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Ca2+ exchanger activation
Life Sciences 89 (2011) 7–14
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Life Sciences j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / l i f e s c i e
Rapamycin (sirolimus) protects against hypoxic damage in primary heart cultures via Na +/Ca 2+ exchanger activation Dalia El-Ani a, b, Hagit Stav a, Victor Guetta b, Michael Arad b, Asher Shainberg a,⁎ a b
The Mina & Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel The Heart Institute, Sheba Medical Center, and Tel-Aviv University Medical School, Tel Aviv, Israel
a r t i c l e
i n f o
Article history: Received 19 January 2011 Accepted 19 April 2011 Keywords: Heart cultures Hypoxia reoxygenation Intracellular calcium Rapamycin (sirolimus) Sodium calcium exchanger (NCX) SR Ca2+ ATPase (SERCA2a)
a b s t r a c t Aims: Rapamycin (sirolimus) is an antibiotic that inhibits protein synthesis through mammalian targeting of rapamycin (mTOR) signaling, and is used as an immunosuppressant in the treatment of organ rejection in transplant recipients. Rapamycin confers preconditioning-like protection against ischemic–reperfusion injury in isolated mouse heart cultures. Our aim was to further define the role of rapamycin in intracellular Ca2+ homeostasis and to investigate the mechanism by which rapamycin protects cardiomyocytes from hypoxic damage. Main methods: We demonstrate here that rapamycin protects rat heart cultures from hypoxic–reoxygenation (H/R) damage, as revealed by assays of lactate dehydrogenase (LDH) and creatine kinase (CK) leakage to the medium, by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) measurements, and desmin immunostaining. As a result of hypoxia, intracellular calcium levels ([Ca 2+]i) were elevated. However, treatment of heart cultures with rapamycin during hypoxia attenuated the increase of [Ca2+]i. Rapamycin also attenuated 45Ca2+ uptake into the sarcoplasmic reticulum (SR) of skinned heart cultures in a dose- and time-dependent manner. KB-R7943, which inhibits the “reverse” mode of Na+/Ca2+ exchanger (NCX), protected heart cultures from H/R damage with or without the addition of rapamycin. Rapamycin decreased [Ca2+]i following its elevation by extracellular Ca2+ ([Ca2+]o) influx, thapsigargin treatment, or depolarization with KCl. Key findings: We suggest that rapamycin induces cardioprotection against hypoxic/reoxygenation damage in primary heart cultures by stimulating NCX to extrude Ca 2+ outside the cardiomyocytes. Significance: According to our findings, rapamycin preserves Ca2+ homeostasis and prevents Ca2+ overload via extrusion of Ca2+ surplus outside the sarcolemma, thereby protecting the cells from hypoxic stress. © 2011 Elsevier Inc. All rights reserved.
Introduction Rapamycin (sirolimus) is an antibiotic that inhibits protein synthesis through mammalian targeting of rapamycin (mTOR) signaling, and is used as an immunosuppressant in the treatment of organ rejection in transplant recipients, including heart transplant recipients (Dunn and Croom, 2006). In addition to attenuating load-induced cardiac hypertrophy (Ha et al., 2005), rapamycin-impregnated stents effectively reduce coronary restenosis (Suttorp et al., 2006). The proposed mechanism for the anti-proliferative effect of rapamycin is based on its ability to bind to its intracellular receptor, the FK506 binding protein 12 (FKBP12) (Marks, 1997). Ryanodine receptors (RyRs), the major intracellular Ca 2+ ([Ca 2+]i) release channel in striated and cardiac muscle (Sharma et al., 2006), play an essential role in excitation–contraction coupling by regulating the delivery of Ca 2+ from the sarcoplasmic reticulum (SR) to the
⁎ Corresponding author. Tel.: + 972 3 5318265; fax: + 972 3 7369231. E-mail address: [email protected] (A. Shainberg). 0024-3205/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2011.04.017
contractile apparatus. Rapamycin reduces the current amplitude of the SR cardiac muscle release channels (Kaftan et al., 1996). Compounds such as rapamycin, which act on ryanodine release channels, have potential therapeutic effects on heart disease (Dulhunty et al., 2007). Xiao et al. (1997) showed that the blockage of FKBP12 prolongs Ca2+ sparks more than 6-fold, potentiates SR Ca2+ release, and induces [Ca 2+]i oscillations. Lehnart et al. (2006)have also shown that the stabilization of cardiac ryanodine receptors prevents intracellular calcium leak and arrhythmias. Khan et al. (2006) report that rapamycin confers preconditioninglike protection against ischemic–reperfusion injury in isolated mouse heart and cardiomyocytes. The authors experimented on adult male ischemic–preconditioned mice treated with rapamycin in a Langendorff model, and concluded that rapamycin induces a potent preconditioning-like effect against myocardial infarction through the opening of mitochondrial KATP channels. How the opening of these channels exerts its protection against stress conditions remains unclear. We demonstrate here that rapamycin protects rat heart cultures from hypoxia–reoxygenation (H/R) damage, as revealed by assays of lactate dehydrogenase (LDH) and creatine kinase (CK)
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leakage to the medium by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5diphenyltetrazolium bromide) measurement and by desmin immunostaining. Rapamycin also attenuated the increase of [Ca 2+]i following hypoxia of heart cultures. On the other hand, it was reported that rapamycin prevented induction of cardioprotection by ischemic preconditioning in isolated reperfused rat hearts (Bose et al., 2007; Hausenloy et al., 2004) and in rabbit hearts in vivo (Raphael et al., 2008). The aim of this study was to further define the role of rapamycin in intracellular Ca 2+ homeostasis, and to investigate the mechanism by which rapamycin protects heart cultures from hypoxic damage. Rapamycin decreased intracellular Ca2+ after the elevation of cytosolic Ca2+ as a result of elevated extracellular calcium [Ca 2+]o to 3.8 mM, following thapsigargin treatment, or as a result of depolarization with KCl. As rapamycin attenuated 45Ca2+ accumulation into the SR, we suggest that rapamycin may activate the Na +/Ca 2+ exchanger in the ”forward” mode, removing cytoplasmic Ca2+ after overload, and that such a mechanism may be involved in cardioprotection against hypoxia.
CO2–95% air at 37 °C. A confluent monolayer exhibiting spontaneous contractions developed within 2 days. The experiments were performed on 4–6-day-old cardiomyocyte cultures. Hypoxic experiments Hypoxia and reoxygenation (H/R) Experiments were performed as previously described (El-Ani et al., 2007). Heart culture dishes were pre-incubated for 20 min with or without 10 μM rapamycin in glucose-free PBS at 37 °C. The dishes were then transferred to hypoxic chambers with a constant flow of argon for 90 min at 37 °C. For re-oxygenation, cultures were transferred with the drug for 2 h to the normoxic incubator under the same conditions as prior to hypoxia. For intracellular Ca 2+ measurements under hypoxic conditions, coverglasses containing cardiomyocytes were placed in a stainless steel chamber attached to a Zeiss epi-fluorescent inverted microscope under argon (100%) flow, as previously described (Zinman et al., 2006).
Materials and methods
LDH or CK measurements
Chemicals All chemicals were obtained from Sigma, unless otherwise mentioned. Rapamycin was obtained from LC Laboratories (USA). KB-R7943 was donated by Kanebo LTD, Osaka, Japan.
Assays were performed as previously described (El-Ani et al., 1997). An amount of 25 μl of the supernatant of growing cells was transferred into a 96-well dish and LDH/CK activity was determined using Sigma LDH-L or CK kits. Experiments were performed with 4–8 replicas each, and were repeated at least 3 times.
