dyes. Most of them THE IMPORTANCE OF
Address for reprint requests and other correspondence: P. Lipp, Inst. for Molecular Cell Biology, Research Ctr. for Molecular Imaging and Screening, Bldg. 61, Medical Faculty, Saarland Univ. Hospital, Saarland Univ., 66421 Homburg/Saar, Germany (e-mail: [email protected]). http://www.ajpheart.org
follow the same basic principle; intramolecularly, Ca2⫹ ions are coordinated by Ca2⫹ binding groups. This binding changes the localization of conjugated electron clouds and thus alters the spectral properties of the indicator. Despite such similarities, the sensors can be separated into ratiometric and nonratiometric molecules with the former sensors such as Fura-2 or Indo-1, allowing an easier and more robust calibration and thus a more quantitative readout of Ca2⫹ concentrations (see below). Although such tasks are also possible with high accuracy using so-called single-excitation, singleemission molecules, e.g., rhod-2, Fluo-3/4 or -8, because of ester loading, the real dye concentration is often not known and therefore the procedure is more complex and often relies on assumptions. For the nonratiometric dyes, several possibilities to circumvent that problem have been described including the combination of two Ca2⫹ sensors (16) or recording of the fluorescence lifetime (14). In addition to such dye properties, another equally important characteristic is the in vivo Ca2⫹ binding/fluorescence relationship, which is plotted in Fig. 1, for a selection of Ca2⫹ sensors. One characteristic property of these curves is the Ca2⫹ dissociation constant (Kd value), which varies between ⬃100 nM (Fura-2) (15) and up to 400 M (Fluo-5N) (18). An almost equally important characteristic is the “steepness” of Ca2⫹ dependence around the Kd value that determines the detectable concentration range, i.e., the shallower the curve, the wider is the measurable range. In addition to spectral considerations, the particular dye that best fits the application of a certain study should be chosen based on the Ca2⫹ concentration range of interest. Even after such considerations, the range of dyes one is left with appears confusing at times. The situation is rendered even more complex because the Kd values not only determine the center of the “usable” Ca2⫹ concentration range for the particular dye but also describe the Ca2⫹ binding kinetics of the small molecular Ca2⫹ indicators. As a rule of thumb, indicators with high Kd values, e.g., in the tens of micromolar range, such as Fluo-4FF or CaOrange-5N, compared with those with lower Kd values, such as Fluo-3/4 (around 350 nM), display substantially faster kinetics for Ca2⫹ release, respectively, as nicely demonstrated by laser spot measurements from Escobar and coworkers (8). In that, the importance to understand and appreciate the Ca2⫹ affinity (Kd value) based on the binding kinetics (on and off rates) for the correct interpretation of fluorescence data from Ca2⫹ indicators becomes obvious. Popular and widely used fluorescent Ca2⫹ probes have “on” rates that vary little from one probe to another. It is the “off” rate that varies widely and dictates the Kd and thereby the affinity to bind Ca2⫹. In living cells, the situation is even more exaggerated because these parameters have been demonstrated to vary between in vitro and in vivo conditions as well as between different subcellular compartments, such as depicted for the cytoplasm and the nucleoplasm in Fig. 1.
0363-6135/14 Copyright © 2014 the American Physiological Society
H1687
Perspectives H1688
DETECTING CALCIUM IN CARDIAC MUSCLE: FLUORESCENCE TO DYE FOR
Fig. 1. In vivo calibration of fluorescent Ca2⫹ indicators. EGTA/CaEGTA solutions were used to construct concentration response curves, illustrating the in situ Ca2⫹dependent fluorescence of Fluo-3 (A), Fluo-4 (B), Oregon Green 488 BAPTA-1 (C), Calcium Green-1 (D), Calcium Orange (E), and Fura Red (F). Black and gray data points represent the nuclear and cytoplasmic fluorescence respectively. This figure is reproduced from Thomas et al. (21) with permission from Elsevier.
