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Am J Physiol Heart Circ Physiol 287: H1987-H1993, 2004. First published July 22, 2004; doi:10.1152/ajpheart.00409.2004
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FKBP12.6 overexpression decreases Ca2+ spark amplitude but enhances [Ca2+]i transient in rat cardiac myocytes

Ana M. Gómez,1,* Iris Schuster,1,* Jérémy Fauconnier,1 Jürgen Prestle,2 Gerd Hasenfuss,2 and Sylvain Richard1

1Institut National de la Santé et de la Recherche Médicale U637-EA3759, Centre Hospitalier Universitaire Arnaud de Villeneuve, 34295 Montpellier, France; and 2Abteilung Kardiologie und Pneumologie, Universität Göttingen, 37099 Göttingen, Germany

Submitted 6 May 2004 ; accepted in final form 15 July 2004


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Ryanodine receptors/Ca2+-release channels (RyR2) from the sarcoplasmic reticulum (SR) provide the Ca2+ required for contraction at each cardiac twitch. RyR2 are regulated by a variety of proteins, including the immunophilin FK506 binding protein (FKBP12.6). FKBP12.6 seems to be important for coupled gating of RyR2 and its deficit and alteration may be involved in heart failure. The role of FKBP12.6 on Ca2+ release has not been analyzed directly, but rather it was inferred from the effects of immunophilins, such us FK506 and rapamycin, which, among other effects, dissociates FKBP12.6 from the RyR2. Here, we investigated directly the effects of FKBP12.6 on local (Ca2+ sparks) and global {intracellular Ca2+ concentration ([Ca2+]i) transients} Ca2+ release in single rat cardiac myocytes. The FKBP12.6 gene was transfected in single myocytes using the adenovirus technique with a reporter gene strategy based on green fluorescent protein (GFP) to check out the success of transfections. Control myocytes were transfected with only GFP (Ad-GFP). Rhod-2 was used as the Ca2+ indicator, and cells were viewed with a confocal microscope. We found that overexpression of FKBP12.6 decreases the occurrence, amplitude, duration, and width of spontaneous Ca2+ sparks. FK506 had diametrically opposed effects. However, overexpression of FKBP12.6 increased the [Ca2+]i transient amplitude and accelerated its decay in field-stimulated cells. The associated cell shortening was increased. SR Ca2+ load, estimated by rapid caffeine application, was increased. In conclusion, FKBP12.6 overexpression decreases spontaneous Ca2+ sparks but increases [Ca2+]i transients, in relation with enhanced SR Ca2+ load, therefore improving excitation-contraction coupling.

calcium (cellular); sarcoplasmic reticulum (function); excitation-contraction coupling; heart

CONTRACTION of the cardiac myocyte is initiated by the Ca2+-induced Ca2+ release mechanism, first proposed by Fabiato and Fabiato (7). Membrane depolarization during an action potential activates voltage-gated L-type Ca2+ channels. These channels, localized mainly in the transverse tubules, open transiently and serve as the major pathway for Ca2+ entry into myocytes. The subsequent local elevation in intracellular Ca2+ concentration ([Ca2+]i) activates Ca2+-release channels [or ryanodine receptors (RyR2) in the heart] in the junctional sarcoplasmic reticulum (SR), providing the amount of Ca2+ required for contraction. This whole sequence of events determines the excitation-contraction (E-C) coupling. Cytoplasmic Ca2+ is then rapidly pumped back into the SR or extruded out of the cell (mainly via the Na+/Ca2+ exchanger) accounting for relaxation. The elementary events that summate to produce this transient elevation in [Ca2+]i are referred to as Ca2+ sparks. These local, rapid, and brief elevations of [Ca2+]i correspond to the opening of a group of RyR2 and can be visualized using confocal microscopy and adequate Ca2+ dyes (4). Ca2+ sparks are therefore useful to analyze the activity of RyR2 in situ.