Solutions
MTT assay
Rapamycin was dissolved in DMSO to 10 mM stock solution. This solution was diluted in phosphate-buffered saline (PBS) to 10 μM before application to the cells (0.1% DMSO). Control cells received diluted vehicle. KB-R7943 was dissolved in DMSO to 100 mM stock solution and diluted in PBS to 10 μM final concentration (0.01% DMSO). Standard phosphate-buffered saline solution (PBS), pH 7.4, contained: 135 mM NaCl, 8.09 mM Na2HPO4, 1.47 mM KH2PO4, 2.68 mM KCl, 0.9 mM CaCl2, 0.48 mM MgCl2, and 5.55 mM glucose. Tyrode– choline solution, pH 7.4, contained: 140 mM choline chloride, 2.68 mM KCl, 1.8 mM CaCl2, 1.05 mM MgCl2, 5.55 mM glucose, and 10 mM HEPES. Tyrode solution, pH 7.4, contained the same components as Tyrode–choline, but with NaCl instead of choline chloride.
Measurements were performed as previously described (El-Ani et al., 2007) according to Sigma protocol. After H/R of cardiomyocytes in 96 well plates, 10 μl of the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5diphenyltetrazoliumbromide) solution (5 mg/ml) was transferred to 100 μl PBS of each well. Following an additional 45 min of reoxygenation, 150 μl fresh solution B was added to each well (solution A: 200 μl HCl [32%] in 50 ml isopropanol; solution B: solution A diluted 3:1 in DMSO). Ten minutes later, absorbance was measured at 600 nm. All experiments were performed at 37 °C with 5% CO2.
Preparation of heart cultures The animals were purchased from Harlan Labs, Jerusalem, Israel. The experiments were carried out in accordance with the guidelines of the Animal Care and Use Committee of Bar-Ilan University, with the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health. Sprague–Dawley rat hearts (2–3 days old) were removed under sterile conditions and washed three times in PBS to remove excess blood cells. The hearts were minced into small fragments and then gently agitated in RDB, a solution of proteolytic enzymes prepared from figtree extract (Biological Institute, Ness-Ziona, Israel). RDB was diluted 1:100 in Ca 2+- and Mg2+-free PBS at 25 °C and incubated with the heart fragments for several cycles of 10 min each, as previously described (El-Ani et al., 2007). Dulbecco's modified Eagle's medium, supplemented with 10% inactivated horse serum (Biological Industries, Kibbutz Beit Haemek, Israel) and 0.5% chick embryo extract, was added to the supernatant containing a suspension of dissociated cells. The mixture was centrifuged at 300 ×g for 5 min. The supernatant was discarded and the cells were resuspended. The cell suspension was diluted to 1.0 × 106 cells/ml, and 1.5 ml of suspension was placed in 35 mm plastic culture dishes or glass cover slips coated with collagen/ gelatin. The cultures were incubated in a humidified atmosphere of 5%
Immunostaining after hypoxia and rapamycin treatment Heart rat cultures were grown on 25-mm-diameter cover slips and subjected to hypoxia and reoxygenation, as described earlier. The cardiomyocytes were subsequently fixed in methanol for 10 min, washed twice in PBS, and permeabilized for 20 min with wash solution containing 0.1% Triton X-100 and 1% BSA (for non-specific antigen blocking). Then the cover slips were incubated for 1.5 h at 24 °C with rabbit polyclonal anti-desmin antibodies (Santa Cruz, CA) that were diluted 1:50 in wash solution. Afterwards, cover slips were washed twice with PBS and incubated with 1:100 dilution in a wash solution of cyan-green Alexa Fluor® 488-conjugated goat anti-rabbit immunoglobulin antibodies (Molecular Probes). Labeling of cardiomyocytes with secondary antibody alone was carried out as a negative control. Nuclei were visualized with addition incubation with propidium iodide (5 μg/ml) for 10 min. Following staining, heart cultures were observed under a Zeiss AxiVision microscope. Skinning procedure and
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Ca 2+ accumulation in the SR
Experiments were performed as previously described (El-Ani et al., 2007). I) Skinning procedure: Cell were incubated in 1 ml skinning solution (140 mM KCl, 20 mM HEPES buffer, 10 mM EGTA, 50 μg/ml saponin, 5 mM NaN3, 5 mM potassium oxalate, 1 μM ruthenium red, pH 7.4) at 25 °C for 20 min. II) Ca 2+accumulation: The skinned heart cultures attached to the dish were washed twice with Ca 2+ uptake solution containing 140 mM KCl, 20 mM HEPES, 0.5 mM EGTA, 0.5 mM
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Fig. 1. Effect of rapamycin on LDH, CK, and MTT following hypoxia and re-oxygenation (H/R). Heart cultures were treated with 10 μM rapamycin (Rp) 20 min before being subjected to 90 min hypoxia and reoxygenation of 2 h (H/R). Rapamycin decreased LDH and CK release (A, B) and increased MTT absorbance (C). (A, B) [* p b 0.05 compared with H/R, ** p b 0.05 compared with control]. Data represent the mean ± SD. Representative results from 6 experiments are shown.
CaCl2, 5 mM NaN3, 5 mM potassium oxalate, 5 mM MgCl2, pH 7.0, at 37 °C. One ml of radioactive uptake solution ( 45Ca 0.5 μCi/ml) was then added to the cells, which were incubated at 37 °C for 10 min, either in the presence or absence of 5 mM ATP. The uptake solution was removed, and the cells were rinsed five times with ice-cold fresh uptake solution. Cells were then lysed with 0.3 ml 1% Triton X-100 and collected in counting vials containing 4 ml scintillation liquid. Radioactivity was determined using a scintillation counter (Packard). Values for specific calcium uptake were derived by subtraction of radioactivity measured for calcium accumulation experiments performed in the absence of ATP. Rapamycin was added to the skinning solution, to the rinsing solution used after skinning, and to the uptake solution. Intracellular Ca 2+ measurements Cellular calcium images of individual cardiomyocytes were obtained from heart cultures preloaded with 3 μM Indo-1 and 1.5 μM pluronic acid for 30 min in glucose-enriched PBS at 25 °C, as previously described (Rickover et al., 2008; Shmist et al., 2005; Zinman et al., 2006). Indo-1 was excited at 340 nm, and the emitted light was then split by a dichroic mirror to two photomultipliers (Hamamatsu Corporation, NJ) with input filters at 410 and 490 nm. The fluorescent signals at 410 and 490 nm acquired every 10 ms were fed to a CAPLAN program written by Dr. D. Kaplan from the Biological Institute (Ness-Ziona, Israel). The increase in the intensity of the fluorescent ratio of 410:490 nm is proportional to the rise in [Ca 2+]i.
Rapamycin Following Hypoxia-Reoxygenation Fig. 2. Desmin immunostaining of hypoxic/reoxygenation (H/R) heart cultures following rapamycin (Rp) treatment. Immunostaining for desmin of heart cultures pretreated with rapamycin 20 min before being subjected to 90 min hypoxia and reoxygenation of 2 h (H/R), showing damaged filaments of cardiomyocytes in H/Rtreated cultures, but not in rapamycin-treated cultures. The red staining is from propidium iodide in the nuclei. Note the cytoplasmic area (green) in the H/R culture, indicating severe damage of desmin filaments compared to H/R heart cultures that were treated with rapamycin or control cultures.
For hypoxic conditions, coverglasses containing heart cultures were placed in a stainless steel chamber under argon (100%) flow in a hypoxic apparatus, as previously described (Zinman et al., 2006). [Ca 2+]i level was determined during the hypoxic treatment from the same group of cells. The drugs were applied to the dish with the cells and remained there until washing. When extracellular calcium [Ca 2+]o was elevated in the medium, the cells were transferred to Tyrode buffer to prevent Ca 2+ precipitation.
