Junctional Cleft [Ca2+]i Measurements Using Novel Cleft-Targeted Ca2+ SensorsNovelty and Significance
Rationale: Intracellular Ca2+ concentration ([Ca2+]i) is regulated and signals differently in various subcellular microdomains, which greatly enhances its second messenger versatility. In the heart, sarcoplasmic reticulum Ca2+ release and signaling are controlled by local [Ca2+]i in the junctional cleft ([Ca2+]Cleft), the small space between sarcolemma and junctional sarcoplasmic reticulum. However, methods to measure [Ca2+]Cleft directly are needed.
Objective: To construct novel sensors that allow direct measurement of [Ca2+]Cleft.
Methods and Results: We constructed cleft-targeted [Ca2+] sensors by fusing Ca2+-sensor GCaMP2.2 and a new lower Ca2+-affinity variant GCaMP2.2Low to FKBP12.6, which binds with high affinity and selectivity to ryanodine receptors. The fluorescence pattern, affinity for ryanodine receptors, and competition by untagged FKBP12.6 demonstrated that FKBP12.6-tagged sensors are positioned to measure local [Ca2+]Cleft in adult rat myocytes. Using GCaMP2.2Low-FKBP12.6, we showed that [Ca2+]Cleft reaches higher levels with faster kinetics than global [Ca2+]i during excitation–contraction coupling. Diastolic sarcoplasmic reticulum Ca2+ leak or sarcolemmal Ca2+ entry may raise local [Ca2+]Cleft above bulk cytosolic [Ca2+]i ([Ca2+]Bulk), an effect that may contribute to triggered arrhythmias and even transcriptional regulation. We measured this diastolic standing [Ca2+]Cleft–[Ca2+]Bulk gradient with GCaMP2.2-FKBP12.6 versus GCaMP2.2, using [Ca2+] measured without gradients as a reference point. This diastolic difference ([Ca2+]Cleft=194 nmol/L versus [Ca2+]Bulk=100 nmol/L) is dictated mainly by the sarcoplasmic reticulum Ca2+ leak rather than sarcolemmal Ca2+ flux.
Conclusions: We have developed junctional cleft-targeted sensors to measure [Ca2+]Cleft versus [Ca2+]Bulk and demonstrated dynamic differences during electric excitation and a standing diastolic [Ca2+]i gradient, which could influence local Ca2+-dependent signaling within the junctional cleft.
Ca2+ is a universal second messenger involved in activation/regulation of cellular processes as diverse as neurotransmitter release, muscle contraction, metabolism, hypertrophic signaling, and cell death. To fulfill such diverse roles and yet be specific enough to trigger distinct responses, information is encoded in the amplitude, temporal properties, and subcellular localization of Ca2+ signals. It is now largely recognized that Ca2+ is regulated and signals differently in various subcellular microdomains.1–3 For example, GABA (γ-aminobutyric acid receptors) inhibition in hippocampal neurons results in spatially confined inhibition of Ca2+ transients shortly after a back-propagating action potential, which may play a key role in regulation of synaptic plasticity.4
Editorial, see p 326
In the heart, Ca2+ release from the sarcoplasmic reticulum (SR) is a key event for excitation–contraction coupling (ECC), ventricular arrhythmias, mitochondrial function, and nuclear transcription.5 Cardiac SR Ca2+ release is controlled by the local [Ca2+] in the junctional cleft ([Ca2+]Cleft), the small, restricted space between the apposing sarcolemma (mostly T-tubules) and junctional SR membrane. Voltage-gated L-type Ca2+ channels (LTCC) and Ca2+-activated Ca2+ channels (or ryanodine receptors [RyRs]) are located at these junctions in the sarcolemma and SR membrane, respectively, and are essential in this local control.6–8 During electric excitation, [Ca2+]Cleft is expected to rise higher and faster than in the bulk cytosol.9–12 Through Ca2+-dependent inactivation, [Ca2+]Cleft feeds back on LTCC and thus plays an important role in shaping the action potential waveform. Several studies12–17 suggest that the Na+/Ca2+ exchanger (NCX), the main route of Ca2+ extrusion in cardiac myocytes, also has access to the cleft and influences [Ca2+]Cleft during both diastole and ECC. In diastole, NCX may help to keep [Ca2+]Cleft low and limit spontaneous RyR activation. Indeed, during stochastic RyR opening events, Ca2+ removal by NCX may limit local Ca2+ release within a given RyRs cluster, which limits Ca2+ sparks and waves, favoring smaller, nonspark Ca2+ release events.18 During an action potential and rapid Na+ influx, local NCX may boost [Ca2+]Cleft and the efficacy of local SR Ca2+ release, especially in the latent period before LTCC opening.19