Another crucial property of the Ca2⫹ sensor, which is independent of the Kd, is it’s dynamic range, i.e., the fold change in fluorescence intensity between Ca2⫹-free and Ca2⫹saturated conditions. This value can vary considerably, e.g., for calcium crimson, it is ⬃3, whereas for Fluo-4, it is ⬃100. This makes it an important parameter that needs to be considered when choosing a dye. In a recent issue of the American Journal of PhysiologyHeart and Circulatory Physiology, Kong and Fast (13) especially addressed the relationship between the Ca2⫹ binding affinity and the apparent fluorescence transients recorded in a multicellular preparation, the coronary perfused left ventricular wedge preparation and compared these fluorescence transients to the optically measured action potentials from the same preparation. The authors employed Ca2⫹ indicators covering a wide range of Kd values of 350 nM (Fluo-4) to around 10 M (Fluo-4FF) and presented a rather comprehensive study of their behavior at various stimulation frequencies. One of the major problems not only for single cell but also for multicellular preparation is the delivery of the dye into the intracellular space of the intact cells. While investigations using intact hearts have loaded the entire heart with the AM-ester form of the dye via coronary perfusion, e.g., Kapur et al. (12), Kong and Fast have developed an elegant technique, the approach of which can be summarized as “only load where you record.” They set up a local perfusion technique for which they combined local solution injection with local optical fiber imaging with the result that they only applied the dye in exactly that tissue portion from which they collected fluorescence signals. In accordance to the particular Kd of the dye used, the authors found characteristic modulations of the fluorescence signals’ amplitude and time course with those indicators with the lowest Kd (Fluo-4) displaying the longest fluorescence transients, especially at higher cycle lengths. In contrast, the indicator with the highest Kd value, Fluo-4FF, showed brief and fast fluorescence transients. It is of particular interest to note that the authors reported biphasic Ca2⫹ transients, especially for the low Kd
indicator Fluo-4 at long cycle lengths. The authors speculated that such secondary Ca2⫹ increases might reflect secondary sarcoplasmic reticulum Ca2⫹ release that did not induce electrogenic Ca2⫹ transporters or channels because the action potentials recorded simultaneously did not display any typical Ca2⫹ dependent distortions. The authors showed that Ca2⫹ recordings in the multicellular preparation were substantially affected by the dyes applied with respect to both amplitude and, in particular, the time course. They claimed that especially low Kd dyes, such as Fluo-4 and Fluo-2MA (also referred to as Fluo-8), result in an overestimation of the Ca2⫹ transient duration because fluorescence transients from these dyes showed a tendency for plateauing. Kong and Fast concluded that low-affinity Ca2⫹ indicators such as Fluo-4FF and Fluo2LA were most suitable for multicellular Ca2⫹ measurements. An alternative interpretation we favor is that low-affinity dyes just detect the peak of the Ca2⫹ transient, as depicted for single cell recordings with TN-XL in Fig. 2D. As mentioned above, the Ca2⫹ dissociation of a particular dye majorly determines the shape of the apparent fluorescence transient obtained, because the relationship between fluorescence intensity and Ca2⫹ concentration is rather nonlinear (compare Fig. 1). Fluorescence data obtained from spatially nonresolved approaches are also tampered by the fact that fluorescence indicators display various Kd values and dynamic ranges (Fmax/Fmin or Rmax/Rmax, whether they are nonratiometric or ratiometric, respectively), depending on the particular intracellular compartment in which they reside (Fig. 1). As long as researchers are only faced by small volume compartments such as the sarcoplasmic reticulum/endoplasmic reticulum or the Golgi apparatus, the convolution of cytosolic and compartment-derived fluorescence signals can be tolerated, but the nucleoplasmic compartment in cardiac myocytes (ventricular cells have 2 nuclei) reflects a significant portion of the fluorescence signal originating from an individual cell. This holds especially true because small molecular dyes are easily accumulated in compartments like the nucleus, particular at
AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00468.2014 • www.ajpheart.org
Perspectives H1689
DETECTING CALCIUM IN CARDIAC MUSCLE: FLUORESCENCE TO DYE FOR
b
1
1.0 Ca2+-bound Fura-2 (µM)
free Ca2+ (µM)
normalized amplitude
measured transients
0.6
1.6
20
14
0.2
calculated transients
Ca2+-bound GCaMP2 (µM)
Aa
free Ca2+ Fura-2/Ca2+-complex GCaMP2/Ca2+-complex
1.0
8
0 0
0
3000
C
Fura-2 ratio
1.2 relative Fura-2 ratio amplitude
B
time (ms)
0.8
0.4
3000
time (ms)
1.2
0.8
0.4 0.0 0.0
0.4
0.8
0
1.2
200
400
600
800
GCaMP2 fluorescence intensity (a.u.)
relative GCaMP2 amplitude (a.u.)
D Ratio
Fura-2
0.28 0.24
YC3.6 Kd = 0.25 µM
TN-XL Kd = 2.5 µM
Ratio 4.6 4.2 Ratio 3.1 3.0
Fig. 2. Comparison between GCaMP2 and Fura-2 in a transgenic mouse line with cardiac-specific expression of GCaMP2 (20). A,a: simultaneous measurements of electrically evoked Ca2⫹ transients with Fura-2 ratio (blue trace) and GCaMP2 (red trace). A,b: calculation of the Fura-2 ratio (blue trace) and GCaMP2 (red trace) readout based on a Ca2⫹ transient as depicted by the dashed line. B: relationship between the relative amplitudes of Ca2⫹ transients measured with Fura-2 and GCaMP2. Amplitudes were calculated by subtracting the resting values from the peak value and dividing by the resting value. It gave a very good correlation between these 2 parameters (P ⬍ 0.0001, 4 mice, 123 cells). Interestingly, there was a population of myocytes that clearly displayed a Fura-2 transient but lacked any detectable GCaMP2 fluorescence changes. AU, arbitrary units. To determine if these cells represented a population of cells displaying only minute GCaMP2 expression, in C, the resting Fura-2 ratio was plotted against GCaMP2 fluorescence. Although there was a broad range of GCaMP2 fluorescence, the number of cells with very low expression was negligible. D: examples of Ca2⫹ transients measured with Fura-2 and two genetically encoded Ca2⫹ indicators and under otherwise identical experimental conditions: distribution of the fluorescence in myocytes (left) and typical train of electrically evoked global Ca2⫹ transients (right). The difference in Kd between YC3.6 and TN-XL becomes evident, since the TN-XL only displays the peak of the Ca2⫹ transients. This figure is reproduced from Kaestner et al. (11), with permission from Wolters Kluwer.