Statistical analysis Statistical analysis was performed by analysis of variance with application of a post hoc Tukey–Kramer test. Results are expressed as mean ± SD (standard deviation). p b 0.05 was accepted as indicating statistical significance.
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Rapamycin
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Time (min) Fig. 3. [Ca2+]i of heart cultures. Heart cultures were loaded with Indo-1 and treated with 10 μM rapamycin (arrow), which decreased [Ca2+]i level. Upon washing and rapamycin removal, [Ca2+]i level was elevated. Representative results from 6 experiments are shown.
Results
Effect of rapamycin on [Ca 2+]i under normoxic and hypoxic conditions
Cardioprotective effect of rapamycin against hypoxia
In order to study the cardioprotective mechanism of rapamycin, the [Ca 2+]i level was analyzed in cells loaded with indo-1 AM. [Ca 2+]i level was given in arbitrary units based on the fluorescent ratio of 410:490 nm. Rapamycin (10 μM) caused an immediate decrease in [Ca 2+]i level. This decrease in [Ca 2+]i lasted as long as rapamycin remained in the dish. Upon the removal of rapamycin, [Ca 2+]i level that was determined from the same cardiomyocytes, immediately rose (Fig. 3). To evaluate the effect of rapamycin on [Ca2+]i, heart cultures were subjected to hypoxia. Rapamycin was administered after 40 min of hypoxia, and the hypoxia continued in the presence of this drug. [Ca2+]i measurements were taken on the same cells subjected to hypoxia in the same chamber. Whereas hypoxia caused gradual, basal (diastolic) [Ca2+]i elevation (Fig. 4B), rapamycin attenuated the further increase of basal [Ca 2+]i as a result of hypoxia (Fig. 4A).
To investigate whether rapamycin can protect cardiomyocytes from hypoxic damage, primary cardiac cells were treated with 10 μM rapamycin for 20 min before being subjected to hypoxia for 90 min and reoxygenation of 2 h (H/R). For evaluation of hypoxic damage, LDH, CK, and MTT were measured 2 h after H/R. Under hypoxic/ reoxygenation conditions, rapamycin (Rp) reduced LDH and CK levels in the medium by 70%, and increased MTT level by 300%, indicating cardioprotection by this drug (Fig. 1A,B,C). Morphological protection by rapamycin against hypoxia To evaluate rapamycin's morphological protection, heart cultures were treated with rapamycin (10 μM) before being subjected to H/R, and the morphology of the cells was then examined. The cells were immunostained for desmin after 2 h of reoxygenation (Fig. 2; green fluorescence) and the nucleus was stained with propidium iodide (red fluorescence). Desmin staining revealed damage in the myofilaments in the cytoplasmic area of the H/R treated cardiomyocytes. However, rapamycin protected the heart fibers from this H/R damage (Fig. 2).
A [Ca2+]i (410/490 nm)
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Rapamycin inhibits SR Ca 2+ATPase (SERCA2a) In order to elucidate the mechanism by which rapamycin reduces [Ca 2+]i, SERCA2a activity was analyzed. Heart cultures were skinned with saponin and then exposed to rapamycin. 45Ca 2+ uptake into the
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Time (min) Fig. 4. Rapamycin attenuated the elevation of [Ca2+]i level following hypoxia. A) Heart cultures were subjected to hypoxia. As a result of hypoxia, [Ca2+]i was elevated. When rapamycin was applied 40 min after the beginning of hypoxia, rapamycin prevented further elevation of [Ca2+]i in the hypoxic heart cultures (H). B) After 10 min in normoxia, the heart cultures were subjected to hypoxia. Increase in [Ca2+]i was detected in heart cultures at various times after exposure to hypoxia. Representative results from 6 experiments are shown.
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SR was performed as described before (El-Ani et al., 2007). It was found that the drug inhibited 45Ca 2+ uptake into the SR in a dose- and time-dependent manner (Fig. 5A, B). As rapamycin inhibited SERCA2a activity under normoxic conditions, there was no rationale to measure the effect of the drug on 45Ca 2+ uptake into the SR under hypoxic conditions. The possibility that SERCA2a is activated by rapamycin was, therefore, ruled out as a mechanism for reducing [Ca 2+]i, and an alternative mechanism was proposed.
Rapamycin stimulates the Na +/Ca2+ exchanger: Effect of rapamycin on [Ca 2+]i in heart cultures pretreated with high extracellular [Ca 2+]o, thapsigargin or following depolarization In the foregoing experiments, it was shown that SERCA2a is not involved in the reduction of [Ca 2+]i by rapamycin. The hypothesis that rapamycin activates the Na +/Ca 2+ exchanger in the “forward” mode was, therefore, proposed. To test this hypothesis, the following experiments were performed: increasing ([Ca 2+]o) from 1.8 to 3.8 mM (arrow) caused an increase of diastolic [Ca 2+]i (Fig. 6A). Application of 10 μM rapamycin (arrow) reduced [Ca 2+]i to baseline in Tyrode buffer containing 140 mM Na + (Fig. 6A). When the experiment was also performed as in Fig. 6A but in Tyrode–choline buffer, in order to prevent Ca 2+ extrusion by the Na +/Ca 2+ exchanger, rapamycin failed to remove Ca 2+ from the cytosol (Fig. 6B). These results are consistent with the hypothesis that rapamycin activates the Na +/Ca 2+ exchanger, since in Tyrode–choline buffer, which inhibited the Na +/Ca 2+ exchanger (Shmist et al., 2005), rapamycin did not reduce [Ca 2+]i after [Ca 2+]i elevation. The effect of rapamycin was then
examined in the presence of thapsigargin, a SERCA2a inhibitor. Five μM thapsigargin increased diastolic [Ca 2+]i of heart cultures (Fig. 7A). This elevation was attenuated by the application of 10 μM rapamycin in 140 mM Na + buffer (Tyrode buffer; Fig. 7A), but not in Na +-free buffer (Tyrode–choline buffer; Fig. 7B). We caused [Ca 2+]i elevation by another method using KCl. When rapamycin was added to the cardiomyocytes following depolarization with 30 mM KCl, which increases [Ca 2+]i (Eble et al., 1998), it decreased [Ca 2+]i (Fig. 8). This result further indicates stimulation of the Na +/Ca 2+ exchanger. As expected, there was no change of [Ca 2+]i after the addition of rapamycin in Tyrode–choline with 30 mM KCl (data not shown). Figs. 6, 7, and 8 indicate that rapamycin activated the “forward” mode of the Na+/Ca2+ exchanger, since rapamycin decreased [Ca 2+]i in Thyrode buffer after its elevation with extracellular Ca2+, thapsigargin, and KCl (Figs. 6A, 7A, 8), a phenomenon that was not detected in Tyrode–choline buffer that inhibited the Na+/Ca2+ exchanger. Cardioprotective effect of KB-R7943 against hypoxia In order to show that rapamycin specifically activates the “forward” mode of the Na +/Ca 2+ exchanger and not the “reverse” mode, KB-R7943 (10 μM), an inhibitor of the “reverse” mode of the Na +/Ca 2+ exchanger (Iwamoto et al., 1996, 2007), was applied. Heart cultures were treated with 10 μM rapamycin and 10 μM KB-R7943 for 20 min before being subjected to hypoxia/reoxygenation. Under H/R conditions, LDH and CK levels in the medium were reduced by 40 and 70% by rapamycin and KB-R7943 (Fig. 9A, B). Although KB-R7943 did
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Fig. 5. Effect of rapamycin on Ca2+ accumulation by the SR. Ca2+ uptake into the SR was decreased by rapamycin in a dose-dependent (A) and time-dependent (B) manner. Data represent the mean ± SD (*p b 0.05, compared to control). Representative results from 6 experiments are shown.