[Ca2+]Cleft could exceed that in bulk cytosol ([Ca2+]Bulk) even during diastole. This is because spontaneous SR Ca2+ release (leak) and Ca2+ influx caused by stochastic openings of LTCCs may create a standing [Ca2+] gradient between the cleft (where Ca2+ influx and release happen) and the bulk cytosol (where most of SR Ca2+ uptake occurs). Indeed, there is functional evidence for elevated [Ca2+]Cleft in NCX knockout cardiac myocytes, which causes reduced LTCC availability and higher ability of the remaining LTCC to trigger SR Ca2+ release.20
Despite its importance, methods to directly measure [Ca2+]Cleft are only beginning to be developed.21,22 Indirect estimates based on the dynamic properties of Ca2+-dependent inactivation of LTCC12,23 and NCX current12,24 during electrically triggered SR Ca2+ release suggest that [Ca2+]Cleft peaks within 10 to 20 ms and reaches tens or even hundreds of micromolar. In contrast, the global Ca2+ transient reaches a maximum of ≈1 µmol/L within ≈70 to 80 ms.6 Although strongly supporting the theory of local control of ECC, such measurements are indirect, difficult to calibrate, depend on the specific membrane localization of these transporters, and provide no information on diastolic [Ca2+]Cleft. Our aim was to construct novel Ca2+ sensors that are specifically targeted to the sarcolemma–SR junctions and thus report local [Ca2+]Cleft. We used these sensors to assess the dynamic changes in [Ca2+]Cleft during ECC and to demonstrate the existence of a standing [Ca2+] gradient during diastole between the cleft and the bulk cytosol.
Detailed Methods are included in the Online Data Supplement.
Mutagenesis of GCaMP2-Based Ca2+ Sensors, Cleft-Targeting, Plasmid Construction, and Expression in Adenoviral Vectors
The genetically encoded Ca2+ sensors GCaMP2.2 and GCaMP2.2Low were constructed by introducing the T203V and, respectively both T203V and D133E mutations in GCaMP2. For targeting to the junctional cleft, GCaMP2.2 and GCaMP2.2Low were fused to the N terminus of FKBP12.6 through a peptide linker. The GCaMP2.2 constructs were subcloned into the pshuttleCMV vector using Bgl II and Not I restriction sites. Subsequent adenovirus generations and amplifications were done using the Adeasy system (Agilent).
Protein Expression and Purification
Tagged and untagged GCaMP2.2Low sensors were cloned into a pRSET expression vector and amplified in BL21 Star (DE3) pLysS cells, and then purified using Profinity IMAC Ni Charged Resins (Bio-Rad) and further subjected to size exclusion chromatography.
Ventricular Myocyte Isolation, Culture, and Adenoviral Infection
All animal protocols were approved by the animal welfare committee at University of California, Davis, and conform to the National Institutes of Health Publication No 85-23 (revised in 1996). Rat ventricular myocytes were isolated by digestion with collagenase, cultured in supplemented M199 media, and infected with adenoviruses expressing one of the GCaMP2.2 sensors. For some experiments, freshly isolated myocytes were permeabilized with 50 µg/mL saponin for 3 minutes. All experiments were done at room temperature.
GCaMP2.2s fluorescence was recorded with a Live-5 laser scanning confocal microscope (excitation, 488 nm; emission, >505 nm). Ca2+ transients were measured in linescan mode (2 ms/line). In other experiments, 2-dimensional images were taken 1 s apart.
The statistical differences between groups were determined using the Student t test.
Low-Affinity GCaMP-Based Genetically Encoded Ca2+ Indicator
GCaMPs are genetically encoded Ca2+ indicators consisting of a circularly permutated, enhanced green fluorescent protein that is flanked by calmodulin at the C terminus and by the calmodulin-binding peptide myosin light-chain kinase M13 at the N terminus (Figure 1A) and have been used to detect [Ca2+] in vivo.25–28 The T203V mutation increases the brightness and dynamic range of GCaMP2 (GCaMP2.2),29 and we used this as a starting point to screen mutations that decrease the Ca2+-binding affinity of GCaMP2.2, while maintaining brightness and dynamic range. EF-hand loop mutational screening identified GCaMP2.2Low, which contains a D133E mutation30 in the EF-hand loop IV of calmodulin (Figure 1A). GCaMP2.2Low has a similar baseline fluorescence and dynamic range as GCaMP2.2, but its affinity for Ca2+ binding is ≈10-fold lower than that of GCaMP2.2 (Kd≈5 µmol/L versus 450 nmol/L; Figure 1B).