temperatures around 37°C. In spatially nonresolved data, this corruption cannot be deconvolved and leads to a prolonged “global” Ca2⫹ transient. It remains unclear to what degree these phenomena contribute to the longer time course of the fluorescence transients reported by Kong and Fast. Due to the
nonlinearity between the fluorescence and Ca2⫹ signal, it appears to be important to provide calibrated Ca2⫹ signals rather than fluorescence transients, especially when comparing and analyzing the time course of transients in great detail, as it is good practice for decades, e.g., Callewaert et al. (6) and
AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00468.2014 • www.ajpheart.org
Perspectives H1690
DETECTING CALCIUM IN CARDIAC MUSCLE: FLUORESCENCE TO DYE FOR
Sipido et al. (19); for a recent review, see Baylor and Hollingworth (3). Although it can be difficult to determine such calibration parameters in the intact tissue, it would have been advantageous to employ in vivo, i.e., in intact cells, properties of the Ca2⫹ indicators where available and/or to perform in vivo calibration of the dyes as described elsewhere (6, 16). Even applying dye properties obtained in non-muscle cells appears more appropriate than using in vitro values (21). Even though the absolute values resulting from such calibration might not exactly reflect the “proper” Ca2⫹ concentration in the multicellular preparations, such fluorescence signal linearization originating from using calibration parameters would substantially help when comparing the properties of Ca2⫹ transients, such as amplitude, shape, and duration, obtained from different fluorescence Ca2⫹ sensors. It is noteworthy to mention that in the study of Kong and Fast, both the voltageCa2⫹ delay and the Ca2⫹ transient rise times were rather independent of the dye used. This finding appears to support the notion that at least for these particular parameters, the kinetics of the dyes were equally well suited for the ventricular multicellular preparation. The study by Kong and Fast clearly highlights that when using Ca2⫹ indicators in multicellular cardiac preparation; the researchers have to carefully consider the kinetics as well as the binding properties of the Ca2⫹ dye(s) applied when interpreting their fluorescence data. For the future, a study similar to the one provided by Kong and Fast but considering in vivo calibration of the fluorescence data might also offer distinct ways solving the problems discussed above. A further significant aspect of multicellular preparations in general that needs a critical consideration is the fact that such recordings cannot discriminate the origin of the fluorescence in terms of the cell type (cytosol of the myocyte and not from endothelial cells, smooth muscle cells, fibroblasts). A putative solution is a totally different approach that should be discussed briefly, the application of genetically encoded Ca2⫹ indicators (GECIs), an exciting and emerging field in the cardiac community (11). This methodology, either through transgenic animals, e.g., Tallini et al. (20), or by viral transduction, e.g., Viero et al. (22), offers the expression of a Ca2⫹ sensor specifically in the cardiac myocyte and in that even targeted to specific subcellular compartments such as the cytosol, mitochondrium, or nucleus. A disadvantage of GECIs compared with small molecular Ca2⫹ fluorophores has been their lower brightness leading to a limited signal-to-noise ratio, but with the latest developments like the GCaMP6 sensors (7), this gap is closed. However, there are remaining disadvantages of GECIs as outlined in Fig. 2, highlighting an altered kinetic response (Fig. 2A) and variability in the expression level (Fig. 2, B and C). Nevertheless, studies that scrutinize fluorescence signals from GECIs are even sparser but will help the progression of the entire field on a longer timescale and as depicted in Fig. 2D; the considerations of the Kd as outlined above remain valid for GECIs. GRANTS P. Lipp and L. Kaestner acknowledge financial support by the HOMFOR program of the Medical Faculty and by the Saarland University. DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the author(s).