Fig. 6. Rapamycin reversed [Ca2+]i elevation by increased extracellular [Ca2+]. A) Increase of extracellular calcium from 1.8 to 3.8 mM (arrow) caused an increase in diastolic [Ca2+]i level. An application of 10 μM rapamycin (arrow) reduced [Ca2+]i to baseline. The experiment was performed in Tyrode buffer containing 140 mM Na+ and 1.8 mM CaCl2. B) This phenomenon was not detected in Na+-free buffer (Tyrode–choline). The experiment was performed as in Fig. 6A but using Tyrode–choline buffer, in which rapamycin failed to decrease [Ca2+]i following extracellular calcium elevation. Representative results from 6 experiments are shown.
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Time (sec) Fig. 7. Rapamycin reversed [Ca2+]i elevation following thapsigargin treatment. A) Five μM thapsigargin increased diastolic [Ca2+]i and then the application of10 μM rapamycin (arrow) reduced [Ca2+]i in 140 mM Na+ Tyrode buffer (A), but not in Na+-free Tyrode– choline buffer (B). Representative results from 6 experiments are shown.
not inhibit the cardioprotective effect of rapamycin against hypoxic damage, KB-R7943 itself exhibited cardioprotection against hypoxic damage (Fig. 9A, B). This implies that either activation of the “forward” mode of the Na +/Ca 2+ exchanger or inhibition of the “reverse” mode of the Na +/Ca 2+ exchanger brings about protection of cardiac cells from hypoxia. Discussion
[Ca2+]i (410/490 nm)
We have demonstrated here that rapamycin protects rat heart cultures from hypoxic reoxygenation damage, as revealed by assays of LDH and CK leakage to the medium (Fig. 1A, B), by MTT absorbance (Fig. 1C) and by desmin immunostaining (Fig. 2). In addition, treatment of heart cultures with rapamycin following hypoxia attenuated the gradual increase of [Ca 2+]i level (Fig. 4A). Our results support the findings of Khan et al. (2006) in a Langendorff model of isolated adult mouse heart, in which rapamycin induced a potent preconditioning-like effect against myocardial infarction. However, whereas Khan et al. (2006) suggested that the protection is via
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Time (sec) Fig. 8. Rapamycin reversed [Ca2+]i elevation following depolarization. Heart cultures in Tyrode buffer were treated with 30 mM KCl, which causes depolarization and [Ca2+]i elevation (arrow). Treatment with rapamycin following depolarization with 30 mM KCl (arrow) decreased [Ca2+]i level. Representative results from 6 experiments are shown.
opening of mitochondrial KATP channels, we propose that rapamycin also activates the sodium calcium exchanger (NCX) in the “forward” mode, thereby reducing calcium overload. It has been generally accepted that Ca 2+ influx through the L-type 2+ Ca channel induces Ca2+ release from the SR via ryanodine channels, which leads to contraction (Eisner et al., 2009). Subsequently, Ca2+ is taken up by the SR Ca2+ATPase, and is also extruded from the cardiomyocyte by the NCX operating in the “forward” mode. Hence, NCX competes with the SR Ca 2+-ATPase in lowering [Ca 2+]i and thereby causing relaxation (Inserte et al., 2009). The NCX can also operate in a “reverse” mode, causing an influx of 1 Ca 2+ ion in exchange for 3 Na + ions during excitation. The “reverse” function of the NCX has been proposed to increase the content of [Ca2+]i in the cardiomyocytes. Preclinical studies have shown that pharmacological interventions targeted against sarcolemmal sodium ion transporters, such as inhibition of the "reverse" mode or activation of the “forward” mode of NCX, are effective in ameliorating heart failure (Baartscheer and van Borren, 2008). Rapamycin caused an immediate [Ca 2+]i decrease (Fig. 3). This reduction of [Ca 2+]i lasted as long as rapamycin remained in the dish. Upon washing, the cells resumed their previous [Ca 2+]i levels (Fig. 3). A similar effect of rapamycin was obtained in the membrane of smooth muscle cells (Weidelt and Isenberg, 2000). Whereas rapamycin reduced the amplitude of the Ca 2+ transients in rabbit heart, it increased their amplitude in mice (Raphael et al., 2008; Su et al., 2003). 45 Rapamycin also decreased ATP-dependent Ca 2+ uptake into the sarcoplasmic reticulum of cardiomyocytes via SERCA2a in a dose- and time-dependent manner (Fig. 5A, B). A similar inhibition of the SR Ca 2+ ATPase (SERCA1a) by rapamycin was observed in skeletal muscle sarcoplasmic reticulum (Bilmen et al., 2002) and in smooth muscle cells (Bultynck et al., 2000). Ischemia and reperfusion are associated with oxidative stress and intracellular Ca 2+ overload (Dhalla et al., 2007). Previous studies have shown different protective mechanisms against hypoxia/ reoxygenation-induced damage, and the involvement of SERCA2a in these mechanisms (El-Ani et al., 2007; Rickover et al., 2008; Shmist et al., 2005; Shneyvays et al., 2005; Zinman et al., 2006). An increase in diastolic [Ca 2+]i following elevation of extracellular calcium could be reversed by the subsequent application of rapamycin in Tyrode solution (Fig. 6A). Likewise, the application of thapsigargin, a SERCA2a inhibitor, increased diastolic [Ca 2+]i in heart cultures, and this increase was reversed by the immediate application of rapamycin (Fig. 7A). Similarly, depolarization caused [Ca 2+]i elevation, which was reduced by rapamycin (Fig. 8). In Na +-free Tyrode–choline buffer, rapamycin did not reduce [Ca 2+]i following exogenic Ca 2+ elevation, thapsigargin application, or following depolarization (Figs. 6B, 7B). Because Ca 2+ uptake into the SR was blocked by thapsigargin, we attribute the extrusion of cytosolic Ca 2+ to the possibility of stimulation of the sarcolemmal NCX by rapamycin. We further hypothesize that this reduction in [Ca 2+]i following exogenic Ca 2+ elevation (Fig. 6A) is probably via stimulation of the “forward” mode of NCX by rapamycin to extrude Ca 2+ out of the cell. Stimulation of NCX is one of the mechanisms for protecting the heart from ischemic insults (Chen et al., 2006; Yeung et al., 2007). The NCX in cardiac myocytes usually operates in the “forward” mode. During the action potential, the NCX can also operate in the “reverse” mode, in which Ca 2+ enters the cells. Conditions such as ischemia/reperfusion-induced elevation of cytosolic calcium can cause heart cell damage. Recently, it has been reported by Yeung et al. (2007) that chronic intermittent hypoxia alters Ca 2+ handling in rat cardiomyocytes by augmenting NCX and ryanodine receptor activity in ischemia-reperfusion by protein kinase A (PKA)- and protein kinase C (PKC)-dependent protection. Despite the impressive inhibition by rapamycin of Ca 2+ uptake into the SR, rapamycin attenuated the amplitude of calcium transients (Fig. 3) and reduced the increase in [Ca 2+]i as a result of hypoxia (Fig. 4A), suggesting a mechanism to prevent intracellular Ca 2+
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LDH Release (OD /min)
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Fig. 9. Effect of rapamycin (Rp) and KB-R7943 on LDH and CK release to the medium following hypoxia and reoxygenation (H/R). Heart cultures were treated with 10 μM rapamycin (Rp) and 10 μM KB-R7943 (KBR) 20 min before being subjected to H/R. Rapamycin and KB-R7943 decreased LDH and CK release after H/R. (A, B) [* p b 0.05 compared with H/R, ** p b 0.05 compared with control]. Data represent the mean ± SD. Representative results from 6 experiments are shown.