GCaMP2.2-FKBP12.6 Ca2+ Sensors Report [Ca2+]Cleft in the Space Between LTCC and RyR
Our strategy for targeting Ca2+ sensors to a specific subcellular microdomain (the cleft) is to attach GCaMP2.2 and our new GCaMP2.2Low to a molecule that specifically targets to that microdomain. On the basis of their Ca2+ affinities (Figure 1B), we expected that GCaMP2.2 would be useful for measuring diastolic [Ca2+]i, whereas GCaMP2.2Low could record the large Ca2+ transients expected in the junctional cleft. To target GCaMP2.2s to the junctions, we fused them to the N terminus of FKBP12.6 (Figure 1A). FKBP12.6 is an endogenous protein that binds with high affinity (Kd≈1 nmol/L), specificity, and 1:1 stoichiometry to RyR2 monomers but does not greatly influence RyR2 function.31 Our previous work showed that fluorescent-tagged FKBP12.6 binds in a striated pattern along T-tubules at the sites of Ca2+ spark initiation, and that binding is prevented by preincubation with nonfluorescent FKBP12.6.31 Linking FKBP12.6 to the GCaMP sensors does not significantly affect their Ca2+ affinity (Figure 1B) or Ca2+-binding kinetics (Online Table I). Saponin-permeabilized myocytes exposed to 100 nmol/L purified untagged GCaMP2.2 show uniform cytosolic distribution of the sensor (Figure 1Ca; Online Figure I shows the entire cell). In contrast, GCaMP2.2-FKBP12.6 has a striated fluorescence pattern (Figure 1Cb) like that seen for purified FKBP12.6 tagged with a small molecule fluorophore,31 which suggests that the sensor is appropriately localized at the z-line where RyRs and clefts are situated. To test the specificity of GCaMP2.2-FKBP12.6 binding at RyRs further, we exposed permeabilized myocytes to GCaMP2.2-FKBP12.6 in the presence of excess nonfluorescent FKBP12.6 to saturate RyRs (Figure 1Cc). In this case, GCaMP2.2-FKBP12.6 shows uniform cytosolic fluorescence. These data suggest that the FKBP12.6-tagged GCaMP2.2 sensors bind with high selectivity to RyRs, which are mainly cleft localized.
We next assessed the affinity of GCaMP2.2-FKBP12.6 binding to RyRs by performing sensor wash-in and wash-out experiments in permeabilized myocytes (Figure 1D). The rate constants for the dissociation (koff) and association (kon) of the sensor from/to RyRs were calculated from exponential fits of the decline in GCaMP2.2-FKBP12.6 fluorescence on removing the sensor from the solution (kdecline=koff), and the GCaMP2.2-FKBP12.6 fluorescence increase on addition of the sensor (kincrease=kon·[GCaMP2.2-FKBP12.6]+koff; Online Figure II). The dissociation constant of the RyR-GCaMP2.2-FKBP12.6 complex (Kd) was calculated as koff/kon and yielded a value of 15.5±3.3 nmol/L. A similar calculation showed that GCaMP2.2Low-FKBP12.6 binds to RyRs with a Kd of 43.8±6.3 nmol/L. Although these affinities are lower than previously reported for FKBP12.6 alone (≈1 nmol/L),31 GCaMP2.2-FKBP12.6 and GCaMP2.2Low-FKBP12.6 bind RyR with relatively high affinity.
We then calibrated the [Ca2+] dependence of the sensors in the myocyte environment (Figure 1E). The dynamic range of the sensors in myocytes was comparable with that measured in vitro (≈6-fold), but the apparent Ca2+ affinity was slightly lower in myocytes for both GCaMP2.2-FKBP12.6 and GCaMP2.2 than in vitro (Kd=1.2 and 1.3 µmol/L, respectively). GCaMP2.2Low and GCaMP2.2Low-FKBP also exhibited roughly a 2-fold decrease in apparent affinity in the myocyte environment (Kd≈11 µmol/L). Although GCaMP2.2-FKBP12.6 could increase Ca2+ buffering in the cleft, such effect is expected to be small and have limited effect for 2 reasons. First, there would be at most 1 GCaMP per RyR, and this is minor when compared with the intense >100:1 buffering in the cleft environment.6 Endogenous Ca2+-binding sites include calmodulin and divalent binding sites on RyR, L-type Ca2+ channels and other cleft proteins, membrane sites, and ATP. Second, buffering in the cleft will not alter the steady state [Ca2+]Cleft at rest and could only slow achievement of steady state by 1 to 5 ms during channel opening or closing.6
After characterizing our junctional cleft-targeted Ca2+ sensors, we expressed them in intact rat cardiac myocytes by adenoviral infection. Myocytes were used for experiments 20 to 24 hours later. Membrane staining with Di-8-ANNEPS indicated that myocytes retain the T-tubules at which most of the junctions reside, under the culture conditions (Online Figure III). Moreover, a stringent test for normal junctional function used transverse line scan images using Fluo-4 (where spatial drop-out or delayed central activation could indicate altered junctional coupling). The results demonstrated that normal transverse synchrony of SR Ca2+ release was well preserved in myocytes expressing the GCaMP sensors after 24 hours culture (Online Figure IV). There was no decrement in either the uniformity of local transverse release activation or rate of rise of the Ca2+ transients. Thus, the clefts where the GCaMP2.2-FKBP12.6 sensors are targeted function normally during ECC.