AUTHOR CONTRIBUTIONS P.L. conception and design of research; P.L. drafted manuscript; P.L. and L.K. edited and revised manuscript; P.L. approved final version of manuscript. REFERENCES 1. Allen DG, Blinks JR. Calcium transients in aequorin-injected frog cardiac muscle. Nature 273: 509 –513, 1978. 2. Ashley CC, Ridgway EB. Simultaneous recording of membrane potential, calcium transient and tension in single muscle fibers. Nature 219: 1168 –1169, 1968. 3. Baylor SM, Hollingworth S. Calcium indicators and calcium signalling in skeletal muscle fibres during excitation-contraction coupling. Prog Biophys Mol Biol 105: 162–179, 2011. 4. Berridge MJ, Lipp P, Bootman MD. The versatility and universality of calcium signalling. Nat Rev Mol Cell Biol 1: 11–21, 2000. 5. Bers DM. Cardiac excitation-contraction coupling. Nature 415: 198 –205, 2002. 6. Callewaert G, Lipp P, Pott L, Carmeliet E. High-resolution measurement and calibration of Ca2⫹-transients using Indo-1 in guinea-pig atrial myocytes under voltage clamp. Cell Calcium 12: 269 –277, 1991. 7. Chen TW, Wardill TJ, Sun Y, Pulver SR, Renninger SL, Baohan A, Schreiter ER, Kerr RA, Orger MB, Jayaraman V, Looger LL, Svoboda K, Kim DS. Ultrasensitive fluorescent proteins for imaging neuronal activity. Nature 499: 295–300, 2013. 8. Escobar AL, Cifuentes F, Vergara JL. Detection of Ca2⫹-transients elicited by flash photolysis of DM-nitrophen with a fast calcium indicator. FEBS Lett 364: 335–338, 1995. 9. Grynkiewicz G, Poenie M, Tsien RY. A new generation of Ca2⫹ indicators with greatly improved fluorescence properties. J Biol Chem 260: 3440 –3450, 1985. 10. Jöbsis FF, O’Connor MJ. Calcium release and reabsorption in the sartorius muscle of the toad. Biochem Biophys Res Commun 25: 246 –252, 1966. 11. Kaestner L, Scholz A, Tian Q, Ruppenthal S, Tabellion W, Wiesen K, Katus HA, Muller OJ, Kotlikoff MI, Lipp P. Genetically encoded Ca2⫹ indicators in cardiac myocytes. Circ Res 114: 1623–1639, 2014. 12. Kapur S, Aistrup GL, Sharma R, Kelly JE, Arora R, Zheng J, Veramasuneni M, Kadish AH, Balke CW, Wasserstrom JA. Early development of intracellular calcium cycling defects in intact hearts of spontaneously hypertensive rats. Am J Physiol Heart Circ Physiol 299: H1843–H1853, 2010. 13. Kong W, Fast VG. The role of dye affinity in optical measurements of Ca2⫹ transients in cardiac muscle. Am J Physiol Heart Circ Physiol 307: i H73–H79, 2014. 14. Lakowicz JR, Szmacinski H, Johnson ML. Calcium imaging using fluorescence lifetimes and long-wavelength probes. J Fluoresc 2: 47–62, 1992. 15. Li Q, Altschuld RA, Stokes BT. Quantitation of intracellular free calcium in single adult cardiomyocytes by fura-2 fluorescence microscopy: calibration of fura-2 ratios. Biochem Biophys Res Commun 147: 120–126, 1987. 16. Lipp P, Niggli E. Ratiometric confocal Ca2⫹-measurements with visible wavelength indicators in isolated cardiac myocytes. Cell Calcium 14: 359 –372, 1993. 17. Miledi R, Parker I, Schalow G. Measurement of calcium transients in frog muscle by the use of arsenazo III. Proc R Soc Lond B Biol Sci 198: 201–210, 1977. 18. Shannon TR, Guo T, Bers DM. Ca2⫹ scraps: local depletions of free [Ca2⫹] in cardiac sarcoplasmic reticulum during contractions leave substantial Ca2⫹ reserve. Circ Res 93: 40 –45, 2003. 19. Sipido KR, Wier WG. Flux of Ca2⫹ across the sarcoplasmic reticulum of guinea-pig cardiac cells during excitation-contraction coupling. J Physiol 435: 605–630, 1991. 20. Tallini YN, Ohkura M, Choi BR, Ji G, Imoto K, Doran R, Lee J, Plan P, Wilson J, Xin HB, Sanbe A, Gulick J, Mathai J, Robbins J, Salama G, Nakai J, Kotlikoff MI. Imaging cellular signals in the heart in vivo: Cardiac expression of the high-signal Ca2⫹ indicator GCaMP2. Proc Natl Acad Sci USA 103: 4753–4758, 2006. 21. Thomas D, Tovey SC, Collins TJ, Bootman MD, Berridge MJ, Lipp P. A comparison of fluorescent Ca2⫹ indicator properties and their use in measuring elementary and global Ca2⫹ signals. Cell Calcium 28: 213–223, 2000. 22. Viero C, Kraushaar U, Ruppenthal S, Kaestner L, Lipp P. A primary culture system for sustained expression of a calcium sensor in preserved adult rat ventricular myocytes. Cell Calcium 43: 59 –71, 2008.
AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00468.2014 • www.ajpheart.org
Perspectives
Detecting calcium in cardiac muscle: fluorescence to dye for Peter Lipp and Lars Kaestner Institute for Molecular Cell Biology and Research Centre for Molecular Imaging and Screening, Medical Faculty, Saarland University, Homburg, Germany Submitted 3 July 2014; accepted in final form 22 September 2014
Ca2⫹ as a second messenger in general and as a mediator between electrical excitation and cellular contraction in the heart is well accepted. Accordingly, numerous Ca2⫹-sensitive fluorescent dyes are available nowadays, rendering the choice of the appropriate dye for a particular application a challenge. It appears to be important to provide calibrated Ca2⫹ signals rather than fluorescence transients, especially when comparing data between studies. In case calibrated signals cannot be provided, then the time course of fluorescence transients should be interpreted with caution and such signals should be referred to as fluorescence transients rather than Ca2⫹ transients. Additionally, we discuss how major properties of the fluorescence dyes should be considered with a special emphasis on their Ca2⫹ dissociation properties. Furthermore, we highlight putative risks of tripping, especially in the context of multicellular recordings, and how to circumvent them. As an ubiquitous intracellular messenger, Ca2⫹ ions play an important role in almost all living cells (4). A particularly dynamic role has been assigned to Ca2⫹ in muscle cells because here they not only control gene expression and other slower signaling processes but serve as a direct transducer of the electrical excitation of the plasma membrane into the activation of the physiological response of the cell, the contraction, by activation of the contractile machinery (5). Almost 50 years ago, Jöbsis and O’Connor (10) reported the measurements of intracellular Ca2⫹ transients in skeletal muscle using the Ca2⫹-sensitive dye murexide, which changes its absorbance upon binding of Ca2⫹ ions. Later, these absorbance dyes were improved and an important member of this family, arsenazo III, was widely used to study intracellular Ca2⫹ in skeletal muscle [see Miledi et al. (17)]. In parallel approaches, studies employing injections of luminescent photoproteins, especially aequorin, led to seminal findings for Ca2⫹ signaling in many cell types, but particularly in cardiac muscle (1,2) (for transduction of genetically encoded sensors, see below). The field of intracellular Ca2⫹ research only took off after Ca2⫹ sensors were synthesized that displayed substantial fluorescence changes upon Ca2⫹ binding (9). When using Ca2⫹ fluorophores, it appears to be important to provide calibrated Ca2⫹ signals rather than fluorescence transients, especially when comparing data between studies. In case calibrated signals cannot be provided, then the time course of fluorescence transients should be interpreted with caution and such signals should be referred to as fluorescence transients rather than Ca2⫹ transients. Nowadays, researchers face a choice of more than 50 different Ca2⫹-sensitive small molecule dyes. Most of them THE IMPORTANCE OF
Address for reprint requests and other correspondence: P. Lipp, Inst. for Molecular Cell Biology, Research Ctr. for Molecular Imaging and Screening, Bldg. 61, Medical Faculty, Saarland Univ. Hospital, Saarland Univ., 66421 Homburg/Saar, Germany (e-mail: [email protected]). http://www.ajpheart.org
follow the same basic principle; intramolecularly, Ca2⫹ ions are coordinated by Ca2⫹ binding groups. This binding changes the localization of conjugated electron clouds and thus alters the spectral properties of the indicator. Despite such similarities, the sensors can be separated into ratiometric and nonratiometric molecules with the former sensors such as Fura-2 or Indo-1, allowing an easier and more robust calibration and thus a more quantitative readout of Ca2⫹ concentrations (see below). Although such tasks are also possible with high accuracy using so-called single-excitation, singleemission molecules, e.g., rhod-2, Fluo-3/4 or -8, because of ester loading, the real dye concentration is often not known and therefore the procedure is more complex and often relies on assumptions. For the nonratiometric dyes, several possibilities to circumvent that problem have been described including the combination of two Ca2⫹ sensors (16) or recording of the fluorescence lifetime (14). In addition to such dye properties, another equally important characteristic is the in vivo Ca2⫹ binding/fluorescence relationship, which is plotted in Fig. 1, for a selection of Ca2⫹ sensors. One characteristic property of these curves is the Ca2⫹ dissociation constant (Kd value), which varies between ⬃100 nM (Fura-2) (15) and up to 400 M (Fluo-5N) (18). An almost equally important characteristic is the “steepness” of Ca2⫹ dependence around the Kd value that determines the detectable concentration range, i.e., the shallower the curve, the wider is the measurable range. In addition to spectral considerations, the particular dye that best fits the application of a certain study should be chosen based on the Ca2⫹ concentration range of interest. Even after such considerations, the range of dyes one is left with appears confusing at times. The situation is rendered even more complex because the Kd values not only determine the center of the “usable” Ca2⫹ concentration range for the particular dye but also describe the Ca2⫹ binding kinetics of the small molecular Ca2⫹ indicators. As a rule of thumb, indicators with high Kd values, e.g., in the tens of micromolar range, such as Fluo-4FF or CaOrange-5N, compared with those with lower Kd values, such as Fluo-3/4 (around 350 nM), display substantially faster kinetics for Ca2⫹ release, respectively, as nicely demonstrated by laser spot measurements from Escobar and coworkers (8). In that, the importance to understand and appreciate the Ca2⫹ affinity (Kd value) based on the binding kinetics (on and off rates) for the correct interpretation of fluorescence data from Ca2⫹ indicators becomes obvious. Popular and widely used fluorescent Ca2⫹ probes have “on” rates that vary little from one probe to another. It is the “off” rate that varies widely and dictates the Kd and thereby the affinity to bind Ca2⫹. In living cells, the situation is even more exaggerated because these parameters have been demonstrated to vary between in vitro and in vivo conditions as well as between different subcellular compartments, such as depicted for the cytoplasm and the nucleoplasm in Fig. 1.
0363-6135/14 Copyright © 2014 the American Physiological Society
H1687
Perspectives H1688
DETECTING CALCIUM IN CARDIAC MUSCLE: FLUORESCENCE TO DYE FOR
Fig. 1. In vivo calibration of fluorescent Ca2⫹ indicators. EGTA/CaEGTA solutions were used to construct concentration response curves, illustrating the in situ Ca2⫹dependent fluorescence of Fluo-3 (A), Fluo-4 (B), Oregon Green 488 BAPTA-1 (C), Calcium Green-1 (D), Calcium Orange (E), and Fura Red (F). Black and gray data points represent the nuclear and cytoplasmic fluorescence respectively. This figure is reproduced from Thomas et al. (21) with permission from Elsevier.
