Differential Ca2+ and Sr2+ regulation of intracellular divalent cations release in ventricular myocytes

Cell Calcium 36 (2004) 119–134

Differential Ca2+ and Sr2+ regulation of intracellular divalent cations release in ventricular myocytes M.E. Zoghbi a , J.A. Copello b , C.A. Villalba-Galea d , P. Vélez c , P.L. Diaz Sylvester b , P. Bolaños a , A. Marcano e , M. Fill b , A.L. Escobar d,∗ a

c

Centro de Biofisica y Bioqu´ımica, Instituto Venezolano de Investigaciones Cient´ıficas, Caracas, Venezuela b Department of Physiology, Loyola University Chicago, Maywood, IL, USA Center for Cellular and Molecular Neuroscience, Faculty of Sciences, University of Valpara´ıso, Valpara´ıso, Chile d Department of Physiology, Texas Tech University Health Science Center, Lubbock, TX 79430, USA e Centro de F´ısica, Instituto Venezolano de Investigaciones Cient´ıficas, Caracas, Venezuela Received 11 September 2003; received in revised form 21 January 2004; accepted 21 January 2004

Abstract The regulation of the Ca2+ -induced Ca2+ release (CICR) from intracellular stores is a critical step in the cardiac cycle. The inherent positive feedback of CICR should make it a self-regenerating process. It is accepted that CICR must be governed by some negative control, but its nature is still debated. We explore here the importance of the Ca2+ released from sarcoplasmic reticulum (SR) on the mechanisms that may control CICR. Specifically, we compared the effect of replacing Ca2+ with Sr2+ on intracellular Ca2+ signaling in intact cardiac myocytes as well as on the function of single ryanodine receptor (RyR) Ca2+ release channels in panar bilayers. In cells, both CICR and Sr2+ -induced Sr2+ release (SISR) were observed. Action potential induced Ca2+ -transients and spontaneous Ca2+ waves were considerably faster than their Sr2+ -mediated counterparts. However, the kinetics of Ca2+ and Sr2+ sparks was similar. At the single RyR channel level, the affinities of Ca2+ and Sr2+ activation were different but the affinities of Ca2+ and Sr2+ inactivation were similar. Fast Ca2+ and Sr2+ stimuli activated RyR channels equally fast but adaptation (a spontaneous slow transition back to steady-state activity levels) was not observed in the Sr2+ case. Together, these results suggest that regulation of the RyR channel by cytosolic Ca2+ is not involved in turning off the Ca2+ spark. In contrast, cytosolic Ca2+ is important in the propagation global Ca2+ release events and in this regard single RyR channel sensitivity to cytosolic Ca2+ activation, not low-affinity cytosolic Ca2+ inactivation, is a key factor. This suggests that the kinetics of local and global RyR-mediated Ca2+ release signals are affected in a distinct way by different divalent cations in cardiac muscle cells. © 2004 Elsevier Ltd. All rights reserved. Keywords: Ryanodine receptor; Ca2+ -induced Ca2+ release; Strontium; Adaptation; Sparks

1. Introduction In cardiac ventricular muscle cells, action potential depolarization activates voltage-dependent dihydropyridine (DHP) sensitive Ca2+ channels located at the transverse tubular system and surface membrane [1]. The influx of Ca2+ through these DHP sensitive Ca2+ channels triggers intracellular Ca2+ release from the sarcoplasmic reticulum (SR) by the mechanism known as Ca2+ -induced Ca2+ release (CICR). Although this mechanism is an inherently self-regenerating process, it is very well controlled and precisely graded in cardiac cells [1]. This tight control of CICR implies that strong negative feedback exists and compen-

∗ Corresponding

author. Tel.: +1-806-743-4059; fax: +1-806-743-1512. E-mail address: [email protected] (A.L. Escobar).

0143-4160/$ – see front matter © 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.ceca.2004.01.023

sates for the inherent positive feedback of CICR. Several potential negative feedback mechanisms have been proposed [2–5]. These mechanisms involve either modulation of the triggering Ca2+ signal [6] and/or the operation of the SR Ca2+ release channel [2,4]. The most popular negative control mechanism proposed to date is Ca2+ dependent inactivation of the ryanodine receptor (RyR) SR Ca2+ release channel. The idea is that there are Ca2+ binding sites on the RyR channel that slowly promote the inactivation of these channels, which terminates the release process [3]. Two types of experimentation, laser scanning confocal microscopy and single RyR channel recording, have made substantial contributions to this field of study. Confocal microscopy has made it possible to image elementary Ca2+ release events (i.e. Ca2+ sparks) in isolated cardiac muscle [7] and skeletal muscle cells [8]. These localized events are thought to be generated by the concerted opening of a

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small number of CICR-competent RyR channels at discrete SR Ca2+ release sites within the cell. The conventional wisdom is that spark recruitment and summation generates the global Ca2+ release phenomena that propagate through the cell and control contraction. Single channel recording has defined various kinetics features of Ca2+ modulation (activation, deactivation, inactivation, adaptation, etc.) [4,54,55] of RyR channel function in vitro. Nevertheless, it is still difficult to compare single RyR channel behavior versus local or global Ca2+ signaling in cells. Consequently, we still lack of a clear understanding of the mechanism that controls local and global RyR-mediated Ca2+ release in cells. Here, we exchange Ca2+ for Sr2+ in cells and define the kinetics of both global and local RyR-mediated Ca2+ and Sr2+ signals. Additionally, we compare how Ca2+ and Sr2+ modulate single RyR channel reconstituted in artificial planar bilayers. It is well documented that Sr2+ can substitute for Ca2+ in a large number of physiological and biochemical processes in cells [9]. For example, Sr2+ can permeate through DHP channels [10–12] and activate the contractile machinery by binding to cardiac troponin-C [13,14]. Strontium can be extruded from the cytosol by the Na+ –Ca2+ exchanger [12,15]. Strontium is actively pumped into the SR [16,17] and can be released by caffeine [13,18]. However, the capacity of Sr2+ to act as a trigger for SR cation release is not clear. Some studies report that Sr2+ is 10-fold less effective than Ca2+ as a SR cation release trigger [13,19]. Other studies report that Sr2+ is unable to induce Sr2+ release from the SR in intact cardiac myocytes [12,18]. Our results clearly show that Sr2+ can indeed trigger 2+ Sr release from the SR in intact myocytes when stimulated by an action potential (AP). We compare the CICR and Sr2+ -induced Sr2+ release (SISR) processes at the molecular (single RyR channel modulation), local (Ca2+ and Sr2+ sparks), global levels (AP-induced Ca2+ - and Sr2+ -transients). These data provide new insights into the mechanisms that may govern RyR-mediated intracellular Ca2+ signaling.