overload. According to our results, this prevention of Ca 2+ overload is not via SERCA2a, but probably via activation of NCX. Our results suggest that rapamycin induces cardioprotection against hypoxic damage by removing Ca2+ from the cytosol through NCX activation. In addition, KB-R7943 – an inhibitor of NCX in the reverse mode (Iwamoto et al., 1996; Yoshitomi et al., 2005) – on the one hand protected the cells from hypoxia, but on the other hand, did not inhibit the cardioprotective effect of rapamycin following hypoxia (Fig. 9A, B). Thus, both treatments – rapamycin and KB-R7943 – prevent intracellular Ca2+ accumulation under hypoxic conditions that leads to activation of proteolytic enzymes and cell damage. Based on the current findings, we suggest that rapamycin induces cardioprotection against hypoxic/reoxygenation damage in heart cultures by stimulating NCX to extrude Ca 2+ from the cardiomyocytes. Thus, rapamycin preserves Ca 2+ homeostasis and prevents Ca 2+ overload via extrusion of surplus Ca 2+ across the sarcolemma, thereby protecting the cells from hypoxic reoxygenation stress. Conflict of interest statement The authors declare that there are no conflicts of interest related to this manuscript.
Acknowledgments We thank A. Isak for technical support. We also thank Sharon Victor for helping to prepare this manuscript for publication. This work was supported in part by an ISF grant no. 876/05 and through the generous support of the Adar Program for the Advancement of Research in Heart Function at Bar-Ilan University. The sponsors were not involved in any way in the making of this paper or the decision to submit it for publication. References Baartscheer A, van Borren MM. Sodium ion transporters as new therapeutic targets in heart failure. Cardiovasc Hematol Agents Med Chem 2008;6:229–36. Bilmen JG, Wootton LL, Michelangeli F. The inhibition of the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase by macrocyclic lactones and cyclosporin A. Biochem J 2002;366:255–63. Bose AK, Mocanu MM, Carr RD, Yellon DM. Myocardial ischaemia-reperfusion injury is attenuated by intact glucagon like peptide-1 (GLP-1) in the in vitro rat heart and may involve the p70s6K pathway. Cardiovasc Drug Ther 2007;21 253–25. Bultynck G, De Smet P, Weidema AF, Ver Heyen M, Maes K, Callewaert G, et al. Effects of the immunosuppressant FK506 on intracellular Ca2+ release and Ca2+ accumulation mechanisms. J Physiol 2000;525(Pt 3):681–93.
Chen L, Lu XY, Li J, Fu JD, Zhou ZN, Yang HT. Intermittent hypoxia protects cardiomyocytes against ischemia-reperfusion injury-induced alterations in Ca2+ homeostasis and contraction via the sarcoplasmic reticulum and Na+/Ca2+ exchange mechanisms. Am J Physiol Cell PH 2006;290:C1221–9. Dhalla NS, Saini HK, Tappia PS, Sethi R, Mengi SA, Gupta SK. Potential role and mechanisms of subcellular remodeling in cardiac dysfunction due to ischemic heart disease. J Cardiovasc Med Hagerstown 2007;8:238–50. Dulhunty AF, Beard NA, Pouliquin P, Casarotto MG. Agonists and antagonists of the cardiac ryanodine receptor: potential therapeutic agents? Pharmacol Ther 2007;113:247–63. Dunn C, Croom KF. Everolimus: a review of its use in renal and cardiac transplantation. Drugs 2006;66:547–70. Eble DM, Cadre BM, Qi M, Bers DM, Samarel AM. Contractile activity modulates atrial natriuretic factor gene expression in neonatal rat ventricular myocytes. J Mol Cell Cardiol 1998;30:55–60. Eisner DA, Kashimura T, Venetucci LA, Trafford AW. From the ryanodine receptor to cardiac arrhythmias. Circ J 2009;73:1561–7. El-Ani D, Jacobson KA, Shainberg A. Regulation of A1 adenosine receptors by electrical stimulation and amiodarone in rat myocardial cells in vitro. Biochem Pharmacol 1997;54:583–7. El-Ani D, Zimlichman R, Mashiach Y, Shainberg A. Adenosine and TNF-alpha exert similar inotropic effect on heart cultures, suggesting a cardioprotective mechanism against hypoxia. Life Sci 2007;81:803–13. Ha T, Li Y, Gao X, McMullen JR, Shioi T, Izumo S, et al. Attenuation of cardiac hypertrophy by inhibiting both mTOR and NFkappaB activation in vivo. Free Radic Biol Med 2005;39:1570–80. Hausenloy DJ, Mocanu MA, Yellon DM. Cross-talk between the survival kinases during early reperfusion: its contribution to ischemic preconditioning. Cardiovasc Res 2004;63:305–12. Inserte J, Barrabes JA, Hernando V, Garcia-Dorado D. Orphan targets for reperfusion injury. Cardiovasc Res 2009;83:169–78. Iwamoto T, Watano T, Shigekawa M. A novel isothiourea derivative selectively inhibits the reverse mode of Na+/Ca2+ exchange in cells expressing NCX1. J Biol Chem 1996;271:22391–7. Iwamoto T, Watanabe Y, Kita S, Blaustein MP. Na+/Ca2+ exchange inhibitors: a new class of calcium regulators. Cardiovasc Hematol Disord Drug Targets 2007;7: 188–98. Kaftan E, Marks AR, Ehrlich BE. Effects of rapamycin on ryanodine receptor/Ca(2+)-release channels from cardiac muscle. Circ Res 1996;78:990–7. Khan S, Salloum F, Das A, Xi L, Vetrovec GW, Kukreja RC. Rapamycin confers preconditioning-like protection against ischemia–reperfusion injury in isolated mouse heart and cardiomyocytes. J Mol Cell Cardiol 2006;41:256–64. Lehnart SE, Terrenoire C, Reiken S, Wehrens XH, Song LS, Tillman EJ, et al. Stabilization of cardiac ryanodine receptor prevents intracellular calcium leak and arrhythmias. Proc Natl Acad Sci U S A 2006;103:7906–10. Marks AR. Intracellular calcium-release channels: regulators of cell life and death. Am J Physiol 1997;272:H597–605. Raphael J, Zuo ZY, Abedat S, Beeri R, Gozal Y. Isoflurane preconditioning decreases myocardial infarction in rabbits via up-regulation of hypoxia inducible factor 1 that is mediated by mammalian target of rapamycin. Anesthesiology 2008;108:415–25. Rickover O, Zinman T, Kaplan D, Shainberg A. Exogenous nitric oxide triggers classic ischemic preconditioning by preventing intracellular Ca2+ overload in cardiomyocytes. Cell Calcium 2008;43:324–33. Sharma MR, Jeyakumar LH, Fleischer S, Wagenknecht T. Three-dimensional visualization of FKBP12.6 binding to an open conformation of cardiac ryanodine receptor. Biophys J 2006;90:164–72.
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Shmist YA, Kamburg R, Ophir G, Kozak A, Shneyvays V, Appelbaum YJ, et al. N, N, N′, N′tetrakis(2-pyridylmethyl)-ethylenediamine improves myocardial protection against ischemia by modulation of intracellular Ca2+ homeostasis. J Pharmacol Exp Ther 2005;313:1046–57. Shneyvays V, Leshem D, Zinman T, Mamedova LK, Jacobson KA, Shainberg A. Role of adenosine A1 and A3 receptors in regulation of cardiomyocyte homeostasis following mitochondrial respiratory chain injury. Am J Physiol Heart C 2005;288:H2792–801. Su Z, Sugishita K, Li F, Ritter M, Barry WH. Effects of FK506 on [Ca2+] i differ in mouse and rabbit ventricular myocytes. J Pharmacol Exp Ther 2003;304:334–41. Suttorp MJ, Laarman GJ, Rahel BM, Kelder JC, Bosschaert MA, Kiemeneij F, et al. Primary stenting of totally occluded native coronary arteries II (PRISON II): a randomized comparison of bare metal stent implantation with sirolimus-eluting stent implantation for the treatment of total coronary occlusions. Circulation 2006;114:921–8. Weidelt T, Isenberg G. Augmentation of SR Ca(2+) release by rapamycin and FK506 causes K(+)-channel activation and membrane hyperpolarization in bladder smooth muscle. Br J Pharmacol 2000;129:1293–300.