Another crucial property of the Ca2⫹ sensor, which is independent of the Kd, is it’s dynamic range, i.e., the fold change in fluorescence intensity between Ca2⫹-free and Ca2⫹saturated conditions. This value can vary considerably, e.g., for calcium crimson, it is ⬃3, whereas for Fluo-4, it is ⬃100. This makes it an important parameter that needs to be considered when choosing a dye. In a recent issue of the American Journal of PhysiologyHeart and Circulatory Physiology, Kong and Fast (13) especially addressed the relationship between the Ca2⫹ binding affinity and the apparent fluorescence transients recorded in a multicellular preparation, the coronary perfused left ventricular wedge preparation and compared these fluorescence transients to the optically measured action potentials from the same preparation. The authors employed Ca2⫹ indicators covering a wide range of Kd values of 350 nM (Fluo-4) to around 10 M (Fluo-4FF) and presented a rather comprehensive study of their behavior at various stimulation frequencies. One of the major problems not only for single cell but also for multicellular preparation is the delivery of the dye into the intracellular space of the intact cells. While investigations using intact hearts have loaded the entire heart with the AM-ester form of the dye via coronary perfusion, e.g., Kapur et al. (12), Kong and Fast have developed an elegant technique, the approach of which can be summarized as “only load where you record.” They set up a local perfusion technique for which they combined local solution injection with local optical fiber imaging with the result that they only applied the dye in exactly that tissue portion from which they collected fluorescence signals. In accordance to the particular Kd of the dye used, the authors found characteristic modulations of the fluorescence signals’ amplitude and time course with those indicators with the lowest Kd (Fluo-4) displaying the longest fluorescence transients, especially at higher cycle lengths. In contrast, the indicator with the highest Kd value, Fluo-4FF, showed brief and fast fluorescence transients. It is of particular interest to note that the authors reported biphasic Ca2⫹ transients, especially for the low Kd
indicator Fluo-4 at long cycle lengths. The authors speculated that such secondary Ca2⫹ increases might reflect secondary sarcoplasmic reticulum Ca2⫹ release that did not induce electrogenic Ca2⫹ transporters or channels because the action potentials recorded simultaneously did not display any typical Ca2⫹ dependent distortions. The authors showed that Ca2⫹ recordings in the multicellular preparation were substantially affected by the dyes applied with respect to both amplitude and, in particular, the time course. They claimed that especially low Kd dyes, such as Fluo-4 and Fluo-2MA (also referred to as Fluo-8), result in an overestimation of the Ca2⫹ transient duration because fluorescence transients from these dyes showed a tendency for plateauing. Kong and Fast concluded that low-affinity Ca2⫹ indicators such as Fluo-4FF and Fluo2LA were most suitable for multicellular Ca2⫹ measurements. An alternative interpretation we favor is that low-affinity dyes just detect the peak of the Ca2⫹ transient, as depicted for single cell recordings with TN-XL in Fig. 2D. As mentioned above, the Ca2⫹ dissociation of a particular dye majorly determines the shape of the apparent fluorescence transient obtained, because the relationship between fluorescence intensity and Ca2⫹ concentration is rather nonlinear (compare Fig. 1). Fluorescence data obtained from spatially nonresolved approaches are also tampered by the fact that fluorescence indicators display various Kd values and dynamic ranges (Fmax/Fmin or Rmax/Rmax, whether they are nonratiometric or ratiometric, respectively), depending on the particular intracellular compartment in which they reside (Fig. 1). As long as researchers are only faced by small volume compartments such as the sarcoplasmic reticulum/endoplasmic reticulum or the Golgi apparatus, the convolution of cytosolic and compartment-derived fluorescence signals can be tolerated, but the nucleoplasmic compartment in cardiac myocytes (ventricular cells have 2 nuclei) reflects a significant portion of the fluorescence signal originating from an individual cell. This holds especially true because small molecular dyes are easily accumulated in compartments like the nucleus, particular at
AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00468.2014 • www.ajpheart.org
Perspectives H1689
DETECTING CALCIUM IN CARDIAC MUSCLE: FLUORESCENCE TO DYE FOR
b
1
1.0 Ca2+-bound Fura-2 (µM)
free Ca2+ (µM)
normalized amplitude
measured transients
0.6
1.6
20
14
0.2
calculated transients
Ca2+-bound GCaMP2 (µM)
Aa
free Ca2+ Fura-2/Ca2+-complex GCaMP2/Ca2+-complex
1.0
8
0 0
0
3000
C
Fura-2 ratio
1.2 relative Fura-2 ratio amplitude
B
time (ms)
0.8
0.4
3000
time (ms)
1.2
0.8
0.4 0.0 0.0
0.4
0.8
0
1.2
200
400
600
800
GCaMP2 fluorescence intensity (a.u.)
relative GCaMP2 amplitude (a.u.)