RyR2 are clustered on the SR membrane and their cytoplasmic domains are in contact. When these clusters are isolated and incorporated into planar lipid bilayers, RyR2 open simultaneously, which is termed coupled gating (18). Several regulatory proteins bind to RyR2, thereby forming a macro-molecular complex. They include FK506 binding protein (FKBP12.6), PKA, protein phosphatases PP1 and PP2A, and mAKAP, a kinase anchoring protein (19). Sorcin can also bind to, and functionally modulate, RyR2 (8, 21). Although controversial (10), it has been proposed that the amount of FKBP12.6 bound to RyR2 is decreased in heart failure and that hyperphosphorylation is involved, thereby promoting instability of the FKBP12.6-RyR2 complex and SR Ca2+ leak (19, 23, 25, 34). It has been suggested that FKBP12.6 removal from the RyR2 functionally uncouples channels gating (18). The role of FKBP12.6 on RyR2 has not been analyzed directly, but rather it was inferred from the effects of FK506 and rapamycin. These immunophylins dissociate FKBP12.6 from the RyR2. In planar lipid bilayers experiments, they increase RyR2 open probability, decrease current amplitude (11, 19), and increase open time (32). In more physiological conditions, such as in single ventricular myocytes, FK506 increases [Ca2+]i transient amplitude in rats and mice, whereas it has the opposite effect in rabbits (20, 27, 32). FK506 also increases Ca2+ sparks frequency (20) but has controversial effects on Ca2+ spark characteristics, ranging from no modification (20) to increased duration (3, 16, 26, 32), with the occurrence of sparks with half-normal amplitude (32). In FKBP12.6 knockout mice, Ca2+ sparks have higher amplitude and duration than those recorded from wild-type animals (33).

Because FKBP12.6 is expected to have effects opposite to those of FK506 and rapamycin, it seems reasonable to postulate that FKBP12.6 decreases Ca2+ spark frequency (20). However, the other effects are unpredictable. Amplitude could be unchanged (20), decreased (33), or increased (32). Similarly, their duration could be unchanged (20) or decreased (3, 16, 26, 32). In line with this reasoning, FKBP12.6 could also be expected to decrease [Ca2+]i transient and contraction (20, 32, 33). Surprisingly, recent evidence show that FKBP12.6 overexpression has a positive, rather than the expected negative, inotropic effect (24).

The aim of this study was to investigate the subcellular effects of FKBP12.6 on Ca2+ release in rat ventricular myocytes. We analyzed elementary Ca2+ release (Ca2+ sparks) and global [Ca2+]i transients in single cells transfected with adenoviruses coding for FKBP12.6 (Ad-FKBP12.6-GFP) and their control (Ad-GFP). We found that FKBP12.6 overexpression reduces the frequency, amplitude, and duration of spontaneous Ca2+ sparks, whereas the amplitude of [Ca2+]i transients is increased.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell preparation. Cardiac ventricular myocytes from male Wistar rats (250–300 g) were isolated by standard enzymatic techniques using collagenase as previously detailed (2). The investigation conformed to the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Pub. No. 85-23, Revised 1996). Myocytes were dispersed in modified culture media (M199, Eurobio) and incubated at 37°C with 100 multiplicity of infection (MOI) adenovirus for 3 h to allow adenoviral infection as described before (24). The viral solution was then discarded, and cells were maintained in the culture media (7,500 cells/ml, suspended in sterile plastic tubes) for 48 h. The culture medium was renewed each 24 h. We used adenoviruses coding for FKBP12.6 plus green fluorescent protein (GFP; Ad-FKBP12.6-GFP) (24). Control myocytes were incubated in the same conditions, but the adenoviruses coded only for GFP (Ad-GFP). We assessed three different MOI: 1, 10, and 100. At MOI of 10, not all myocytes were transfected (data not shown), but at 100 MOI, 100% of rod-shaped myocytes were transfected, as assessed by the green fluorescence (Fig. 1A). Experiments were performed on rod-shaped cardiac myocytes at room temperature (23 ± 2°C).



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  		Fig. 1. Ca2+ spark frequency is decreased in FK506 binding protein (FKBP12.6)-overexpressing cardiomyocytes. A: images of rat cardiac myocytes 48 h after transfection. The green fluorescence, shown in the image (right), indicates green fluorescent protein (GFP) expression. B: line-scan images of cardiac myocytes 48 h after transfection with Ad-GFP (control; top) and Ad-GFP-FKBP12.6 (FKBP12.6; bottom). C: bar graph comparing Ca2+ spark frequency measured in 21 Ad-GFP cells (control) and 15 FKBP12.6-overexpressing cells. *P < 0.005.