Xiao RP, Valdivia HH, Bogdanov K, Valdivia C, Lakatta EG, Cheng H. The immunophilin FK506-binding protein modulates Ca2+ release channel closure in rat heart. J Physiol 1997;500(Pt 2):343–54. Yeung HM, Kravtsov GM, Ng KM, Wong TM, Fung ML. Chronic intermittent hypoxia alters Ca2+ handling in rat cardiomyocytes by augmented Na+/Ca2+ exchange and ryanodine receptor activities in ischemia–reperfusion. Am J Physiol Cell PH 2007;292:C2046–56. Yoshitomi O, Akiyama D, Hara T, Cho S, Tomiyasu S, Sumikawa K. Cardioprotective effects of KB-R7943, a novel inhibitor of Na+/Ca2+ exchanger, on stunned myocardium in anesthetized dogs. J Anesth 2005;19:124–30. Zinman T, Shneyvays V, Tribulova N, Manoach M, Shainberg A. Acute, nongenomic effect of thyroid hormones in preventing calcium overload in newborn rat cardiocytes. J Cell Physiol 2006;207:220–31.
Contents lists available at ScienceDirect
Life Sciences j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / l i f e s c i e
Rapamycin (sirolimus) protects against hypoxic damage in primary heart cultures via Na +/Ca 2+ exchanger activation Dalia El-Ani a, b, Hagit Stav a, Victor Guetta b, Michael Arad b, Asher Shainberg a,⁎ a b
The Mina & Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel The Heart Institute, Sheba Medical Center, and Tel-Aviv University Medical School, Tel Aviv, Israel
a r t i c l e
i n f o
Article history: Received 19 January 2011 Accepted 19 April 2011 Keywords: Heart cultures Hypoxia reoxygenation Intracellular calcium Rapamycin (sirolimus) Sodium calcium exchanger (NCX) SR Ca2+ ATPase (SERCA2a)
a b s t r a c t Aims: Rapamycin (sirolimus) is an antibiotic that inhibits protein synthesis through mammalian targeting of rapamycin (mTOR) signaling, and is used as an immunosuppressant in the treatment of organ rejection in transplant recipients. Rapamycin confers preconditioning-like protection against ischemic–reperfusion injury in isolated mouse heart cultures. Our aim was to further define the role of rapamycin in intracellular Ca2+ homeostasis and to investigate the mechanism by which rapamycin protects cardiomyocytes from hypoxic damage. Main methods: We demonstrate here that rapamycin protects rat heart cultures from hypoxic–reoxygenation (H/R) damage, as revealed by assays of lactate dehydrogenase (LDH) and creatine kinase (CK) leakage to the medium, by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) measurements, and desmin immunostaining. As a result of hypoxia, intracellular calcium levels ([Ca 2+]i) were elevated. However, treatment of heart cultures with rapamycin during hypoxia attenuated the increase of [Ca2+]i. Rapamycin also attenuated 45Ca2+ uptake into the sarcoplasmic reticulum (SR) of skinned heart cultures in a dose- and time-dependent manner. KB-R7943, which inhibits the “reverse” mode of Na+/Ca2+ exchanger (NCX), protected heart cultures from H/R damage with or without the addition of rapamycin. Rapamycin decreased [Ca2+]i following its elevation by extracellular Ca2+ ([Ca2+]o) influx, thapsigargin treatment, or depolarization with KCl. Key findings: We suggest that rapamycin induces cardioprotection against hypoxic/reoxygenation damage in primary heart cultures by stimulating NCX to extrude Ca 2+ outside the cardiomyocytes. Significance: According to our findings, rapamycin preserves Ca2+ homeostasis and prevents Ca2+ overload via extrusion of Ca2+ surplus outside the sarcolemma, thereby protecting the cells from hypoxic stress. © 2011 Elsevier Inc. All rights reserved.
Introduction Rapamycin (sirolimus) is an antibiotic that inhibits protein synthesis through mammalian targeting of rapamycin (mTOR) signaling, and is used as an immunosuppressant in the treatment of organ rejection in transplant recipients, including heart transplant recipients (Dunn and Croom, 2006). In addition to attenuating load-induced cardiac hypertrophy (Ha et al., 2005), rapamycin-impregnated stents effectively reduce coronary restenosis (Suttorp et al., 2006). The proposed mechanism for the anti-proliferative effect of rapamycin is based on its ability to bind to its intracellular receptor, the FK506 binding protein 12 (FKBP12) (Marks, 1997). Ryanodine receptors (RyRs), the major intracellular Ca 2+ ([Ca 2+]i) release channel in striated and cardiac muscle (Sharma et al., 2006), play an essential role in excitation–contraction coupling by regulating the delivery of Ca 2+ from the sarcoplasmic reticulum (SR) to the
⁎ Corresponding author. Tel.: + 972 3 5318265; fax: + 972 3 7369231. E-mail address: [email protected] (A. Shainberg). 0024-3205/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2011.04.017
contractile apparatus. Rapamycin reduces the current amplitude of the SR cardiac muscle release channels (Kaftan et al., 1996). Compounds such as rapamycin, which act on ryanodine release channels, have potential therapeutic effects on heart disease (Dulhunty et al., 2007). Xiao et al. (1997) showed that the blockage of FKBP12 prolongs Ca2+ sparks more than 6-fold, potentiates SR Ca2+ release, and induces [Ca 2+]i oscillations. Lehnart et al. (2006)have also shown that the stabilization of cardiac ryanodine receptors prevents intracellular calcium leak and arrhythmias. Khan et al. (2006) report that rapamycin confers preconditioninglike protection against ischemic–reperfusion injury in isolated mouse heart and cardiomyocytes. The authors experimented on adult male ischemic–preconditioned mice treated with rapamycin in a Langendorff model, and concluded that rapamycin induces a potent preconditioning-like effect against myocardial infarction through the opening of mitochondrial KATP channels. How the opening of these channels exerts its protection against stress conditions remains unclear. We demonstrate here that rapamycin protects rat heart cultures from hypoxia–reoxygenation (H/R) damage, as revealed by assays of lactate dehydrogenase (LDH) and creatine kinase (CK)
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leakage to the medium by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5diphenyltetrazolium bromide) measurement and by desmin immunostaining. Rapamycin also attenuated the increase of [Ca 2+]i following hypoxia of heart cultures. On the other hand, it was reported that rapamycin prevented induction of cardioprotection by ischemic preconditioning in isolated reperfused rat hearts (Bose et al., 2007; Hausenloy et al., 2004) and in rabbit hearts in vivo (Raphael et al., 2008). The aim of this study was to further define the role of rapamycin in intracellular Ca 2+ homeostasis, and to investigate the mechanism by which rapamycin protects heart cultures from hypoxic damage. Rapamycin decreased intracellular Ca2+ after the elevation of cytosolic Ca2+ as a result of elevated extracellular calcium [Ca 2+]o to 3.8 mM, following thapsigargin treatment, or as a result of depolarization with KCl. As rapamycin attenuated 45Ca2+ accumulation into the SR, we suggest that rapamycin may activate the Na +/Ca 2+ exchanger in the ”forward” mode, removing cytoplasmic Ca2+ after overload, and that such a mechanism may be involved in cardioprotection against hypoxia.