D Ratio
Fura-2
0.28 0.24
YC3.6 Kd = 0.25 µM
TN-XL Kd = 2.5 µM
Ratio 4.6 4.2 Ratio 3.1 3.0
Fig. 2. Comparison between GCaMP2 and Fura-2 in a transgenic mouse line with cardiac-specific expression of GCaMP2 (20). A,a: simultaneous measurements of electrically evoked Ca2⫹ transients with Fura-2 ratio (blue trace) and GCaMP2 (red trace). A,b: calculation of the Fura-2 ratio (blue trace) and GCaMP2 (red trace) readout based on a Ca2⫹ transient as depicted by the dashed line. B: relationship between the relative amplitudes of Ca2⫹ transients measured with Fura-2 and GCaMP2. Amplitudes were calculated by subtracting the resting values from the peak value and dividing by the resting value. It gave a very good correlation between these 2 parameters (P ⬍ 0.0001, 4 mice, 123 cells). Interestingly, there was a population of myocytes that clearly displayed a Fura-2 transient but lacked any detectable GCaMP2 fluorescence changes. AU, arbitrary units. To determine if these cells represented a population of cells displaying only minute GCaMP2 expression, in C, the resting Fura-2 ratio was plotted against GCaMP2 fluorescence. Although there was a broad range of GCaMP2 fluorescence, the number of cells with very low expression was negligible. D: examples of Ca2⫹ transients measured with Fura-2 and two genetically encoded Ca2⫹ indicators and under otherwise identical experimental conditions: distribution of the fluorescence in myocytes (left) and typical train of electrically evoked global Ca2⫹ transients (right). The difference in Kd between YC3.6 and TN-XL becomes evident, since the TN-XL only displays the peak of the Ca2⫹ transients. This figure is reproduced from Kaestner et al. (11), with permission from Wolters Kluwer.
temperatures around 37°C. In spatially nonresolved data, this corruption cannot be deconvolved and leads to a prolonged “global” Ca2⫹ transient. It remains unclear to what degree these phenomena contribute to the longer time course of the fluorescence transients reported by Kong and Fast. Due to the
nonlinearity between the fluorescence and Ca2⫹ signal, it appears to be important to provide calibrated Ca2⫹ signals rather than fluorescence transients, especially when comparing and analyzing the time course of transients in great detail, as it is good practice for decades, e.g., Callewaert et al. (6) and
AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00468.2014 • www.ajpheart.org
Perspectives H1690
DETECTING CALCIUM IN CARDIAC MUSCLE: FLUORESCENCE TO DYE FOR
Sipido et al. (19); for a recent review, see Baylor and Hollingworth (3). Although it can be difficult to determine such calibration parameters in the intact tissue, it would have been advantageous to employ in vivo, i.e., in intact cells, properties of the Ca2⫹ indicators where available and/or to perform in vivo calibration of the dyes as described elsewhere (6, 16). Even applying dye properties obtained in non-muscle cells appears more appropriate than using in vitro values (21). Even though the absolute values resulting from such calibration might not exactly reflect the “proper” Ca2⫹ concentration in the multicellular preparations, such fluorescence signal linearization originating from using calibration parameters would substantially help when comparing the properties of Ca2⫹ transients, such as amplitude, shape, and duration, obtained from different fluorescence Ca2⫹ sensors. It is noteworthy to mention that in the study of Kong and Fast, both the voltageCa2⫹ delay and the Ca2⫹ transient rise times were rather independent of the dye used. This finding appears to support the notion that at least for these particular parameters, the kinetics of the dyes were equally well suited for the ventricular multicellular preparation. The study by Kong and Fast clearly highlights that when using Ca2⫹ indicators in multicellular cardiac preparation; the researchers have to carefully consider the kinetics as well as the binding properties of the Ca2⫹ dye(s) applied when interpreting their fluorescence data. For the future, a study similar to the one provided by Kong and Fast but considering in vivo calibration of the fluorescence data might also offer distinct ways solving the problems discussed above. A further significant aspect of multicellular preparations in general that needs a critical consideration is the fact that such recordings cannot discriminate the origin of the fluorescence in terms of the cell type (cytosol of the myocyte and not from endothelial cells, smooth muscle cells, fibroblasts). A putative solution is a totally different approach that should be discussed briefly, the application of genetically encoded Ca2⫹ indicators (GECIs), an exciting and emerging field in the cardiac community (11). This methodology, either through transgenic animals, e.g., Tallini et al. (20), or by viral transduction, e.g., Viero et al. (22), offers the expression of a Ca2⫹ sensor specifically in the cardiac myocyte and in that even targeted to specific subcellular compartments such as the cytosol, mitochondrium, or nucleus. A disadvantage of GECIs compared with small molecular Ca2⫹ fluorophores has been their lower brightness leading to a limited signal-to-noise ratio, but with the latest developments like the GCaMP6 sensors (7), this gap is closed. However, there are remaining disadvantages of GECIs as outlined in Fig. 2, highlighting an altered kinetic response (Fig. 2A) and variability in the expression level (Fig. 2, B and C). Nevertheless, studies that scrutinize fluorescence signals from GECIs are even sparser but will help the progression of the entire field on a longer timescale and as depicted in Fig. 2D; the considerations of the Kd as outlined above remain valid for GECIs. GRANTS P. Lipp and L. Kaestner acknowledge financial support by the HOMFOR program of the Medical Faculty and by the Saarland University. DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the author(s).