2.2. Steady state single RyR channel function Studies of steady state single RyR channel function were carried out following the procedures outlined in Copello et al. [22]. Bilayers were formed using a mixture of phosphatidylethanolamine, phosphatidylserine and phosphatidylcholine (5:4:1 ratio by weight, 50 mg/ml in decane) over a 100 ␮m hole in a Teflon septum. The septum were separated into two compartments (termed cis and trans). Microsomes (5–10 ␮g protein per ml) were added to the cis chamber and were fused into the bilayer in the presence of 0.5–1 M CsCl and 5 mM CaCl2 while stirring. Fusion was detected by the sudden appearance of unitary Cl− currents. The cis chamber was then perfused with 20 ml of recording solution. The recording solution in the cis chamber contained 250 mM HEPES and 125 mM Tris (pH 7.4) unless otherwise stated. The recording solution in the trans chamber contained 250 mM HEPES and 53 mM Ca2+ (pH 7.4) unless otherwise stated. To examine the divalent dependency of single channel open probability (Po ), solutions with different free Ca2+ or Sr2+ concentrations were used. After perfusion of the recording solution, the cis free Ca2+ concentration was adjusted using different mixtures of dibromo-BAPTA (1 mM) and Ca2+ . The cis free Sr2+ concentration was adjusted in the same way utilizing BAPTA or EGTA. The compositions of these solutions were calculated using the MAXChelator (version 6.5) software [23] with critical stability constants obtained from Martell and Smith [24] or from Harrison and Bers [25]. The free Ca2+ and Sr2+ concentrations were verified using divalent cation sensitive electrodes [53]. The electrodes were fashioned from polyethylene tubing and divalent selective membranes manufactured using the ionophore ETH 129 (Fluka Chemical Corporation, Ronkonkoma, NY, USA) as described elsewhere [26]. Mean Po was calculated from single channel current recordings of 3–5 min duration. The EC50 s reported represent half maximal response and were estimated as previously described [22]. For Po values obtained at low cis Ca2+ or Sr2+ concentrations (10 nM to 200 ␮M), the following equation was applied:

2. Materials and methods 2.1. Sarcoplasmic reticulum microsome preparation Heavy microsomes enriched in Type II RyR were obtained from canine ventricular cardiac muscle [20]. Briefly, hearts were removed from dogs anaesthetized with sodium pentobarbital (20 mg/kg). The cardiac tissue was kept in a saline solution (154 mM NaCl, 10 mM Tris–malate, pH 6.8) at a temperature of 4 ◦ C. Pieces of the left ventricle were chopped and then homogenized. Heavy SR microsomes were obtained by differential centrifugation and stored at −70 ◦ C in the same saline solution [56], which contained 300 mM sucrose. These heavy microsomes were then reconstituted in planar lipid bilayers [21].

Po = A +

Po max 1 + (EC50 /[X2+ ])n

(1)

where Po is the open probability, [X2+ ] is the Ca2+ or Sr2+ concentration, A is the activity (Po ) of at 10 nM [X2+ ] (always ≤0.05), Po max is the maximum open probability, and n is the Hill coefficients for Ca2+ or Sr2+ activation. The equation does not consider the steady state RyR inhibition that occurs at much higher Ca2+ or Sr2+ concentrations (i.e. mM level). The IC50 s of the steady state RyR inhibition at high cytosolic Ca2+ or Sr2+ concentrations (200 ␮M to 10 mM) were estimated in a similar fashion. The differences between the mean values were statistically evaluated by a t-test (Origin, version 6, Microcal, OR, USA).

M.E. Zoghbi et al. / Cell Calcium 36 (2004) 119–134

2.3. Non-stationary single RyR channel function Reconstitution of single RyR channels for flash photolysis was performed as previously described [27]. Bilayers (200–400 pF electrical capacitance) were formed across holes in Delrin cups with a mixture (50 mg/ml in decane) of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) (Avanti Polar Lipids, Pelham, AL, USA) in a 7:3 relation. The microsomes were added to the cis side of the bilayer. The trans side was held at virtual ground. The standard solution here was 20 mM CsCH3 SO3 , 20 ␮M CaCl2 , 20 mM HEPES, pH 7.4. Microsome fusion was promoted by stirring in an osmotic gradient. The orientation of reconstituted RyR channels was such that their cytoplasmic face was in the cis compartment (i.e. defined by sidedness of ATP sensitivity). After channel incorporation, the trans-CsCH3 SO3 concentration was adjusted to 420 mM reversing the osmotic gradient. Flash photolysis experiments were performed on single RyR channels as previously described [4,28]. Briefly, 3 mM DM-Nitrophen (Calbiochem, San Diego, CA, USA [29]) was added to the cis compartment. The free Ca2+ or Sr2+ concentration was adjusted to 100 nM by addition of CaCl2 or SrCl2 , respectively. An intense UV light pulse (10 ns; 355 nm) from a frequency tripled Nd:YAG laser (Quanta-Ray DCR-11 and GCR-100; Spectra Physics, Mountain View, CA, USA) was used to induce photorelease of Ca2+ or Sr2+ only in the microenvironment of the channel. Laser light was directed at the bilayer through a 450 ␮m diameter fused-silica optic fiber (Fiberguide Industries, Stirling, NJ, USA). Photolyzed solution was replaced with unphotolyzed solution by stirring the bath. Cation selective electrodes [53] were used to determine the steady state free Ca2+ or Sr2+ before and after photolysis. The slow response time of these electrodes precludes measurement of the time course of the true free Ca2+ or Sr2+ change applied. It should be noted that the applied free Ca2+ or Sr2+ changes were not a simple step. Instead, they contained a very fast (<0.5 ms) overshoot that is due to rebinding of the divalent cation to the free DM-Nitrophen present. Changes in Po are illustrated by ensemble currents generated by summation of several single channel data sweeps. 2.4. Ventricular myocyte dissociation Rat ventricular myocytes were enzymatically dissociated using the Langendorff coronary retroperfusion technique [30]. Briefly, the hearts were placed on a Petri dish containing a Ca2+ -free Tyrode solution (in mM: 140 NaCl, 5.4 KCl, 1 MgCl2 , 0.33 Na2 HPO4 , 10 HEPES, 10 glucose, pH 7.4) at 37 ◦ C. The aorta was cannulated and the coronary arteries were washed with Tyrode solution for 6 min. The heart was then perfused with Tyrode solution containing 2 mg/ml collagenase 257 U/mg (Worthington Biochemical Corporation, NJ, USA) and 0.1 mg/ml of protease 4.4 U/mg (Pronase E, SIGMA, St. Louis, MO, USA). The hearts were then per-

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fused with Tyrode solutions containing 0.2 and then 2 mM CaCl2 to finish the dissociation procedure. After dissociation, cells were loaded (60 min, room temperature) with the acetoxymethyl ester (AM) form of Rhod-2 (Molecular Probes Inc., Eugene, OR, USA). Loading solution contained 20 ␮M Rhod-2 (AM) in Tyrode containing 2 mM CaCl2 . Indicator loaded cells were rinsed continuously for 15 min before use. 2.5. Ventricular myocyte Ca2+ –Sr2+ replacement Substitution of Ca2+ by Sr2+ was accomplished using a nominal Ca2+ -free Tyrode solution (<10 ␮M free Ca2+ ) containing 2 mM SrCl2 . Rhod-2 loaded cells were washed three times with the 2 mM SrCl2 Tyrode solution. To promote substitution of Ca2+ by Sr2+ within intracellular stores, cells were incubated in the Ca2+ -free solution for 30 min. In some experiments, cells were incubated in Tyrode solutions containing 100 ␮M ryanodine before recording to block intracellular divalent release. 2.6. Permeabilized ventricular myocytes Rodent ventricular myocytes were permeabilized using the technique described elsewhere [31,32]. Briefly, the myocytes were incubated in an internal solution containing (in mM): 132 K-aspartate, 5.4 KCl, 0.811 MgCl2 , 3 MgATP, phosphocreatine 10, 0.5 EGTA, creatine phosphokinase 5 U/ml, 10 MOPS, pH 7.2. Permeabilization was induced by 30 s exposure of the myocytes on internal solution containing 0.01% of saponin. Subsequently, the myocytes were washed with an internal solution free of saponin. Fluorescence recordings were obtained in permeabilized myocytes by placing the cell in the internal solution containing 50 ␮M of the potassium salt of the fluorescent indicator Fluo-4. Finally, the free Ca 2+ or Sr2+ concentration was adjusted to 100 nM or 1 ␮M, respectively. 2.7. Optical measurements on ventricular myocytes The cells were placed on the stage of an inverted fluorescence microscope (Nikon Diaphot, Tokyo, Japan) modified for confocal spot detection [33]. The cells were field stimulated with current pulses (SD9, Grass, USA) applied through platinum wires. A Planapo 63× (NA 1.4) oil immersion objective (Zeiss, Gottingen, Germany) was used to visualize the cells. An Argon laser (Model 80, Lexel, Fremont, CA, USA) was used to illuminate the cells with a 0.5 ␮m spot of light. The local fluorescence transients were detected by a Ptype -Intermediate-Ntype photodiode (PIN-PD) connected to a patch clamp amplifier (Axopatch 200B, Axon Instruments, Union City, CA USA). Snap shot images were obtained by epilluminating a small field (30–50 ␮m diameter) on the cell with 7 ns light pulses. In these studies, the 532 nm line of a Nd:YAG laser (Spectra Physics) was used as a light source, and the images were acquired with a