CO2–95% air at 37 °C. A confluent monolayer exhibiting spontaneous contractions developed within 2 days. The experiments were performed on 4–6-day-old cardiomyocyte cultures. Hypoxic experiments Hypoxia and reoxygenation (H/R) Experiments were performed as previously described (El-Ani et al., 2007). Heart culture dishes were pre-incubated for 20 min with or without 10 μM rapamycin in glucose-free PBS at 37 °C. The dishes were then transferred to hypoxic chambers with a constant flow of argon for 90 min at 37 °C. For re-oxygenation, cultures were transferred with the drug for 2 h to the normoxic incubator under the same conditions as prior to hypoxia. For intracellular Ca 2+ measurements under hypoxic conditions, coverglasses containing cardiomyocytes were placed in a stainless steel chamber attached to a Zeiss epi-fluorescent inverted microscope under argon (100%) flow, as previously described (Zinman et al., 2006).
Materials and methods
LDH or CK measurements
Chemicals All chemicals were obtained from Sigma, unless otherwise mentioned. Rapamycin was obtained from LC Laboratories (USA). KB-R7943 was donated by Kanebo LTD, Osaka, Japan.
Assays were performed as previously described (El-Ani et al., 1997). An amount of 25 μl of the supernatant of growing cells was transferred into a 96-well dish and LDH/CK activity was determined using Sigma LDH-L or CK kits. Experiments were performed with 4–8 replicas each, and were repeated at least 3 times.
Solutions
MTT assay
Rapamycin was dissolved in DMSO to 10 mM stock solution. This solution was diluted in phosphate-buffered saline (PBS) to 10 μM before application to the cells (0.1% DMSO). Control cells received diluted vehicle. KB-R7943 was dissolved in DMSO to 100 mM stock solution and diluted in PBS to 10 μM final concentration (0.01% DMSO). Standard phosphate-buffered saline solution (PBS), pH 7.4, contained: 135 mM NaCl, 8.09 mM Na2HPO4, 1.47 mM KH2PO4, 2.68 mM KCl, 0.9 mM CaCl2, 0.48 mM MgCl2, and 5.55 mM glucose. Tyrode– choline solution, pH 7.4, contained: 140 mM choline chloride, 2.68 mM KCl, 1.8 mM CaCl2, 1.05 mM MgCl2, 5.55 mM glucose, and 10 mM HEPES. Tyrode solution, pH 7.4, contained the same components as Tyrode–choline, but with NaCl instead of choline chloride.
Measurements were performed as previously described (El-Ani et al., 2007) according to Sigma protocol. After H/R of cardiomyocytes in 96 well plates, 10 μl of the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5diphenyltetrazoliumbromide) solution (5 mg/ml) was transferred to 100 μl PBS of each well. Following an additional 45 min of reoxygenation, 150 μl fresh solution B was added to each well (solution A: 200 μl HCl [32%] in 50 ml isopropanol; solution B: solution A diluted 3:1 in DMSO). Ten minutes later, absorbance was measured at 600 nm. All experiments were performed at 37 °C with 5% CO2.
Preparation of heart cultures The animals were purchased from Harlan Labs, Jerusalem, Israel. The experiments were carried out in accordance with the guidelines of the Animal Care and Use Committee of Bar-Ilan University, with the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health. Sprague–Dawley rat hearts (2–3 days old) were removed under sterile conditions and washed three times in PBS to remove excess blood cells. The hearts were minced into small fragments and then gently agitated in RDB, a solution of proteolytic enzymes prepared from figtree extract (Biological Institute, Ness-Ziona, Israel). RDB was diluted 1:100 in Ca 2+- and Mg2+-free PBS at 25 °C and incubated with the heart fragments for several cycles of 10 min each, as previously described (El-Ani et al., 2007). Dulbecco's modified Eagle's medium, supplemented with 10% inactivated horse serum (Biological Industries, Kibbutz Beit Haemek, Israel) and 0.5% chick embryo extract, was added to the supernatant containing a suspension of dissociated cells. The mixture was centrifuged at 300 ×g for 5 min. The supernatant was discarded and the cells were resuspended. The cell suspension was diluted to 1.0 × 106 cells/ml, and 1.5 ml of suspension was placed in 35 mm plastic culture dishes or glass cover slips coated with collagen/ gelatin. The cultures were incubated in a humidified atmosphere of 5%
Immunostaining after hypoxia and rapamycin treatment Heart rat cultures were grown on 25-mm-diameter cover slips and subjected to hypoxia and reoxygenation, as described earlier. The cardiomyocytes were subsequently fixed in methanol for 10 min, washed twice in PBS, and permeabilized for 20 min with wash solution containing 0.1% Triton X-100 and 1% BSA (for non-specific antigen blocking). Then the cover slips were incubated for 1.5 h at 24 °C with rabbit polyclonal anti-desmin antibodies (Santa Cruz, CA) that were diluted 1:50 in wash solution. Afterwards, cover slips were washed twice with PBS and incubated with 1:100 dilution in a wash solution of cyan-green Alexa Fluor® 488-conjugated goat anti-rabbit immunoglobulin antibodies (Molecular Probes). Labeling of cardiomyocytes with secondary antibody alone was carried out as a negative control. Nuclei were visualized with addition incubation with propidium iodide (5 μg/ml) for 10 min. Following staining, heart cultures were observed under a Zeiss AxiVision microscope. Skinning procedure and
45
Ca 2+ accumulation in the SR
Experiments were performed as previously described (El-Ani et al., 2007). I) Skinning procedure: Cell were incubated in 1 ml skinning solution (140 mM KCl, 20 mM HEPES buffer, 10 mM EGTA, 50 μg/ml saponin, 5 mM NaN3, 5 mM potassium oxalate, 1 μM ruthenium red, pH 7.4) at 25 °C for 20 min. II) Ca 2+accumulation: The skinned heart cultures attached to the dish were washed twice with Ca 2+ uptake solution containing 140 mM KCl, 20 mM HEPES, 0.5 mM EGTA, 0.5 mM
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Fig. 1. Effect of rapamycin on LDH, CK, and MTT following hypoxia and re-oxygenation (H/R). Heart cultures were treated with 10 μM rapamycin (Rp) 20 min before being subjected to 90 min hypoxia and reoxygenation of 2 h (H/R). Rapamycin decreased LDH and CK release (A, B) and increased MTT absorbance (C). (A, B) [* p b 0.05 compared with H/R, ** p b 0.05 compared with control]. Data represent the mean ± SD. Representative results from 6 experiments are shown.
CaCl2, 5 mM NaN3, 5 mM potassium oxalate, 5 mM MgCl2, pH 7.0, at 37 °C. One ml of radioactive uptake solution ( 45Ca 0.5 μCi/ml) was then added to the cells, which were incubated at 37 °C for 10 min, either in the presence or absence of 5 mM ATP. The uptake solution was removed, and the cells were rinsed five times with ice-cold fresh uptake solution. Cells were then lysed with 0.3 ml 1% Triton X-100 and collected in counting vials containing 4 ml scintillation liquid. Radioactivity was determined using a scintillation counter (Packard). Values for specific calcium uptake were derived by subtraction of radioactivity measured for calcium accumulation experiments performed in the absence of ATP. Rapamycin was added to the skinning solution, to the rinsing solution used after skinning, and to the uptake solution. Intracellular Ca 2+ measurements Cellular calcium images of individual cardiomyocytes were obtained from heart cultures preloaded with 3 μM Indo-1 and 1.5 μM pluronic acid for 30 min in glucose-enriched PBS at 25 °C, as previously described (Rickover et al., 2008; Shmist et al., 2005; Zinman et al., 2006). Indo-1 was excited at 340 nm, and the emitted light was then split by a dichroic mirror to two photomultipliers (Hamamatsu Corporation, NJ) with input filters at 410 and 490 nm. The fluorescent signals at 410 and 490 nm acquired every 10 ms were fed to a CAPLAN program written by Dr. D. Kaplan from the Biological Institute (Ness-Ziona, Israel). The increase in the intensity of the fluorescent ratio of 410:490 nm is proportional to the rise in [Ca 2+]i.