FKBP12.6-tagged sensors express in a striated pattern, with intensity maxima spaced ≈2 µm apart (Figure 2A; full images are shown in Online Figure V). This strongly supports the conclusion that the FKBP12.6-tagged sensors are targeted to RyR at the z-line, as in permeabilized cells. In contrast, the untagged sensors show a rather uniform cytosolic distribution (Figure 2A). To test the Ca2+ responsiveness of the new sensors when expressed in intact cells, we first depleted myocytes of Ca2+ (using ionomycin and Ca2+-free solution), to determine the minimum sensor fluorescence (Fmin; Figure 2Bb versus Figure 2Ba). Then, myocytes were Ca2+-overloaded by restoring extracellular Ca2+ in Na+-free solution to assess maximum fluorescence (Fmax) just as hypercontracture begins (Figure 2Bc). Importantly, GCaMP2.2Low and GCaMP2.2Low-FKBP12.6 have a similar dynamic range, Fmax/Fmin, in myocytes (5.5±0.6; n=7 for GCaMP2.2 and 5.7±0.5; n=9 for GCaMP2.2-FKBP12.6). These numbers agree well with those in Figure 1E. Thus, our novel FKBP12.6-tagged Ca2+ indicators are highly sensitive to Ca2+ and are positioned to measure local cleft [Ca2+]Cleft versus untagged sensors reporting bulk cytosolic [Ca2+]Bulk in intact myocytes.
Larger and Faster Local Ca2+ Transients in the Junctional Space Versus Bulk Cytosol
We expressed the low-affinity untagged and FKBP12.6-tagged sensors to measure Ca2+ transients in the bulk cytosol and junctional cleft, respectively (Figure 3). Myocytes were field-stimulated at 0.5 Hz, and Ca2+ transients were measured in linescan mode using a laser scanning confocal microscope. Figure 3A shows representative Ca2+ transients reported by GCaMP2.2Low and GCaMP2.2Low-FKBP12.6. Junctional cleft transients recorded by GCaMP2.2Low-FKBP12.6 are ≈2-fold larger (Figure 3B) and have a much faster upstroke (time-to-peak, 46±5 versus 90±7 ms; Figure 3C) and decay (decay τ=254±19 versus 421±42 ms; Figure 3D) when compared with global Ca2+ transients measured by the untargeted GCaMP2.2Low. This is encouraging (see Discussion section of this article for inferred [Ca2+] values), but based on theoretical models11 and indirect cleft Ca2+ transient assessments,12,24 one would expect a much larger difference in both amplitude and kinetics between the targeted and the untargeted sensor. An obvious explanation is that the kinetics of local [Ca2+] change in the cleft are likely to be fast, and GCaMP sensors lack the temporal resolution to detect such rapid kinetics. The dissociation rate constant for GCaMP2.2Low (≈6 s−1; Online Table I) is consistent with this notion. Although this new low-affinity Ca2+-sensor is not fast enough to report [Ca2+]Cleft dynamics during ECC accurately, it may still be useful in determining relative changes in [Ca2+]Cleft with acute treatments (eg, slower or larger). Targeted GCaMP2.2Low may also provide more accurate values of local [Ca2+]i in microdomains with local Ca2+ flux rates that are lower than in striated muscle junctions, where local Ca2+ flux rates may be among the highest in nature.