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cooled CCD camera (MCD 600; Spectra Source, Houston, TX, USA). Trans-illumination images were captured using the light from a halogen lamp. The sampling protocol applies sampling in equivalent time of multiple electrical stimulations (i.e. a single image was taken after each electrical stimulation), in contrast to sampling in real time (i.e. many images are taken during the same fluorescent transient). Successive images were taken at different delays following an AP stimulus. A computer was used to digitally control the delay between stimulus and image acquisition. The laser, CCD camera, A/D conversion system and the micropositioning of the cells were managed by a LabVIEW (version 4.0, National Instruments, Austin, TX, USA) based program. 2.8. Fluorescent indicator calibration with Ca2+ and Sr2+ The Ca2+ or Sr2+ concentration was calculated from fluorescence measurements ( F/F) using the following equation: [X2+ ] =

Kd [( F/Fmax )/( F/F)] − 1

(2)

where the dissociation constant (Kd ) for Rhod-2 is 2.7 ␮M for Ca2+ and 116 ␮M for Sr 2+ , F/Fmax was 30.6 for Ca2+ , 8 for Sr2+ and [X2+ ] the Ca2+ or Sr2+ concentration. The values of F/Fmax and Kd were determined in vitro by measuring with Ca2+ and Sr2+ standard solutions. These solutions were prepared under identical condition to the one used for the steady state bilayer experiments with the addition of the salt form of Rhod-2 (Molecular Probes Inc.) at a concentration of 20 ␮M. A 500 ␮l microcuvette was loaded with the different pCa or pSr solutions and the fluorescence at 525 nm was measured in a spectrofluorometer (Photon Technology International, Lawrenceville, NJ, USA). 2.9. Sparks and waves optical measurements Local optical measurements were performed with a scanning confocal system (MRC-1024ES, BioRad Laboratories, Hercules, CA, USA) using an Olympus 60× oil immersion with 1.4 NA objective. The size of the selected pixel was 246 nm and the sampling frequency 160 Hz (6.2 ms per line). Local Ca2+ or Sr2+ release events (sparks) were obtained from line scanned images were converted to an ASCII format with the program “ImageJ” (version 1.25, National Institutes of Health, Bethesda, MD, USA). Finally, the fluorescent traces were analyzed with our own software written in LabVIEW (National Instruments). The time course of the sparks was fitted with the following function that assumes that the rising phase of the spark is monotonic and the decaying phase can be represented by the sum of two exponential functions: f(t) = A(1 − e−(t−t0 )/τ1 )(B e−(t−t0 )/τ2 +(1 − B) e−(t−t0 )/τ3 )

(3)

where A is the maximum amplitude of the spark, B and 1−B are the weights of the first and the second components of the exponential decay. The parameter τ 1 corresponds to the time constant of the rising phase and τ 2 and τ 3 are the time constants for the decaying phase of the fluorescent signal.

3. Results 3.1. Evoked intracellular Ca2+ - and Sr2+ -transients Intracellular Ca2+ or Sr2+ release initiated by an AP was measured in isolated cardiac ventricular myocytes. In the Ca2+ case, the AP evoked an inward Ca2+ current that triggered SR Ca2+ release (i.e. CICR [3]). In the Sr2+ case, the AP evoked an inward Sr2+ current that triggered SR Sr2+ release (i.e. SISR). Here, changes in Rhod-2 fluorescence due to changes in intracellular free divalent concentration were measured. The fluorescent transients recorded when Ca2+ or Sr2+ was released will be referred to as Ca2+ - or Sr2+ -transients, respectively. A typical AP-induced Ca2+ -transient in a rat ventricular myocyte is shown in Fig. 1A. The time-to-peak was 92.7 ± 9.4 ms (mean ± S.E.; n = 14) and the maximum value reached is equivalent to a free Ca2+ concentration of 1.2 ␮M. The relaxation phase of the Ca2+ -transient was best fit with a single exponential function with a time constant of 104.7 ± 4.8 ms (mean ± S.E.; n = 14). The action of 100 ␮M ryanodine on the AP-induced Ca2+ -transient is also shown in Fig. 1A. The amplitude of the Ca2+ -transient was about one-third of the size in the presence of ryanodine. Ryanodine at this concentration is thought to completely block RyR Ca2+ release channels. Thus, the difference between the two traces is the contribution of RyR-mediated Ca2+ release to the transient. The time-to-peak of the ryanodine-modified transient (86.5 ± 6.8 ms; n = 12) was not significantly different compared to control (92.7 ms). However, the relaxation time constant increased substantially (∼60% change; 104.7 ± 4.8 ms versus. 166 ± 17.7 ms; also see Table 1). The AP-induced fluorescent transients were also measured after Ca2+ was replaced by Sr2+ . Typical AP-induced Sr2+ -transients are considerably slower than Ca2+ -transients as shown in Fig. 1B. The time-to-peak of the Sr2+ -transients was 369.4 ± 17.8 ms (n = 12) and maximum value reached is equivalent to a free Sr2+ concentration of 14 ␮M. This is roughly 10 times larger than the peak Ca2+ reached during a Ca2+ -transient (see Fig. 1A). The average time constant of the decaying phase of the Sr2+ -transient was 363.6 ± 4.9 ms. Fig. 1C shows normalized AP-induced Ca2+ - and Sr2+ -transients to better illustrate the kinetic differences between them. The time-to-peak and relaxation time constant of the Ca2+ -transient was about four times faster than that of the Sr2+ -transient. When myocytes were incubated with 100 ␮M ryanodine, the amplitude of the AP-induced Sr2+ -transient was decreased about three-fold (Fig. 1B) similar to that

Table 1 Time-to-peak and decay time constants for Ca2+ and Sr2+ fluorescence transients in presence and absence of ryanodine Ca2+ -transients

Ca2+ + ryanodine

Sr2+ -transients

Sr2+ + ryanodine

Time-to-peak (ms)

Decay time constant (ms)

Cell number

Time-to-peak (ms)

Decay time constant (ms)

Cell number

Time-to-peak (ms)