 
Confocal images. Transfected myocytes were loaded with the membrane-permeant Ca2+ dye rhod 2 AM. Cells were placed in Tyrode solution [containing (in mM) 140 NaCl, 4 KCl, 1.8 CaCl2, 1 MgCl2, 5.5 glucose, and 5 HEPES; pH 7.4 with NaOH] and loaded with 5 µM rhod 2 AM at room temperature during 30 min. Cells were then resuspended in the same solution but without dye. Experiments started after an additional 30-min waiting period to allow for deesterification of the dye. Cells were superfused with the Tyrode solution during image acquisition. Confocal images (Zeiss LSM 510) were acquired in the line-scan mode at 1.5 ms/line as detailed previously (12). Rhod 2 was excited using the 550-nm line of an argon ion laser. Emission was collected at >560 nm. In a set of experiments, spontaneous Ca2+ sparks were recorded in quiescent cells. In another set of experiments, cells were first field stimulated through two parallel platinum electrodes at 1 Hz during 1–2 min to allow for steady-state [Ca2+]i transients. The line scan was selected parallel to the longitudinal cell axis to be able to measure the associated cell shortening. Stimulation was then stopped, and Ca2+ sparks were recorded. After this, cells were field stimulated again, and caffeine (10 mM) was added to estimate the SR Ca2+ load. Images recorded in freshly isolated myocytes (not transfected with GFP) were loaded with the Ca2+ fluorescence dye fluo-3. The 488-nm line of the argon laser was used to illuminate, and emission was collected at >505 nm.

Images were corrected for the background fluorescence. The fluorescence values (F) were then normalized to the basal fluorescence (from a region of the image without Ca2+ sparks, or before each electrical stimulation; F0) to obtain the fluorescence ratio (F/F0). All images were processed and analyzed using IDL (RSI) software and homemade routines. Ca2+ sparks were detected using an automated detection method (5) and using, as a criterion, amplitudes of fluorescence fourfold the standard deviation of the image. This criterion limited the detection of false events while detecting most sparks (5). Rising time was measured as the time between maximum and minimum values of the second derivative of the fluorescence transient corresponding to the Ca2+ spark.

Statistics. Data are expressed as means ± SE. Unpaired t-test was used to compare data obtained in the control versus test. Results were considered significant with P < 0.05.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Properties of spontaneous Ca2+ sparks in FKBP12.6-overexpressing myocytes. To analyze elementary Ca2+ release through RyR2 channels, we recorded spontaneous Ca2+ sparks after 48-h transfection with the adenovirus coding for GFP only (Ad-GFP) as a control and for FKBP12.6 + GFP (Ad-FKBP12.6-GFP). Transfection was verified in each individual cell by green fluorescence (Fig. 1A). Spontaneous Ca2+ sparks were less frequent in Ad-FKBP12.6-GFP-infected myocytes than in Ad-GFP-infected control cells. This is illustrated in Fig. 1B, which shows typical line-scan images of Ca2+ sparks recorded 48 h after transfection. The bar graph in Fig. 1C shows the significant decrease of Ca2+ spark frequency in the Ad-FKBP12.6-GFP-infected cells.

We further characterized the Ca2+ sparks by comparing their properties both in Ad-GFP-infected control cells and in Ad-FKBP12.6-GFP infected cells. The insets in Fig. 2, A–D, show the averaged characteristics of Ca2+ sparks in Ad-GFP-infected control cells and in Ad-FKBP12.6-GFP-infected cells. FKBP12.6 overexpression caused a significant decrease in the averaged peak amplitude (Fig. 2A) and reductions in full width at half-maximum amplitude (FWHM; Fig. 2B), rising time (Fig. 2C), and full duration at half-maximum peak (FDHM; Fig. 2D) of Ca2+ sparks.



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  		Fig. 2. Ca2+ spark characteristics in control and FKBP12.6-overexpressing cardiomyocytes. A: frequency histogram of the amplitude [measured as the peak value of fluorescence (F) normalized by the basal fluorescence (F0)] of spontaneous Ca2+ sparks. Inset, bar graph showing averaged Ca2+ spark amplitude in Ad-GFP (C; n = 533) and Ad-FKBP12.6-GFP cells (FKBP; n = 118). B: frequency histogram of Ca2+ sparks width at half their maximal amplitude (FWHM). Inset, bar graph showing that averaged width is decreased by FKBP12.6. C: frequency histogram of the time to peak. Inset, bar graph showing that averaged time to peak is decreased by FKBP12.6. D: frequency histogram of Ca2+ sparks duration at half maximum (FDHM). Inset, bar graph showing that averaged duration is decreased by FKBP12.6. Lines represent the best fits with the y = y0 + A x exp{–0.5[ln(x/xc)/w]2} function (see text for definition of variables). In all graphs, the solid circles and lines indicate Ad-GFP and the gray circles and lines indicate Ad-FKBP12.6-GFP. *P < 0.05; **P < 0.01.