AUTHOR CONTRIBUTIONS P.L. conception and design of research; P.L. drafted manuscript; P.L. and L.K. edited and revised manuscript; P.L. approved final version of manuscript. REFERENCES 1. Allen DG, Blinks JR. Calcium transients in aequorin-injected frog cardiac muscle. Nature 273: 509 –513, 1978. 2. Ashley CC, Ridgway EB. Simultaneous recording of membrane potential, calcium transient and tension in single muscle fibers. Nature 219: 1168 –1169, 1968. 3. Baylor SM, Hollingworth S. Calcium indicators and calcium signalling in skeletal muscle fibres during excitation-contraction coupling. Prog Biophys Mol Biol 105: 162–179, 2011. 4. Berridge MJ, Lipp P, Bootman MD. The versatility and universality of calcium signalling. Nat Rev Mol Cell Biol 1: 11–21, 2000. 5. Bers DM. Cardiac excitation-contraction coupling. Nature 415: 198 –205, 2002. 6. Callewaert G, Lipp P, Pott L, Carmeliet E. High-resolution measurement and calibration of Ca2⫹-transients using Indo-1 in guinea-pig atrial myocytes under voltage clamp. Cell Calcium 12: 269 –277, 1991. 7. Chen TW, Wardill TJ, Sun Y, Pulver SR, Renninger SL, Baohan A, Schreiter ER, Kerr RA, Orger MB, Jayaraman V, Looger LL, Svoboda K, Kim DS. Ultrasensitive fluorescent proteins for imaging neuronal activity. Nature 499: 295–300, 2013. 8. Escobar AL, Cifuentes F, Vergara JL. Detection of Ca2⫹-transients elicited by flash photolysis of DM-nitrophen with a fast calcium indicator. FEBS Lett 364: 335–338, 1995. 9. Grynkiewicz G, Poenie M, Tsien RY. A new generation of Ca2⫹ indicators with greatly improved fluorescence properties. J Biol Chem 260: 3440 –3450, 1985. 10. Jöbsis FF, O’Connor MJ. Calcium release and reabsorption in the sartorius muscle of the toad. Biochem Biophys Res Commun 25: 246 –252, 1966. 11. Kaestner L, Scholz A, Tian Q, Ruppenthal S, Tabellion W, Wiesen K, Katus HA, Muller OJ, Kotlikoff MI, Lipp P. Genetically encoded Ca2⫹ indicators in cardiac myocytes. Circ Res 114: 1623–1639, 2014. 12. Kapur S, Aistrup GL, Sharma R, Kelly JE, Arora R, Zheng J, Veramasuneni M, Kadish AH, Balke CW, Wasserstrom JA. Early development of intracellular calcium cycling defects in intact hearts of spontaneously hypertensive rats. Am J Physiol Heart Circ Physiol 299: H1843–H1853, 2010. 13. Kong W, Fast VG. The role of dye affinity in optical measurements of Ca2⫹ transients in cardiac muscle. Am J Physiol Heart Circ Physiol 307: i H73–H79, 2014. 14. Lakowicz JR, Szmacinski H, Johnson ML. Calcium imaging using fluorescence lifetimes and long-wavelength probes. J Fluoresc 2: 47–62, 1992. 15. Li Q, Altschuld RA, Stokes BT. Quantitation of intracellular free calcium in single adult cardiomyocytes by fura-2 fluorescence microscopy: calibration of fura-2 ratios. Biochem Biophys Res Commun 147: 120–126, 1987. 16. Lipp P, Niggli E. Ratiometric confocal Ca2⫹-measurements with visible wavelength indicators in isolated cardiac myocytes. Cell Calcium 14: 359 –372, 1993. 17. Miledi R, Parker I, Schalow G. Measurement of calcium transients in frog muscle by the use of arsenazo III. Proc R Soc Lond B Biol Sci 198: 201–210, 1977. 18. Shannon TR, Guo T, Bers DM. Ca2⫹ scraps: local depletions of free [Ca2⫹] in cardiac sarcoplasmic reticulum during contractions leave substantial Ca2⫹ reserve. Circ Res 93: 40 –45, 2003. 19. Sipido KR, Wier WG. Flux of Ca2⫹ across the sarcoplasmic reticulum of guinea-pig cardiac cells during excitation-contraction coupling. J Physiol 435: 605–630, 1991. 20. Tallini YN, Ohkura M, Choi BR, Ji G, Imoto K, Doran R, Lee J, Plan P, Wilson J, Xin HB, Sanbe A, Gulick J, Mathai J, Robbins J, Salama G, Nakai J, Kotlikoff MI. Imaging cellular signals in the heart in vivo: Cardiac expression of the high-signal Ca2⫹ indicator GCaMP2. Proc Natl Acad Sci USA 103: 4753–4758, 2006. 21. Thomas D, Tovey SC, Collins TJ, Bootman MD, Berridge MJ, Lipp P. A comparison of fluorescent Ca2⫹ indicator properties and their use in measuring elementary and global Ca2⫹ signals. Cell Calcium 28: 213–223, 2000. 22. Viero C, Kraushaar U, Ruppenthal S, Kaestner L, Lipp P. A primary culture system for sustained expression of a calcium sensor in preserved adult rat ventricular myocytes. Cell Calcium 43: 59 –71, 2008.
AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00468.2014 • www.ajpheart.org