Decay time constant (ms)

Cell number

Time-to-peak (ms)

Decay time constant (ms)

1 2 3 4 5 6 7 8 9 10 11 12 13 14

129 110 81 60 112 130 60 40 62 38 92 124 127 133

105 117 107 89 135 139 115 92 100 85 96 91 78 117

15 16 17 18 19 20 21 22 23 24 25 26 – –

300 298 382 339 470 324 440 405 365 460 340 310 – –

330 387 370 360 386 358 385 351 365 344 363 365 – –

27 28 19 30 31 32 33 34 35 36 37 38

60 80 75 130 97 122 58 86 110 65 82 74

96 163 105 127 136 212 265 114 197 241 237 100

39 40 41 42 43 44 45 46 47 – – –

504 425 269 462 464 300 280 510 483 – – –

445 457 391 439 351 341 396 391 411 – – –

369.4 ± 17.8 12

363.6 ± 4.9 12

410.8 ± 33.1 9

402.4 ± 13.4 9

Mean ± S.E. N

92.7 ± 9.4 14

104.7 ± 4.8 14

86.6 ± 6.8 12

6.8 ± 17.8 12

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Cell number

Both time-to-peaks and decay time constants for Ca2+ and Sr2+ were statistically different (P < 0.01). N represents the number of cells.

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(A)

Ca2+ Transients Control

400 nM 200 ms

+Ryanodine

(B)

Sr2+ Transients Control +Ryanodine

(C)

3.5 µM 500 ms

Controls Normalized 500 ms

Fig. 1. Action potential triggered release in single ventricular cardiac myocytes. Ventricular myocytes were incubated at room temperature with Tyrode solution containing either 2 mM CaCl2 or SrCl2 . The cells were loaded with Rhod-2 (see Section 2) The fluorescence trace shows the time course of an AP-triggered Ca2+ -transient and the effect of 100 ␮M ryanodine on the release process (A). Similarly, we show the time course of an AP-triggered Sr2+ -transient and the effect of 100 ␮M ryanodine on the Sr2+ release process (B). Ryanodine reduced both the amplitude and rise time of the transient. Comparison between the time courses of Ca2+ and Sr2+ -transients induced by an action potential. Superimposed and normalized Ca2+ - and Sr2+ -transients (C). In the Sr2+ case, the transient had a slower time-to-peak and slower relaxation kinetics.

observed in the Ca2+ -transient case (Fig. 1A). This observation verifies that Sr2+ was indeed loaded in and released from the SR (n = 9). The time-to-peak of control and ryanodine-modified Sr2+ -transients were similar. However, the decay time constant of the ryanodine-modified Sr2+ -transient was slightly increased (∼9% change; 363.6± 4.9 ms versus 402.4 ± 13.4 ms). In contrast, the decay time constant of the ryanodine-modified Ca2+ -transient increased ∼60%. The ryanodine-modified transients provide some indication of the Ca2+ and Sr2+ entry that occurs through the sarcolemma during the AP. A comparison of the duration of ryanodine-modified Ca2+ - and Sr2+ -transients indicates the Sr2+ influx is approximately three times longer than the Ca2+ influx. This is consistent with the longer duration of Sr2+ inward current reported for L-type Ca2+ channels in cardiac muscle cells [12]. 3.2. Spatial distribution of sub-sarcomeric Ca2+ or Sr2+ The fluorescence transients shown in Fig. 1 report global (i.e. whole cell) changes in Ca2+ and Sr2+ concentration.

Those transients provide little information about the spatial distribution of the local free Ca2+ or Sr2+ concentrations that occur near the SR release sites. The spatiotemporal local Ca2+ or Sr2+ distributions during the AP were defined using a snapshot imaging technique [34]. Images of local Ca2+ distribution in Rhod-2 loaded ventricular cardiac myocyte at different times following an AP are shown in Fig. 2. The trans-illumination image shows the characteristic dark striations at ∼2 ␮m intervals are associated with the sarcomeric structure of the cell (Fig. 2A). The following series of snap-shot images were obtained from a small optical field of the cell that was epi-illuminated with a very short (∼7 ns) excitation light pulse. Snapshot fluorescence images were collected just prior to the AP (control) and then at different times (20, 50, 100, 150, 200, and 500 ms) after its initiation. The control image shows that there is a homogenous fluorescence distribution prior to the AP. The fluorescence increase after AP initiation results from an increase in the intracellular Ca2+ concentration. At 20 ms after the AP, there is small but detectable increase in fluorescence. The increase in fluorescence peaks at the 100 ms mark. After peaking, the fluorescence decreases back to baseline levels. In the series of images shown in Fig. 2A, the fluorescence increase in ventricular myocytes in response to the AP does not show clear indications of intrasarcomeric fluorescent gradients. The failure to detect them here could be due to many factors (see [34]). One factor is the relatively short sarcomeric spacing (∼2 ␮m) in these cells. Nevertheless, this same imaging method applied to skeletal muscle fibers (sarcomere spacing: ∼2.1 ␮m) was able to resolve clear sub-sarcomeric spatial Ca2+ heterogeneities (i.e. Ca2+ gradients) [34]. Thus, the failure to detect them here does not mean that they do not exist. It may just mean that the optical resolution of this far field optical technique does not have enough spatial bandwidth to resolve spatial fluorescence gradients under these experimental conditions. The time course of the AP-triggered intracellular fluorescence transient could be reconstituted from the snapshot images (Fig. 2B). Maximum fluorescence at the same central region in the cell was determined in all images (including data not shown in Fig. 2A). The time course of the reconstructed Ca2+ -transient (Fig. 2B) is quite similar to that obtained when the global fluorescence Ca2+ -transient was measured using a photodiode (Fig. 1A). Fig. 3A shows a sequence of snapshot images acquired from a Rhod-2 loaded ventricular myocyte in which Ca2+ was substituted for Sr2+ . Images reveal intracellular Sr2+ distribution before (control) and after AP stimulation (150, 500, 750, 1000, 1500, and 2000 ms). In the control image, the uniform fluorescence again indicates that the dye was homogenously distributed. The fluorescence increase after AP initiation results from an increase in the intracellular Sr2+ concentration. The increase in fluorescence is visible 50 ms after stimulation (data not shown) and peaks at ∼500 ms. After that, it decreases back to baseline levels. As found for Ca2+ release (Fig. 2), no clear spatial Sr2+ heterogeneities

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Fig. 2. Sequences of fluorescence images reporting changes in intracellular free Ca2+ recorded with the flash laser imaging technique. A trans-illumination image of a rat ventricular cardiac myocyte incubated with Tyrode solution containing 2 mM Ca2+ , followed by a series of snap shot flash laser images is shown (A). The delay times between the electrical stimulation and the time the image was captured is marked on the top of each fluorescence image. The time course of the change in the free Ca2+ concentration obtained from this type of snap shot imaging is shown (B).