On β-adrenergic activation with isoproterenol (ISO; Online Figure VI), untargeted GCaMP2.2Low readily detects the expected large increase in Ca2+ transient amplitude and 2-fold acceleration of [Ca2+]i decline because of enhanced SR Ca2+ uptake. The cleft-targeted sensor Ca2+ transient decays much faster than the untargeted sensor under control conditions but fails to accelerate with ISO, despite a similar doubling of amplitude. Because [Ca2+]Cleft is expected to sense released Ca2+ and diffusion from the cleft to cytosol, rather than SR Ca2+ uptake rate (which is distributed throughout the cytosol), this is exactly what one would expect. Thus, the cleft-targeted GCaMP2.2Low is more sensitive to Ca2+ release at the cleft than to transport by SR Ca2+-ATPase (SERCA) and differs qualitatively from bulk [Ca2+]i sensors. This relates to Ca2+ sparks that decay primarily via diffusion from the source, with minor influence by SERCA function,32 despite much of the indicator signal coming from outside the cleft. In contrast, global [Ca2+]i decline is dictated almost entirely by Ca2+ transport out of the cytosol.6
Standing Diastolic [Ca2+] Gradient Between the Junctional Space and Bulk Cytosol
During diastole, Ca2+ leak from the SR or Ca2+ influx across sarcolemma may raise local [Ca2+]Cleft above [Ca2+]Bulk (distant from Ca2+ sources). Moreover, the high density of both L-type Ca2+ channels and RyR2 at the cleft when compared with the broader distribution of the transporters that remove Ca2+ from the cytosol (SR Ca2+-ATPase and Na+/Ca2+ exchange) could allow even modest Ca2+ leak through these channels to produce a standing [Ca2+] gradient (Figure 4A, left). Increases in SR Ca2+ leak have been implicated in pathological states, where [Ca2+]Cleft could be preferentially elevated. We used our targeted GCaMP2.2 to test for such diastolic standing [Ca2+] gradients between the cleft and the cytosol directly.
On blocking Ca2+ fluxes into the cleft, [Ca2+] should decline in both compartments, and importantly, any [Ca2+]Cleft versus [Ca2+]Bulk gradient should dissipate (Figure 4A, right). This maneuver is valuable so that we can define a point (F0) where both the cytosolic and targeted GCaMP2.2 sense the same [Ca2+]i. Figure 4B and 4C shows how [Ca2+]Cleft and [Ca2+]Bulk decline when we abruptly block RyRs with 1 mmol/L tetracaine and both LTCC and NCX by rapidly switching the external solution to a 0Na+/0 Ca2+ solution. Both [Ca2+]Cleft and [Ca2+]Bulk dropped rapidly after the application of tetracaine and 0Na+/0 Ca2+ solution, reaching the F0 steady state (where [Ca2+]Cleft=[Ca2+]Bulk) in ≈1 minute (Figure 4B and 4C, transition time from a to b). Note that [Ca2+] decline was much larger for GCaMP2.2-FKBP12.6. These results suggest that the initial diastolic [Ca2+]Cleft was higher (F/F0=1.8) than [Ca2+]Bulk (F/F0=1.2). In both cases, fluorescence was restored on return to the normal bath solution (time point c in Figure 4B and 4C).
Mean data confirm that the inhibition of SR and sarcolemmal Ca2+ fluxes in the cleft produces a significantly larger decline in the fluorescence of GCaMP2.2-FKBP12.6, which reports local [Ca2+]Cleft, versus the untagged GCaMP2.2, which reports [Ca2+]Bulk (−ΔF/F0=0.55±0.10 for GCaMP2.2-FKBP12.6 versus 0.13±0.02 for GCaMP2.2; Figure 4D). This indicates that there is indeed a standing [Ca2+]i gradient during diastole between the junctional cleft and the bulk cytosol. Assuming that diastolic [Ca2+]Bulk=100 nmol/L (before tetracaine, 0Na+/0Ca2+) and using data in Figure 1E, we calculated that diastolic [Ca2+]Cleft is 194±28 nmol/L.
Another way of reducing diastolic SR Ca2+ leak is to block SERCA with thapsigargin (Figure 5A). SERCA inhibition slowly unloads the SR, which is paralleled by a slow decrease in SR Ca2+ leak.33 As expected, with 10 µmol/L thapsigargin and 0Na+/0 Ca2+ solution, the fluorescence signal in myocytes expressing either GCaMP2.2 or GCaMP2.2-FKBP12.6 declined more slowly, reaching a new steady state in ≈10 minutes (Figure 5B). A similar time course was previously demonstrated for thapsigargin-induced cessation of SR Ca2+ leak, measured as the rate of decline in SR [Ca2+].33,34 Similar to RyR blockade with tetracaine, the fluorescence decline was significantly larger for GCaMP2.2-FKBP12.6 versus untagged GCaMP2.2 (−ΔF/F0=0.77±0.11 versus 0.41±0.06; Figure 5C). The overall decline in [Ca2+]i was larger for thapsigargin versus tetracaine (presumably because of longer time in Ca2+-free solution), but the difference between cleft and bulk was similar for both methods (−ΔF/F0=0.36 versus 0.42).