 
The distributions of these characteristics were asymmetrical due to detection limits of the smaller Ca2+ sparks. Broad modal distribution of Ca2+ spark amplitudes has been described earlier (8, 12, 17, 29, 35) and differs from the monotonic decaying distribution theoretically predicted (5), probably because the present method restricted the "out-of-focus" events. Ca2+ spark distribution has been previously adjusted to a simple (20), bimodal (31), or multicomponent (30) Gauss function. We fitted our data using the function derived from lognormal: y = y0 + A x exp{–0.5[ln(x/xc)/w]2} used earlier by other authors to describe the distribution of randomly acquired Ca2+ sparks (17), where y and x are the dependent and independent variables, respectively, y0 is offset, xc is center, w is width, and A is amplitude. This function can also be used to fit multipeak data. Figure 2A shows the frequency histogram of Ca2+ spark amplitudes (peak F/F0) in Ad-GFP-infected control cells and in Ad-FKBP12.6-GFP-infected cells. The distribution of Ca2+ spark amplitudes in Ad-GFP-infected cells could be reasonably well fitted (R2 = 0.98) by the function with a center at about 1.70 (peak F/F0). The distribution of Ca2+ sparks recorded in Ad-FKBP12.6-GFP-infected cells was best fitted by a double peak function (R2 = 0.99), evidencing a bimodal distribution of two distinct populations: one with a peak amplitude of 1.48 (peak F/F0) and a second, very small, population of larger Ca2+ sparks with a peak at 1.63 (peak F/F0), a value very similar to the population recorded in Ad-GFP-infected control cells. Even though there is not an absolute uniqueness of fitting by two peak functions, and although a reasonable correlation could also be obtained by fitting data by a single function (R2 = 0.90, peak = 1.5), the distribution, however, suggested that the Ca2+ sparks recorded in Ad-FKBP12.6-GFP cells have smaller amplitude. Indeed, one major effect of FKBP12.6 overexpression was to depress the population of larger amplitude Ca2+ sparks recorded in control myocytes and to induce the appearance of Ca2+ sparks of small amplitude (Fig. 2A). The Ca2+ spark FWHM distribution was also bimodal, with two populations exhibiting peaks at 1.87 and 3.81 µm (R2 = 0.96), respectively, for Ad-GFP and one population with a peak at 1.87 µm (R2 = 0.87) for Ad-FKBP12.6-GFP cells (Fig. 2B). This indicated that the wider Ca2+ sparks are markedly reduced by FKBP12.6 overexpression. Similarly, FDHM distribution was also fitted by functions with peaks at 32.98 ms for Ad-GFP (R2 = 0.92) and at 27.8 ms for Ad-FKBP12.6-GFP (R2 = 0.90), indicating reduction in the duration of Ca2+ spark following FKBP12.6 overexpression.

Properties of spontaneous Ca2+ sparks in the presence of FK506. Because overexpression of FKBP12.6 decreases Ca2+ sparks frequency, amplitude, duration, and width, it would be expected that dissociating FKBP12.6 from RyR2 with a pharmacological tool had the opposite effects. We assessed this hypothesis on freshly isolated myocytes. Figure 3A shows line-scan images of a normal myocyte before (top) and during perfusion with 5 µM FK506 (bottom). FK506 significantly increased the occurrence of Ca2+ sparks (Fig. 3B) and also their averaged amplitude (Fig. 3C), FWHM (Fig. 3D), and FDHM (Fig. 3E). Hence, the functional effects of FK506 and FKBP12.6 overexpression were diametrically opposed, suggesting that they reflect a genuine regulation of RyR activity, i.e., occurring via changes in the binding of FKBP12.6 to RyR2. FKBP has been shown to stabilize the closed state of RyRs incorporated into planar lipid bilayers (1, 32). It is worth to note that Ca2+ spark occurrence was lower in freshly isolated cells than after 48 h in culture, presumably because cultured cells undergo phenotypic remodeling. However, because in the FKBP12.6-overexpressing experiments control myocytes were also maintained in culture and transfected in the same conditions as the FKBP12.6-overexpressing cells, the effects found should be due to FKBP12.6 overexpression.