(i.e. no sub-sarcomeric gradients) were evident (Fig. 3A). The global Sr2+ -transient (Fig. 3B) was reconstructed from the snapshot fluorescence images and its time course was similar to that measured using the photodiode (Fig. 1B). Like the Ca2+ case (Fig. 2), these data (Fig. 3) show that the AP induces relatively uniform local Sr2+ release across the cell. 3.3. Spontaneous Ca

2+

and Sr

2+

release

One consequence of the Ca2+ /Sr2+ ionic replacements is that the different divalents can alter the time course of the cardiac action potential. This in turn could influence the time course of the AP-evoked intracellular Ca2+ - and Sr2+ -transients. Thus, the kinetics of spontaneous SR Ca2+ and Sr2+ release phenomena (waves and sparks) were also determined in intact Rhod-2 loaded cardiac myocytes. Fig. 4 shows spontaneous Ca2+ and Sr2+ waves, a spontaneous global release phenomenon, measured using a standard confocal microscope in line scan mode. The half duration of the observed Ca2+ waves was 398.9 ± 29.7 ms (n = 3 cells). The half duration of the observed Sr2+ waves was substantially longer (2211 ± 58.9 ms; n = 3 cells). This implies

that the lengthening of Sr2+ -mediated global release phenomenon is not solely a consequence of a longer AP in Sr2+ loaded cells. Instead, it indicates that the mechanism(s) involved in terminating spontaneous waves are much slower when Ca2+ is replaced by Sr2+ . We also observed that the frequency of Sr2+ waves (0.01 ± 0.01 s−1 ; n = 3 cells) in Sr2+ loaded cells was also substantially lower than that of Ca2+ waves (0.06 ± 0.01 s−1 ; n = 3 cells) in Ca2+ loaded cells. Spontaneous local Ca2+ release events (i.e. sparks) were also recorded (in six different experiments) in Ca2+ loaded cells. A summary of Ca2+ spark properties can be found in Table 2. In intact cells, the activation phase of the Ca2+ spark (Fig. 5A) was well described by a single exponential function having an average time constant of 3.7 ± 0.97 ms (n = 106; Fig. 5C, top panel, open bar). The decay phase of the Ca2+ sparks was best described by the sum of two exponential functions with average time constants of 28.9 ± 5.85 ms and 282.3 ± 120.6 ms (n = 106; Fig. 5C, middle panel, open bar). Numerous attempts were made to record Sr2+ sparks in Sr2+ loaded intact cells. None were observed. Consequently, spark studies were continued in

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Fig. 3. Sr2+ changes measured Flash Laser fluorescence Images induced by action potential stimulation. Series of snap shot fluorescence images when Ca2+ was replaced by Sr2+ as described in Section 2 (A). The delay times between the electrical stimulation and the time the image was captured is marked on the top of each fluorescence image. The time course of the change in the free Sr2+ concentration obtained from this type of snap shot imaging is shown (B). Table 2 Properties of Ca2+ sparks in intact ventricular myocytes Experiment number

[Ca2+ ] (mM)

n (sparks)

τ 1 (ms)

07212001 07262001 08102001 08312001-a 08312001-b

2 2 2 2 2

35 24 16 16 15

3.54 3.56 4.58 4.44 2.16

saponin-permeabilized cells [1]. The amplitude in F/F of the Ca2+ sparks was 0.21 ± 0.016. The time course of the Ca2+ sparks in intact and permeabilized cells are compared in Fig. 5A. The Ca2+ sparks in permeabilized cells had a

± ± ± ± ±

τ 2 (ms)

B 0.652 0.980 0.729 0.797 2.073

0.668 0.796 0.865 0.854 0.721

± ± ± ± ±

0.014 0.01 4 0.008 0.009 0.036

35.8 29.7 32.0 26.7 20.3

± ± ± ± ±

τ 3 (ms) 2.38 2.26 1.76 1.76 3.24

245.9 277.3 464.9 293.7 129.5

± ± ± ± ±

9.59 18.8 41.5 22.3 12.3

slower rise (time constant = 11.3 ± 2.5 ms; n = 94) and faster decay kinetics (time constant = 11.6 ± 2.4 ms and 97.9 ± 12.9 ms; n = 94), having a full duration at half maximum (FDHM) of 23.75 ± 4.27 ms (n = 94) (Table 3). In

Table 3 Properties of Ca2+ and Sr2+ sparks in permeabilized ventricular myocytes Experiment number

Divalent

n (sparks)

τ 1 (ms)

10182001-a 10182001-b 10232001 10182001-c 10182001-d 10182001-e

Ca2+

15 42 37 19 9 29

10.16 9.48 14.13 10.14 4.21 14.13

Ca2+ Ca2+ Sr2+ Sr2+ Sr2+

± ± ± ± ± ±

τ 2 (ms)

B 6.326 4.230 13.703 9.876 4.74 16.08

0.951 0.930 0.960 0.948 0.916 0.969

± ± ± ± ± ±

0.017 0.021 0.003 0.037 0.039 0.0197

13.78 11.90 9.03 9.34 11.50 8.85

± ± ± ± ± ±

τ 3 (ms) 2.60 1.47 1.41 1.91 3.78 0.91

102.0 108.3 83.5 98.5 110.9 103.9

± ± ± ± ± ±

21.91 9.20 12.9 12.6 37.9 15.9

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Fig. 4. Ca2+ and Sr2+ waves. Myocytes were incubated in Tyrode solution containing either Ca2+ or Sr2+ (right and left panels, respectively). In presence of Ca2+ the waves have duration of 400 ms (left side). Notice the presence of Ca2+ sparks in the line scan image. When Ca2+ was substituted by Sr2+ for more than 40 min, a huge change in kinetics can be observed (right side). The differences in the time course are significant.

order to characterize the spatial properties of these sparks the full-width at half-maximum (FWHM) was computed, given a value of 3.1 ± 0.5 ␮m (n = 7). To record Sr2+ sparks, permeabilized cells were incubated in an internal solution containing 1 ␮M Sr2+ . At lower cytosolic Sr2+ levels, very low Sr2+ sparks frequency made experimentation impractical. The amplitude in F/F of the Sr2+ sparks was 0.32 ± 0.016. The time courses of typical Ca2+ and Sr2+ sparks are superimposed in Fig. 5B and the average properties of Sr2+ sparks summarized in Fig. 5C. Interestingly, Ca2+ and Sr2+ spark in permeabilized myocytes did not have statistically different (P > 0.35, t-test) amplitude or kinetics. The Sr2+ sparks had a mono-exponential rise of 9.5 ± 5.0 ms (time constant, n = 57), and a bi-exponential decay with time constants 9.9 ± 1.4 ms and 104.4 ± 6.2 ms (n = 57) (Table 3). Additionally the FDHM was 21.05 ± 2.73 ms (n = 57) and the FWHM 2.4 ± 0.5 ␮m (n = 11). 3.4. Steady-state single RyR2 channel Ca2+ or Sr2+ regulation Single RyR2 channels were reconstituted into planar lipid bilayers and sample single channel recordings are shown in

Fig. 6. The current carrier in these studies was either Ca2+ (Fig. 6A) or Sr2+ (Fig. 6B) and net current was in the lumen to cytosol (trans to cis) direction. Recordings at three different cytosolic steady-state free Ca2+ and Sr2+ concentrations are shown (Fig. 6A and B). The relationship between Po and the cytosolic divalent concentration are shown at bottom (Fig. 6). Connected points represent data from the same channel. In the case of Ca2+ (Fig. 6A), all channels tested were closed at <0.1 ␮M Ca2+ and were maximally active at 10 ␮M Ca2+ . Fit of the activation phase of these Po data using Eq. (1) (see Section 2) revealed an average EC50 of 1.9 ± 0.5 ␮M (n = 6) with a Hill coefficient of 2.8 ± 0.4. Typically, the RyR2 channels were substantially inhibited at 10 mM (average IC50 = 3.6 ± 1.0 ␮M). These Ca2+ data are in good agreement with previous reports [22,35–37]. All RyR2 channels tested were also activated by Sr2+ (Fig. 6B). However, larger cytosolic Sr2+ concentrations (compared to Ca2+ ) were required to activate the channels. The EC50 of Sr2+ activation was near 45 ± 6 ␮M (n = 6) with a Hill coefficient of 3.1 ± 0.6. As in the Ca2+ case described above, single RyR2 channel inhibition was observed at millimolar Sr2+ concentrations. The IC50 of Sr2+ inhibition was 4.6 ± 0.9 mM. The EC50 s for single RyR2 channel