To assess the relative contribution of the SR and sarcolemmal Ca2+ fluxes to Ca2+ entry into the cleft, we did separate experiments where only sarcolemmal Ca2+ fluxes are blocked (0Na+/0 Ca2+ solution). In this case, the decrease in the sensor fluorescence was similar for the FKBP12.6-tagged and the untagged sensor (−ΔF/F0=0.22±0.03 for GCaMP2.2-FKBP12.6 versus 0.17±0.02 for GCaMP2.2; Figure 5C). In contrast, inhibition of SR Ca2+ leak alone resulted in a significantly larger drop in the GCaMP2.2-FKBP12.6 fluorescence versus the untagged sensor (−ΔF/F0=0.55±0.11 versus 0.24±0.07; Figure 5C). These results indicate that most of the Ca2+ entering the junctional cleft during diastole comes from the SR and SR Ca2+ leak is the main factor in setting a diastolic [Ca2+]i gradient between the cleft and the bulk cytosol.
In many cell types, [Ca2+]i is regulated and signals differently in various subcellular microdomains, which greatly enhances its versatility as a secondary messenger. Recognition of such subcellular regulation stimulated us to develop methods for measuring local [Ca2+]i in microdomains. Here, we attached the genetically encoded Ca2+ sensor GCaMP2.2 and a newly developed lower Ca2+ affinity variant GCaMP2.2Low to the N terminus of FKBP12.6 to construct novel Ca2+ sensors that bind specifically to RyRs. Our data indicate that GCaMP2.2-FKBP12.6 and GCaMP2.2Low-FKBP12.6 have a high affinity and selectivity for RyRs although slightly reduced when compared with untagged FKBP12.6. In cardiac myocytes, RyRs are localized predominantly in the junctional SR membrane, where the SR comes into close proximity of the external sarcolemma (the dyads). Because of this specific targeting, the FBP12.6-tagged sensors report local [Ca2+]i in the small dyadic cleft, [Ca2+]Cleft. This local [Ca2+]Cleft controls and is influenced by both RyRs and L-type Ca2+ channels. Therefore, [Ca2+]Cleft critically affects myocyte function at rest (during diastole) and during electric excitation. Despite its importance, methods to measure [Ca2+]Cleft directly are only now becoming available.21,22 This key parameter has typically been inferred from the dynamics of NCX12,24 and L-type Ca2+ currents12,23 in electrically stimulated myocytes, but no such estimates could be made for diastolic [Ca2+]Cleft. Our novel Ca2+ sensors allow direct measurement of [Ca2+]Cleft, both during ECC (the low-affinity sensor GCaMP2.2Low-FKBP12.6) and at rest (GCaMP2.2-FKBP12.6). However, the extremely rapid changes of [Ca2+]Cleft expected during ECC are too fast to be captured accurately by even the 10 µmol/L Kd sensor although they are likely to be useful to assess relative changes in [Ca2+]Cleft during perturbations that might alter the amplitude or kinetics of [Ca]cleft changes during ECC (as we have seen for ISO stimulation).
We use FKBP12.6 to target the sensors because of the high affinity and selectivity for RyR2 in cardiac myocytes.31 A limitation with this approach is that FKBP12.6 can also influence RyR channel gating, stabilizing the closed state.35 FKBP12.6 overexpression in myocytes can reduce spontaneous Ca2+ spark frequency and either increase or decrease Ca2+ transient amplitude.36,37 Thus, using FKBP12.6 to target these Ca2+ sensors may reduce diastolic RyR2 opening. However, in studies measuring simultaneously FKBP12.6 binding to RyR2 and Ca2+ sparks, saturating RyR2 with FKBP12.6 only reduced Ca2+ spark frequency by 18%.31 This effect would mean that we are probably slightly underestimating diastolic [Ca2+]Cleft.