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  		Fig. 3. Ca2+ spark characteristics in the presence of FK506. A: line-scan images of a freshly isolated cardiac myocyte before (top) and during (bottom) perfusion with 5 µM FK506. B: bar graph showing Ca2+ spark frequency measured in 35 myocytes before (open bar) and during (blue bar) FK506 application. ***P < 0.001. C: Ca2+ spark amplitude (peak F/F0, measured as in Fig. 2A) before (n = 225) and after (n = 853) FK506. ***P < 0.001. D: reduction of Ca2+ spark width at half its maximal amplitude by FK506. *P < 0.05. E: reduction of Ca2+ spark duration by FK506. *P < 0.05.

 
Effect of FKBP12.6 on [Ca2+]i transients and contraction in beating cells. Previous reports showed that FK506 increases both [Ca2+]i transient and contraction (20, 32). Because FK506 dissociates FKBPs from RyRs, one could hypothesize that FKBP12.6 decreases both [Ca2+]i transients and contraction. However, contraction is increased in rabbit myocytes overexpressing FKBP12.6 (24). We thus investigated the effect of FKBP12.6 on both the [Ca2+]i transient and associated contraction here in rat ventricular cells. The line-scan images in Fig. 4A show representative confocal line-scan images recorded from an Ad-GFP-infected control myocyte and from an Ad-FKBP12.6-GFP-infected myocyte that were field stimulated at 1 Hz. The corresponding [Ca2+]i transients are shown above each image. It is clear that the amplitude of the [Ca2+]i transient recorded in the Ad-FKBP12.6-GFP myocyte is larger that the one in the Ad-GFP-infected control myocyte. Both the averaged contraction (measured as percent shortening) and the averaged [Ca2+]i transient amplitude (peak F/F0) were significantly higher in FKBP12.6-overexpressing myocytes (Fig. 4, B and C), suggesting that increased contraction reflects enhanced Ca2+ release during a twitch. The analysis of the decay time constant of the [Ca2+]i transient, which was best fit by a monoexponential function, was significantly faster in the FKBP12.6-overexpressing cells (Fig. 4D).



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  		Fig. 4. Intracellular Ca2+ concentration ([Ca2+]i) transient is increased in FKBP12.6-overexpressing myocytes. A: examples of line-scan images in an Ad-GFP (control; left) and a FKBP12.6-overexpressing myocyte (right) field stimulated at 1 Hz. The corresponding fluorescence [Ca2+]i transients are shown on top. B: fractional shortening is increased in Ad-FKBP12.6-GFP cardiomyocytes (FKBP12.6; red bars, n = 16) compared with Ad-GFP myocytes (control; open bars, n = 13). ***P < 0.001. C: peak amplitude of the fluorescence [Ca2+]i transient is also increased. *P < 0.05. D: decay time constant ({tau}) of the [Ca2+]i transient is decreased in FKBP12.6-overexpressing cells. *P < 0.05.

 
It was rather surprising to find an increase in the amplitude of the global [Ca2+]i transient because the amplitude of individual Ca2+ sparks is decreased. During a normal twitch, the RyR2 are activated and Ca2+ release will depend on the amount of Ca2+ stored in the SR. Hence, the amplitude of the global [Ca2+]i transient depends highly on the SR Ca2+ load. To assess the SR Ca2+ load, we used rapid caffeine application. Caffeine keeps the RyR2 open and all the Ca2+ stored in the SR is released. Figure 5A shows representative examples of line-scan images of caffeine-evoked [Ca2+]i transients in Ad-infected control cells and Ad-FKBP12.6-infected cells. The FKBP12.6-overexpressing cells presented higher SR Ca2+ load (Fig. 5B).



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  		Fig. 5. Ca2+ load is increased in FKBP12.6-overexpressing myocytes. A: line-scan images during caffeine application in Ad-GFP (control; top) and FKBP12.6-overexpressing myocytes (bottom). B: peak amplitude of the caffeine-evoked fluorescence [Ca2+]i transient is increased in FKBP12.6-overexpressing myocytes (n = 7) compared with control myocytes (n = 7). *P < 0.05.