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Fig. 5. Sparks recorded in intact and permeabilized myocytes. (A) Effect of permeabilzation on Ca2+ sparks. (B) Comparison between Ca2+ sparks and Sr2+ in permeabilized cells. (C) Bar graph shows the kinetic characteristics of Ca2+ and Sr2+ sparks recorded on intact and permeabilized myocytes. The chart shows that although there are significant differences between sparks recorded on intact and permeabilized cells, the were no kinetic differences between sparks recorded on permeabilized in presence of Ca2+ and Sr2+ (P < 0.01).

Ca2+ and Sr2+ activation were statistically different (P < 0.01, t-test) but the IC50 s were not. It has been suggested that physiological levels of Mg2+ may compete with Ca2+ for occupancy of the Ca2+ activation site. Intuitively, Mg2+ should also compete with Sr2+ for occupancy of this site. Of course, the effectiveness of Mg2+ competition may be different with Sr2+ or Ca2+ . Thus, the Sr2+ or Ca2+ EC50 s were also defined in the presence of a physiologically relevant free Mg2+ concentration (1 mM). The capacity of Ca2+ and Sr2+ to activate single RyR2 channels in the presence of physiological levels of free Mg2+ and total ATP is shown in Fig. 7. Channels were closed at low cytosolic Ca2+ levels (<1 ␮M) and were activated when cytosolic Ca2+ levels exceeded the 1 ␮M mark (Fig. 7A). The average EC50 of Ca2+ activation here was 12 ± 2 ␮M (n = 5), which was significantly higher from that in the absence of ATP and Mg2+ (1.9 ␮M, P < 0.01). In the Sr2+ case (Fig. 7B), the average EC50 of Sr2+ activation was 61 ± 20 ␮M (n = 4), which was not significantly different to that in the absence of ATP and Mg2+ (45 ␮M). Thus, Mg2+ does compete differently with Sr2+ or Ca2+ at the cytosolic activation site. However, there remains a substantial difference in the Ca2+ and Sr2+ EC50 s for the RyR2 channel.

3.5. Non-stationary single RyR2 channel Ca2+ and Sr2+ regulation Single RyR channel behavior in response to fast Ca2+ and Sr2+ changes was also examined. Fast Ca2+ and Sr2+ changes were produced by flash photolysis of DM-Nitrophen. This is an EDTA-based compound that does not differentiate substantially between divalents. Nevertheless, its divalent binding affinity is highly photosensitive and consequently flash photolysis of divalent-bound DM-Nitrophen can generate rapid elevations of the free divalent concentration. In this study, fast elevations of Ca2+ or Sr2+ were applied to single RyR2 channels in planar bilayers. In Fig. 8, representative single channel recordings from a single RyR2 channel following photo-release of Ca2+ (Fig. 8A) and Sr2+ (Fig. 8C) are shown. Four single channel sweeps are aligned with the flash (arrow) in each case. Between flashes, resting conditions were re-established. Corresponding ensemble currents (i.e. generated by summing multiple single channel sweeps) are also shown (Fig. 8B and D). The time course of channel activity (Po ) following the fast Ca2+ and Sr2+ change is best illustrated by these ensemble currents. Single RyR2 channel activity rapidly rose

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Fig. 6. Regulation of single RyR2 channels by cytosolic Ca2+ and Sr2+ . Single RyR2 channels were reconstituted into planar lipid bilayers. The solutions contained HEPES–Tris (cytosolic side) and HEPES/Ca2+ or HEPES/Sr2+ (luminal side). Channel activity was recorded at 0 mV holding potential. (A) Representative unitary Ca2+ currents at different cytosolic Ca2+ concentrations. Open events are shown as downward deflections. The zero current level is marked. Lower panel illustrates Po as a function of cytosolic Ca2+ concentration of six different single channel experiments. Data points from the same channel are connected by lines. Averaged EC50 for activation was 1.9 ± 0.5 ␮M (range: 1.3–4 ␮M∗ , n = 6) and n (Hill coefficient) = 2.8 ± 0.4 (range: 2–5). Averaged IC50 for inactivation (n = 4) was 3.6 ± 1.0 ␮M (range: 1.8–6.2 mM) and 2.0 ± 0.3 (range: 1.5–3.0). In two cases, IC50 was >10 mM (out of the range of our measurements). Line at the beginning of traces indicates the closed level of the channel. (B) Representive unitary Sr2+ currents at different cytosolic Sr2+ concentrations. Lower panel illustrates Po as a function of cytosolic Sr2+ concentration of six different single channel experiments. Averaged EC50 for activation was 45 ± 6 ␮M∗ (range: 29–70 ␮M, n = 6) and n (Hill coefficient) = 3.1 ± 0.6 (range: 2–5). Averaged IC50 for inactivation (n = 5) was 4.6 ± 0.9 mM (range: 2.1–6.4 mM) and n (Hill coefficient) = 2.6 ± 0.6 (range: 1.0–4.7). (*: EC50 values in Sr2+ solutions were significantly higher than those in Ca2+ solutions; P < 0.01.)

following the fast stimulus. In the Ca2+ case (Fig. 8B), channel activity peaked and then spontaneously decayed. In the Sr2+ case (Fig. 8D), channel activity peaked and then remained constant (i.e. no spontaneous decay was evident). Thus, fast application of either Ca2+ or Sr2+ rapidly activates the RyR2 channel but the sustained response following the initial fast activation is different.

4. Discussion It is well known that Sr2+ can substitute for Ca2+ in many intracellular Ca2+ signaling processes in frog skeletal fibers [13] and cardiac myocytes [12,14,18]. The Sr2+ ion is, therefore, a potentially useful probe to study the mechanisms that control cardiac EC coupling. Here, we exchanged intra- and extracellular Ca2+ for Sr2+ and define the changes in AP-evoked and spontaneous intracellular RyR2-mediated release phenomena (i.e. transients, waves and sparks). Additionally, how Ca2+ for Sr2+ modulates single RyR2 channels in vitro was also defined. We found clear evidence of SR2+ -induced RyR2-mediated 2+ release in intact ventricular myocytes. Global Sr

RyR2-mediated Sr2+ release phenomena (evoked transients or spontaneous waves) were substantially slower than their Ca2+ counterparts. In saponin-permeabilized myocytes, the kinetics of spontaneous RyR2-mediated Sr2+ sparks was similar to that of classical Ca2+ sparks. Snapshot fluorescence imaging confirmed that evoked Ca2+ or Sr2+ release occurred through out the across section of the cell. Single RyR2 channel studies showed that the Ca2+ and Sr2+ EC50 s were significantly different (1.9 ± 0.5 ␮M versus 45 ± 6 ␮M; n = 6) but their IC50 s were not (3.6 ± 1.0 ␮M versus 4.6 ± 0.9 ␮M; n = 6). Although fast Ca2+ and Sr2+ stimuli rapidly activated single RyR2 channels, the sustained response to these stimuli differed. Some possible interpretations of these experimental observations are discussed in the following sections. 4.1. Evidence for Sr2+ -induced Sr2+ release Application of ryanodine reduced the amplitude of both Ca2+ and Sr2+ AP-evoked transients in the intact myocytes. In the Ca2+ case, the AP-evoked Ca2+ -transient is largely the result of RyR2-mediated Ca2+ release that was