A fraction (≈15%–20%) of RyRs are located outside the sarcolemma–SR junctions in rat myocytes.38 Thus, the GCaMP2.2-FKBP12.6 signal is not generated entirely by [Ca2+]Cleft, but is slightly contaminated by [Ca2+] at such nonjunctional RyRs. Of note, local [Ca2+] at nonjunctional RyRs may also be relevant, even if less influential on sarcolemmal currents. The adenoviral expression of GCaMP sensors in cardiac myocytes could risk alteration of dyad structure or function. Although cleft dimensions were not directly measured (eg, via electron microscopy), the FKBP12.6-targeted sensors are highly localized along T-tubules (of which ≈50% are in dyads in rat myocytes)6 and normal synchronous transverse activation during ECC was maintained (Online Figure IV). Therefore, we assume that the ultrastructure of the junctional cleft was not appreciably altered by the experimental conditions. FKBP12.6-GCaMP2.2 also has to be studied in a practical range of protein expression. If one waits too long or transfects too aggressively, one can express enough FKBP12.6-targeted protein to more than saturate the ≈1 µmol/L of RyR-binding sites. In that case, the excess sensor will not be effectively targeted. Any amount of untargeted GCaMP2.2-FKBP12.6 (as above) would cause underestimation of the difference between [Ca2+]Cleft and [Ca2+]Bulk. This means that our [Ca2+]Cleft measurements and the diastolic [Ca2+] gradient are lower limit estimates. True [Ca2+]Cleft would be larger. On balance, with awareness of these challenges with respect to interpretation, we think that the advantages of targeting sensors to the cleft this way outweigh the limitations and do not affect any of our conclusions. Potential enhancements in live cell imaging resolution that selectively visualize such local domains would complement approaches like ours with locally targeted sensors.22
[Ca2+]Cleft and [Ca2+]Bulk in Cardiac Myocytes
Our novel cleft-targeted sensor GCaMP2.2Low-FKBP12.6 reported a larger and faster rise in [Ca2+]Cleft versus [Ca2+]Bulk during electric excitation of myocytes, in good agreement with previous estimates from indirect measurements.9–12 Weber et al24 inferred the submembrane [Ca2+] near NCX during a normal action potential as peaking at >3.2 µmol/L within <32 ms (versus a global Ca2+ transient that peaks at 1.1 µmol/L in 81 ms). However [Ca2+]Cleft ought to be much higher than submembrane [Ca2+]i. Acsai et al12 estimated the Ca2+ transient near Ca2+ release sites from the kinetics of L-type Ca2+ current and from a fraction of NCX current. They calculated that this local Ca2+ transient has 10 to 15 µmol/L in amplitude, peaks at ≈10 ms after depolarization and recovers within ≈50 ms. In contrast, the global Ca2+ transient reached a maximum of ≈1 µmol/L within ≈70 to 100 ms. Estimates of [Ca2+]Cleft based on geometry, diffusion, and Ca2+ flux measurements suggest even higher (100 µmol/L) and faster [Ca2+]Cleft changes during electric excitation.11 On the basis of measured diastolic [Ca2+]Cleft and [Ca2+]Bulk (194 and 100 nmol/L, respectively; Figure 4E), the characteristics of GCaMP2.2Low and GCaMP2.2Low-FKBP response to Ca2+ in myocytes (Figure 1E), and the Ca2+ transient amplitude (Figure 3C), we calculated that peak [Ca2+]Bulk is 560±73 nmol/L, whereas peak [Ca2+]Cleft is ≈2-fold larger (1272±199 nmol/L). This peak [Ca2+]Cleft is much smaller than previous estimates. The time-to-peak of [Ca2+]Cleft was 46±5 ms (versus 90±7 ms for [Ca2+]Bulk), slightly longer than the estimates based on NCX and L-type Ca2+ current. We think that these differences in the amplitude and dynamics of cleft Ca2+ transients versus previous estimates are mostly because of the relatively slow kinetics of Ca2+ binding and unbinding of the GCaMP2.2 sensors (Online Table I). Thus, although GCaMP2.2Low-FKBP12.6 detects larger and faster cleft Ca2+ transients versus the global sensor and can detect changes in amplitude and kinetics (eg, with ISO), much faster indicators will be required for accurate [Ca2+]Cleft measurement during the dynamics of active SR Ca2+ release in heart.