 
Caffeine was applied on periodically stimulated myocytes. Thus increased SR Ca2+ load in Ad-FKBP12.6 cells can explain enhanced [Ca2+]i transients. However, the Ca2+ sparks presented in Figs. 1 and 2 were measured in quiescent cells, and it is hard to know whether FKBP12.6 would also induce a reduction of Ca2+ sparks in conditions more comparable to the working heart. We also recorded Ca2+ sparks just after field stimulating the cell for 1–2 min, so in conditions similar to caffeine measurements. In these conditions, Ca2+ sparks were modified similarly as in quiescent cells. The peak amplitude of Ca2+ sparks recorded in FKBP12.6-overexpressing cells (F/F0: 1.74 ± 0.02, n = 309) was also significantly smaller (P = 0.02) than those recorded in control myocytes (F/F0: 1.8 ± 0.01, n = 291).


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Specific overexpression of FKBP12.6 by adenoviral gene transfer was used in rat ventricular cells to study the functional effects of this RyR2-associated regulatory protein at both the subcellular (Ca2+ sparks) and the cellular ([Ca2+]i transients) levels. Two main effects were observed: 1) FKBP12.6 decreased the occurrence and the amplitude, width, and duration of spontaneous Ca2+ sparks in quiescent myocytes; and 2) FKBP12.6 increased the amplitude of the [Ca2+]i transient in field-stimulated cells.

Regulation of Ca2+ sparks. Data obtained from isolated RyRs incorporated into planar lipid bilayers have shown that FK506 increases the opening probability of the RyR2 Ca2+ release channel and induces longer openings in a subconductance state (1, 32). Here, we find that overexpression of FKBP12.6 in single rat ventricular cells decreases the frequency and duration of Ca2+ sparks (Figs. 1 and 2), consistently with reports showing opposite effects by FK506 (Fig. 3, B and D) (3, 16, 20, 26, 32). According to the theory of coupled gating of RyR2 (18), it is predicted that FKBP12.6 allows all RyR2 forming a cluster to open and close simultaneously, thereby producing shorter Ca2+ sparks (26). This theory explains the reduced spatial size of individual Ca2+ sparks here after FKBP12.6 overexpression (Fig. 2B) because faster closing of coordinated RyR2 would limit Ca2+ spread from the point of release. It has been predicted that the width of the distribution of Ca2+ spark characteristics decreases as the number of RyRs increases (26). As seen in the frequency histograms of Fig. 2, the distribution of Ca2+ sparks recorded in Ad-FKBP12.6-GFP-infected myocytes is narrower than that of Ca2+ sparks recorded in Ad-GFP-infected cells. This indicates that the number of RyR2 that open simultaneously to produce one Ca2+ spark is increased by FKBP12.6, which supports the role of FKBP12.6 on coupled gating. However, one intriguing result here was the concomitant reduction of Ca2+ spark amplitude. It is possible that, because FKBP12.6 decreases RyR2 open time (16, 32), less Ca2+ is released per opening, leading to a decrease in Ca2+ spark amplitude. Therefore, faster closing of the FKBP12.6-RyR2 complex would explain both shorter rising time of the Ca2+ sparks and decreased amplitude in FKBP12.6-overexpressing cells (Fig. 2C).

Positive inotropy and Ca2+ handling. FK506 increases [Ca2+]i transients and contractions in rat and mouse ventricular myocytes (20, 27, 32) but decreases [Ca2+]i transients in the rabbit (27), evidencing species differences. Other authors have shown that overexpression of FKBP12.6 increases contraction and [Ca2+]i transients in rabbit ventricular myocytes (14, 24). In the rat, although both FK506 and FKBP12.6 increase global [Ca2+]i transients, they have opposite effects on Ca2+ sparks. In the case of FK506, effects on targets other than RyR2, such as potassium channels, may underlie positive inotropism (6). Therefore, the effects of FKBP12.6 on [Ca2+]i transient and contraction are not easy to predict from experiments with FK506. Here, we report that direct manipulation of FKBP12.6, by overexpression of the protein, increases cellular contraction in rat ventricular cells. We provide direct evidence that an increase in the [Ca2+]i transient (Fig. 4C) amplitude accounts for the increase in contraction (Fig. 4B).