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Fig. 7. Single RyR2 channel activation by cytosolic Ca2+ and Sr2+ in the presence of Mg and ATP. Conditions are similar to those in the legend of Fig. 6, except that in all cases 5.6 mM Mg2+ and 5 mM ATP were added to the cytosolic solutions (free [Mg2+ ] ∼ 1 mM). (A) Single channel recording of RyR2 channels in lumenal Ca2+ (50 mM) and with various cytosolic Ca2+ concentrations. Open events are shown as downward deflections. The zero current level is marked. Lower panel illustrates Po as a function of cytosolic Ca2+ concentration of five different single channel experiments. Data points from the same channel are connected by lines. The EC50 for Ca2+ activation was 12 ± 2 ␮M∗ with a Hill coefficient of 2.6 ± 0.6. (B) Single channel recording of RyR2 channels in lumenal Sr2+ (50 mM) and with various cytosolic Sr2+ concentrations. Lower panel illustrates Po as a function of cytosolic Sr2+ concentration of four different single channel experiments. The EC50 for Sr2+ activation was 61 ± 20 ␮M∗ with a Hill coefficient of 2.9 ± 0.8. (*: EC50 values in Sr2+ solutions were significantly higher than those obtained in Ca2+ solutions; P < 0.01.)

triggered by Ca2+ entry during the AP stimulus. Ryanodine blocks the RyR2-mediated component of the transient reducing its amplitude. There is good consensus in the field for this interpretation [1]. Substitution of Ca2+ by Sr2+ was accomplished by placing the cells in Ca2+ -free, Sr2+ -containing solutions for 30 min prior to experimental. This Sr2+ replacement protocol gave similar results compared to one in which cells were preconditioned by multiple 1 mM caffeine pulses in a divalent-free Tyrode to deplete the intracellular stores [18,38]. Undoubtedly, the differences in the time course between Ca2+ and Sr2+ will be dependent on both the SR uptake and buffering. Nevertheless, our results suggest that Sr2+ was loaded in the SR and released by the applied AP stimulus. Since the observed Sr2+ -transients were obtained in the presence of 2 mM of extracellular Sr2+ (i.e. absence of extracellular Ca2+ ), the most likely situation is that the observed Sr2+ -transient is due to RyR2-mediated Sr2+ release triggered by Sr2+ entry during the AP stimulus. This is consistent with our demonstration that single RyR2 channels conduct Sr2+ and are activated by the Sr2+ ion. Thus, the data presented here (whole cell and single channel) indicate that Sr2+ can replace Ca2+ in both triggering intracellular release and as the released ion (i.e. Sr2+ -induced Sr2+ release).

4.2. Slower kinetics of AP-evoked Sr2+ -transients In intact ventricular myocytes, there were clear differences in the time course of the AP-evoked Sr2+ -transients compared to their Ca2+ counterparts. For example, the relaxation of the Sr2+ -transient was 3.6 times slower than the relaxation of Ca2+ -transient. Instead, the longer duration is likely due to differences in intracellular Ca2+ /Sr2+ handling or release. In the cell, the exponential decay during the relaxation phase of the Ca2+ -transient is primarily attributed to active Ca2+ removal by the SR uptake [39–41]. Thus, the slower relaxation of the Sr2+ -transient may simply reflect less effective Sr2+ uptake into the SR. Indeed, the SR pump has substantially (25-fold) lower affinity for Sr2+ than Ca2+ [13]. The peak Ca2+ and Sr2+ concentrations reached after AP stimulation were also quite different for Ca2+ - and Sr2+ -transients (∼2 ␮M versus ∼14 ␮M, respectively). This difference could also be explained by less efficient Sr2+ uptake by the SR. Another contributing factor could be lower intracellular Sr2+ buffer capacity compared to Ca2+ case. For example, the affinity of troponin-C for Sr2+ is two to four times lower than its affinity for Ca2+ [14]. Thus, the slow time course of the evoked Sr2+ -transients is likely due to a compilation of factors like less efficient SR Sr2+ pumping and/or cytosolic Sr2+ buffering.

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Fig. 8. Single RyR2 channel activation by fast Ca2+ and Sr2+ elevations. (A) Response of single RyR2 channel to a fast photolytically induced Ca2+ stimulus. Four sequential single channel recordings are shown. Resting conditions were re-established between recordings. Opening events are shown as upward deflections. The time of the flash is marked. (B) The ensemble behavior of a single RyR2 channel to the fast Ca2+ stimulus. The ensemble data were generated by summing single channel recordings. (C) Response of single RyR2 channel to a fast photolytically induced Sr2+ stimulus. (D) The ensemble behavior of a single RyR2 channel to the fast Sr2+ stimulus.

A decrease in the buffer capacity, however, cannot explain the different rise times (i.e. time-to-peak) of the Ca2+ - and Sr2+ -transients. The time-to-peak of the Sr2+ -transient was 4 times slower than the Ca2+ -transient (369 ms versus 92 ms, respectively). Lower Sr2+ buffering capacity would increase, not decrease, transient rise time and Bassani et al. [40] showed that the time-to-peak of the Ca2+ -transient is not modified by blocking the SR Ca2+ ATPase. How then can the differences in time-to-peak be explained? Time-to-peak will clearly depend on the duration of the net DHPR-mediated sarcolemma cation influx that triggered by the AP. Thus, the difference could be due to AP prolongation when Ca2+ is

substituted for Sr2+ [12,42,43]. The AP is prolonged because there is slower inactivation of the inward divalent cation current [11,44,45] and blockade of outward K+ current by Sr2+ [46]. The consequence is a long inward Sr2+ current that would lengthen the time-to-peak and extend the duration of Sr2+ release from the SR. Still another factor that could prolong the SR release process is that cytosolic Ca2+ and Sr2+ may not participate in terminating RyR-mediated release equally. If Sr2+ were less effective at terminating release, then release would be prolonged. Again, the slower time-to-peak of the AP-evoked Sr2+ -transients is likely due to a compilation of factors. In addition, differences in the