Using GCaMP2.2-FKBP12.6, we could directly assess diastolic [Ca2+]Cleft, a parameter not previously measured. We demonstrated that during diastole, [Ca2+]Cleft is >90 nmol/L higher than [Ca2+]Bulk. This standing diastolic [Ca2+] gradient indicates that Ca2+ extrusion from the cleft (via junctionally located NCX and diffusion into bulk cytosol) cannot keep up with Ca2+ entry into the cleft. Our data show that diastolic cleft Ca2+ influx is caused mainly by the SR Ca2+ leak (versus entry through LTCC). At the same time, elevated diastolic [Ca2+]Cleft can reduce LTCC availability (via Ca2+-dependent inactivation) and alter RyR gating to increase their activity by direct RyR activation and also indirectly via activation of CaMKII followed by CaMKII-dependent RyR phosphorylation.39 Increased RyR opening leads to a larger SR Ca2+ leak and a higher risk for generation of propagating Ca2+ waves, which are a known risk for arrhythmias. The balance of Ca2+ fluxes in the cleft is altered under pathological conditions (eg, in heart failure SR Ca2+ leak and Na+/Ca2+ exchange function are increased).40
The high diastolic [Ca2+]Cleft may also have important effects on local signaling cascades beyond the sphere of ECC. Indeed, there is much indirect evidence that local [Ca2+]i in these environments may be important in the activation of calcineurin or CaMKII that can signal to the nucleus and trigger alterations in gene expression.41 In pathological conditions where SR Ca2+ leak is known to be enhanced, the higher local level of diastolic [Ca2+]Cleft may have an important effect on these (and other) broader signaling pathways.
In summary, we have developed sensors for measuring [Ca2+]Cleft versus [Ca2+]Bulk and demonstrated dynamic differences during ECC. We have also provided measures of a normal standing [Ca2+]i gradient during diastole between the junctional cleft and the bulk cytosol and showed that this gradient is set mainly by the SR Ca2+ leak.
We thank Dr Khafaga, Ms Dao, and Ms Lee for help in preliminary experiments and Dr Ginsburg for technical support.
Sources of Funding
This work was supported by the National Institutes of Health (R01-HL109501 to S. Despa; R01-HL081526, R37-30077, and R01-HL092097 to D.M. Bers; and 5-P01-HL095488 to M.I. Kotlikoff).
In April 2014, the average time from submission to first decision for all original research papers submitted to Circulation Research was 14.38 days.
The online-only Data Supplement is available with this article at http://circres.ahajournals.org/lookup/suppl/doi:10.1161/CIRCRESAHA.115.303582/-/DC1.
- Nonstandard Abbreviations and Acronyms
- intracellular Ca2+ concentration
- local [Ca2+] in the junctional cleft
- [Ca2+] in the bulk cytosol
- excitation–contraction coupling
- L-type Ca2+ channels
- Na+/Ca2+ exchanger
- ryanodine receptors
- sarcoplasmic reticulum
- Received January 29, 2014.
- Revision received May 24, 2014.
- Accepted May 28, 2014.
- © 2014 American Heart Association, Inc.
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Novelty and Significance
What Is Known?
Ca2+ is regulated and signals differently in various subcellular microdomains, which greatly enhances its second messenger versatility.
In the heart, sarcoplasmic reticulum Ca2+ release is controlled by local [Ca2+]i in the junctional cleft, the small space between sarcolemma and junctional sarcoplasmic reticulum.
This local cleft [Ca2+]i is thought to be important in not only the regulation of excitation–contraction coupling but also to downstream Ca2+-dependent signaling (eg, via calmodulin, calcineurin and CaMKII).
What New Information Does This Article Contribute?
We constructed novel Ca2+-sensors that are targeted to microdomains rich in ryanodine receptors.
In cardiac myocytes, the novel Ca2+-sensors are positioned to measure local Ca2+ in the junctional cleft.
There is a standing Ca2+ gradient during diastole between the junctional cleft and bulk cytosol in cardiac myocytes, which could influence local Ca2+-dependent signaling within the cleft.
Ca2+ is a universal second messenger involved in activation/regulation of diverse cellular processes. To allow such versatility, Ca2+ is regulated and signals differently in various subcellular microdomains, which generated a large interest in developing methods for measuring local rather than bulk Ca2+. Here, we targeted novel Ca2+-sensors to microdomains rich in ryanodine receptors. This was accomplished by fusing the genetically encoded Ca2+-sensor GCaMP2.2 and a lower Ca2+-affinity variant GCaMP2.2Low to FKBP12.6, a protein that binds with high affinity and selectivity to ryanodine receptors. In cardiac myocytes, these sensors report local [Ca2+] in the small junctional cleft between the plasmalemma and junctional sarcoplasmic reticulum ([Ca2+]Cleft), a parameter that critically affects heart function and dysfunction. Using these sensors, we demonstrate that [Ca2+]cleft reaches higher levels with faster kinetics than global [Ca2+]i during excitation–contraction coupling. We also show that there is a substantial standing diastolic [Ca2+] gradient between [Ca2+]Cleft and bulk [Ca2+]i, mainly because of ryanodine receptor–dependent Ca2+ leak. Such [Ca2+]Cleft characteristics may have important effect on local Ca2+-dependent signaling.