The question thus arises as to how FKBP12.6 overexpression can enhance [Ca2+]i transients while decreasing Ca2+ sparks frequency and amplitude. The reduction of spontaneous Ca2+ spark frequency and amplitude might prevent Ca2+ leak during diastole and thereby increase SR Ca2+ content. The higher SR Ca2+ content would underlie the higher [Ca2+]i transient amplitude. Moreover, because of the role of FKBP12.6 on RyR-coupled gating, it is possible that the number of RyR2 that open simultaneously and early during excitation is increased in FKBP12.6-overexpressing myocytes, which is expected to favor larger peak amplitude of the [Ca2+]i transient.

It has been shown that drugs that just modify the open probability of RyRs have only transient effects on SR Ca2+ release (28). The reason is that increased Ca2+ release tends to empty SR Ca2+, thereby counterbalancing the effect of enhanced release, and vice versa (15). Here, however, we find a persistent increase of SR Ca2+ load in FKBP12.6-overexpressing cells (Fig. 5). Because SR load is the result of net Ca2+ fluxes, other Ca2+ transport mechanisms could be concurrently altered by FKBP12.6. For example, Ca2+ release through RyR2 and Ca2+ extrusion through the Na+/Ca2+ exchanger tend to decrease SR Ca2+ load, whereas increased Ca2+ current (ICa) and SERCA activity have opposite effects. The decrease of the [Ca2+]i transient decay time constant in FKBP12.6-overexpressing myocytes (Fig. 4D) could potentially reflect an effect of FKBP12.6 on the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA). However, to our knowledge, there is no evidence that FKBP12.6 binds to SERCA. Alternatively, the acceleration of [Ca2+]i transient decay could be due to the stabilizing effect of FKBP12.6 on the closed state of the RyR2. In other words, if most openings occur at the beginning of the depolarization, fewer openings at the end of the action potential would result in early termination of SR Ca2+ release. It was shown that the duration of the [Ca2+]i transient evoked by field stimulation depends on the synchrony of Ca2+ sparks at the beginning of the stimulation (13).

Neither ICa nor Ca2+ extrusion through the Na+/Ca2+ exchanger is altered by FKBP12.6 overexpression (14) or FK506 (27). We also find no difference in the decay time of the caffeine-evoked [Ca2+]i transient (data not shown), which reflects Ca2+ extrusion through the Na+/Ca2+ exchanger. However, Ca2+ extrusion by the Na+/Ca2+ exchanger could be indirectly modulated by FKBP12.6 (20). The shorter [Ca2+]i transients in FKBP12.6-overexpressing myocytes limit Ca2+ extrusion out of the cell through the Na+/Ca2+ exchanger, thereby favoring SR Ca2+ load. In fact, the Ca2+ loss through the Na+/Ca2+ exchanger can be measured as the integral of the Na+/Ca2+ exchanger current, which mirrors the [Ca2+]i transient (9), and thus it is dependent on its length. So, even if the peak [Ca2+]i transient is higher in FKBP12.6-overexpressing cells, it is possible that the net Ca2+ loss is not enhanced because the transient is shortened (22).

In summary, overexpression of FKBP12.6 has differential effect on the RyR2 during diastole and systole in isolated rat cardiac myocytes. On one hand, FKBP12.6 decreases the occurrence, amplitude, width, and duration of spontaneous Ca2+ sparks. On the other hand, FKBP12.6 increases the peak of [Ca2+]i transients, which involves increased SR Ca2+ load and probably synchronized RyR2 openings early during the depolarization. Both mechanisms contribute to improve SR Ca2+ load and systolic function. Therefore, this differential effect is beneficial for E-C coupling. Thus FKBP12.6 arises as a potential pharmacological target to treat cardiac diseases in which diastolic and systolic Ca2+ is impaired, as in heart failure.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by Fondation pour la Recherche Médicale Grant INE20001117051 (to S. Richard), the Fondation de France, the Deutsche Forschungsgemeinschaft (to J. Prestle and G. Hasenfuss), and fellowships from the Ministère de la Recherche et de la Technologie (to J. Fauconnier) and Fondation pour la Recherche Médicale (to I. Schuster). S. Richard and A. M. Gómez hold Centre National de la Recherche Scientifique positions.


   ACKNOWLEDGMENTS
 
We thank P. Fontanaud for helpful discussion on data analysis.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. Richard, Institut National de la Santé et de la Recherche Médicale U-637, Centre Hospitalier Universitaire Arnaud de Villeneuve, 34295 Montpellier, France (E-mail: srichard{at}montp.inserm.fr)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

* A. M. Gómez and I. Schuster contributed equally to this work. Back


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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