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association and dissociation rate constants of the dye for Ca2+ and Sr2+ always can be involved in promoting differences in the changes of the free divalent concentration time courses inside the cell. It is clear that the big difference in the dissociation constant of Rhod-2 for Ca2+ and Sr2+ (2.7 ␮M for Ca2+ and 116 ␮M for Sr2+ ) suggest differences in the rate constants. But, the fact that the sparks measured in Ca2+ and Sr2+ have very similar time courses implies that the differences observed in the time courses of the action potential induced transients and the spontaneous waves are not generated by the different bandwidth of the indicator for Ca2+ and Sr2+ . 4.3. Spatial distribution of Ca2+ and Sr2+ release The spatial pattern of local distribution of AP-evoked Ca2+ and Sr2+ release was also defined using snapshot fluorescence imaging [33,34,47]. Since release site localization in the cell has a regular periodicity [1], fast synchronous release at these sites should generate clear sub-sarcomeric Ca2+ and/or Sr2+ heterogeneities immediately following initiation of release. Diffusion will reduce these sub-sarcomeric heterogeneities over time. Indeed, this is exactly what was observed previously in skeletal muscle using this snapshot technique [34]. However, no detectable sub-sarcomeric Ca2+ or Sr2+ heterogeneities were observed here in these cardiac muscle cells. The inability to resolve sub-sarcomeric heterogeneities may simply mean that: (1) the gradients in cardiac myocytes are smaller (due to slower and/or less synchronous release) and/or, (2) spacing between release sites may be smaller [34]. Despite the absence of resolvable sub-sarcomeric heterogeneities, the snap shot data illustrate the spatial distribution of RyR2-mediated Ca2+ or Sr2+ release after an AP is similar. This indicates that the Ca2+ for Sr2+ exchange was uniform and complete throughout the cross-section of the cell. 4.4. Spontaneous Sr2+ release events Spontaneous Sr2+ waves had much longer duration than Ca2+ waves. These waves were not triggered by an AP and thus the longer duration of Sr2+ waves cannot be attributed to differences in ionic divalent fluxes at the plasma membrane level. Instead, the longer duration is likely due to differences in the rates of intracellular Ca2+ /Sr2+ release and reuptake. Possibly these very same differences prolong the relaxation phase of the AP-evoked Sr2+ transient. To our knowledge, this study reports the first measurement of a spark mediated by an ion other than Ca2+ . Thus, these studies provide some unique insights into the underpinnings of this phenomenon. The first interesting observation is that Sr2+ -mediated sparks were not observed in intact ventricular cardiac myocytes. The reason is probably the lower Sr2+ affinity of the RyR2 channel’s activation site makes it less

likely to spontaneously open at the low resting Sr2+ present in the intact cell. Consistent with this explanation is that Sr2+ sparks were only observed in permeabilized myocytes after cytosolic Sr2+ concentrations were elevated to the 1 ␮M free Sr2+ mark. The second interesting observation was that the time course of Ca2+ and Sr2+ sparks was similar in permeabilized cell. This is surprising because the Ca2+ for Sr2+ replacement slowed the time-to-peak and relaxation phases of the AP-evoked transients as well as substantially prolonged spontaneous waves. Thus, the slower kinetics of these global phenomena (transients and waves) is not due to differences spark kinetics. Instead, they are likely due to differences in spark recruitment and/or propagation. The implication is that Ca2+ and Sr2+ differentially modulate the mechanism(s) that govern spark recruitment/propagation but not the mechanism(s) that control individual spark kinetics. 4.5. Ca2+ and Sr2+ modulation of single RyR2 channels Our single channel data shows that the RyR2 channel from dog heart is regulated in vitro by both high-affinity (activating) and low-affinity (inhibitory) Ca2+ binding sites, These sites display affinity for Ca2+ that is undistinguishable from those measured in RyR2 channels from rat heart [48]. It appears that the same activating and inhibitory sites modulate the channel when they are occupied by Sr2+ . However, the apparent affinities of the activation site for Ca2+ and Sr2+ are significantly different. It takes substantially more Sr2+ to activate the channel. Studies of [3 H] ryanodine binding that have forwarded a similar conclusion (e.g. [49]). Also in line with this is that fact that RyR1 channels in the presence of Mg2+ and ATP are less sensitive to Sr2+ than Ca2+ [19]. The lower RyR2 sensitivity to Sr2+ activation observed in bilayers is consistent with our observation that Sr2+ sparks were observed in permeabilized cells only after increasing cytosolic Sr2+ concentrations to ∼1 ␮M. The higher Sr2+ levels required to activate RyRs are also consistent with slower waves due to slower Sr2+ spark recruitment/propagation in Sr2+ loaded intact cells. Interestingly, the apparent affinities and efficacies of Ca2+ and Sr2+ at the RyR2 channel’s low-affinity cytosolic inhibition site are the same. This also has interesting implications. It means that differential Ca2+ /Sr2+ action at this site is not responsible for the longer time-to-peak of the AP-evoked Sr2+ -transient, slower relaxation of the AP-evoked Sr2+ -transient or the prolongation of Sr2+ waves in the intact cells. Thus, these results suggest that the low-affinity Ca2+ inhibition commonly observed in single RyR2 channels studies in vitro is not responsible for turning off global SR Ca2+ release phenomena. Fast Ca2+ or Sr2+ elevations were also applied to single RyR2 channels in bilayers. The channels activated rapidly in response to either a fast Ca2+ or Sr2+ elevation. This fast activation is consistent with previous reports [4,27]. In the Ca2+ case, channel activity peaked at high Po values and then spontaneously decayed (also see [4]). In the Sr2+

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case, channel activity increased rapidly to a plateau level and remained constant over the measured period. It is important to note that the Ca2+ or Sr2+ stimuli were not identical. The Ca2+ stimuli were applied from a resting Ca2+ level of 100 nM while the Sr2+ stimuli were applied from a resting Sr2+ level of 1 ␮M. This was by design. These resting concentrations represent similar regions of the steady-state Ca2+ and Sr2+ dose–response relationships (see Fig. 6). They are about an order of magnitude below the average activation threshold in each case. The difference in resting Ca2+ and Sr2+ levels may impact the nature of the applied stimulus. Photolysis of DM-Nitrophen can produce a large fast free Ca2+ (or possible Sr2+ ) overshoot on the leading edge of the stimulus [29,50,51]. The amplitude and duration of this overshoot theoretically depends on the resting Ca2+ (or Sr2+ ) concentration. Unfortunately, there is not enough information available about the Sr2+ –DM-Nitrophen interaction to accurately predict the nature of the Sr2+ overshoot (if any). Nevertheless, it is reasonable to assume that the overshoot would be constrained to the first millisecond of the stimulus. We have previously argued that this type of overshoot is unlikely to impact how a channel behaves over the next several seconds [52]. However, an overshoot is likely to impact how the channel initially responds to the stimulus. For example, an overshoot could accelerate the initial closed to open transition. Interpretations of the non-stationary RyR2 channel results should keep these issues in mind. Here, single RyR2 channels were activated equally fast (within 1 ms) by the Ca2+ and Sr2+ stimuli. Our interpretation is that both fast Ca2+ and Sr2+ can rapidly and robustly activate single RyR2 channels. This is consistent with the similar rise times of Ca2+ and Sr2+ sparks. The spontaneous decay following the Ca2+ stimulus is thought to reflect a transient re-equilibration between RyR2 gating modes [52]. The absence of a spontaneous decay following the Sr2+ stimulus suggests that this does not occur in the Sr2+ case. Thus, re-equilibration of gating modes following a fast Ca2+ stimulus (i.e. adaptation) does not likely play a role in defining Ca2+ spark kinetics. Specifically, the results presented in this paper indicates that adaptation [4] is not involved in the termination of local the Ca2+ release events as Ca2+ sparks. However, it could be important in controlling the kinetics of global SR Ca2+ release phenomena.

Acknowledgements We thank Dr. Carlo Caputo for helpful comment during this work and Dr. Hugo Arias, Dr. Narine Sarvazyan, Dr. Guillermo Perez and Dr. Raul Martinez-Zaguilan for reviewing the manuscript. The single channel studies were supported by NIH HL57832 and HL64210 to M.F. and by AHA Grant 0130142N and Muscular Dystrophy Grant to J.A.C. The fluorescence studies were supported TTUHSC SEED grant to A.L.E.

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