Rapamycin Following Hypoxia-Reoxygenation Fig. 2. Desmin immunostaining of hypoxic/reoxygenation (H/R) heart cultures following rapamycin (Rp) treatment. Immunostaining for desmin of heart cultures pretreated with rapamycin 20 min before being subjected to 90 min hypoxia and reoxygenation of 2 h (H/R), showing damaged filaments of cardiomyocytes in H/Rtreated cultures, but not in rapamycin-treated cultures. The red staining is from propidium iodide in the nuclei. Note the cytoplasmic area (green) in the H/R culture, indicating severe damage of desmin filaments compared to H/R heart cultures that were treated with rapamycin or control cultures.
For hypoxic conditions, coverglasses containing heart cultures were placed in a stainless steel chamber under argon (100%) flow in a hypoxic apparatus, as previously described (Zinman et al., 2006). [Ca 2+]i level was determined during the hypoxic treatment from the same group of cells. The drugs were applied to the dish with the cells and remained there until washing. When extracellular calcium [Ca 2+]o was elevated in the medium, the cells were transferred to Tyrode buffer to prevent Ca 2+ precipitation.
Statistical analysis Statistical analysis was performed by analysis of variance with application of a post hoc Tukey–Kramer test. Results are expressed as mean ± SD (standard deviation). p b 0.05 was accepted as indicating statistical significance.
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Rapamycin
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Time (min) Fig. 3. [Ca2+]i of heart cultures. Heart cultures were loaded with Indo-1 and treated with 10 μM rapamycin (arrow), which decreased [Ca2+]i level. Upon washing and rapamycin removal, [Ca2+]i level was elevated. Representative results from 6 experiments are shown.
Results
Effect of rapamycin on [Ca 2+]i under normoxic and hypoxic conditions
Cardioprotective effect of rapamycin against hypoxia
In order to study the cardioprotective mechanism of rapamycin, the [Ca 2+]i level was analyzed in cells loaded with indo-1 AM. [Ca 2+]i level was given in arbitrary units based on the fluorescent ratio of 410:490 nm. Rapamycin (10 μM) caused an immediate decrease in [Ca 2+]i level. This decrease in [Ca 2+]i lasted as long as rapamycin remained in the dish. Upon the removal of rapamycin, [Ca 2+]i level that was determined from the same cardiomyocytes, immediately rose (Fig. 3). To evaluate the effect of rapamycin on [Ca2+]i, heart cultures were subjected to hypoxia. Rapamycin was administered after 40 min of hypoxia, and the hypoxia continued in the presence of this drug. [Ca2+]i measurements were taken on the same cells subjected to hypoxia in the same chamber. Whereas hypoxia caused gradual, basal (diastolic) [Ca2+]i elevation (Fig. 4B), rapamycin attenuated the further increase of basal [Ca 2+]i as a result of hypoxia (Fig. 4A).
To investigate whether rapamycin can protect cardiomyocytes from hypoxic damage, primary cardiac cells were treated with 10 μM rapamycin for 20 min before being subjected to hypoxia for 90 min and reoxygenation of 2 h (H/R). For evaluation of hypoxic damage, LDH, CK, and MTT were measured 2 h after H/R. Under hypoxic/ reoxygenation conditions, rapamycin (Rp) reduced LDH and CK levels in the medium by 70%, and increased MTT level by 300%, indicating cardioprotection by this drug (Fig. 1A,B,C). Morphological protection by rapamycin against hypoxia To evaluate rapamycin's morphological protection, heart cultures were treated with rapamycin (10 μM) before being subjected to H/R, and the morphology of the cells was then examined. The cells were immunostained for desmin after 2 h of reoxygenation (Fig. 2; green fluorescence) and the nucleus was stained with propidium iodide (red fluorescence). Desmin staining revealed damage in the myofilaments in the cytoplasmic area of the H/R treated cardiomyocytes. However, rapamycin protected the heart fibers from this H/R damage (Fig. 2).
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Rapamycin inhibits SR Ca 2+ATPase (SERCA2a) In order to elucidate the mechanism by which rapamycin reduces [Ca 2+]i, SERCA2a activity was analyzed. Heart cultures were skinned with saponin and then exposed to rapamycin. 45Ca 2+ uptake into the
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Time (min) Fig. 4. Rapamycin attenuated the elevation of [Ca2+]i level following hypoxia. A) Heart cultures were subjected to hypoxia. As a result of hypoxia, [Ca2+]i was elevated. When rapamycin was applied 40 min after the beginning of hypoxia, rapamycin prevented further elevation of [Ca2+]i in the hypoxic heart cultures (H). B) After 10 min in normoxia, the heart cultures were subjected to hypoxia. Increase in [Ca2+]i was detected in heart cultures at various times after exposure to hypoxia. Representative results from 6 experiments are shown.
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SR was performed as described before (El-Ani et al., 2007). It was found that the drug inhibited 45Ca 2+ uptake into the SR in a dose- and time-dependent manner (Fig. 5A, B). As rapamycin inhibited SERCA2a activity under normoxic conditions, there was no rationale to measure the effect of the drug on 45Ca 2+ uptake into the SR under hypoxic conditions. The possibility that SERCA2a is activated by rapamycin was, therefore, ruled out as a mechanism for reducing [Ca 2+]i, and an alternative mechanism was proposed.
Rapamycin stimulates the Na +/Ca2+ exchanger: Effect of rapamycin on [Ca 2+]i in heart cultures pretreated with high extracellular [Ca 2+]o, thapsigargin or following depolarization In the foregoing experiments, it was shown that SERCA2a is not involved in the reduction of [Ca 2+]i by rapamycin. The hypothesis that rapamycin activates the Na +/Ca 2+ exchanger in the “forward” mode was, therefore, proposed. To test this hypothesis, the following experiments were performed: increasing ([Ca 2+]o) from 1.8 to 3.8 mM (arrow) caused an increase of diastolic [Ca 2+]i (Fig. 6A). Application of 10 μM rapamycin (arrow) reduced [Ca 2+]i to baseline in Tyrode buffer containing 140 mM Na + (Fig. 6A). When the experiment was also performed as in Fig. 6A but in Tyrode–choline buffer, in order to prevent Ca 2+ extrusion by the Na +/Ca 2+ exchanger, rapamycin failed to remove Ca 2+ from the cytosol (Fig. 6B). These results are consistent with the hypothesis that rapamycin activates the Na +/Ca 2+ exchanger, since in Tyrode–choline buffer, which inhibited the Na +/Ca 2+ exchanger (Shmist et al., 2005), rapamycin did not reduce [Ca 2+]i after [Ca 2+]i elevation. The effect of rapamycin was then
examined in the presence of thapsigargin, a SERCA2a inhibitor. Five μM thapsigargin increased diastolic [Ca 2+]i of heart cultures (Fig. 7A). This elevation was attenuated by the application of 10 μM rapamycin in 140 mM Na + buffer (Tyrode buffer; Fig. 7A), but not in Na +-free buffer (Tyrode–choline buffer; Fig. 7B). We caused [Ca 2+]i elevation by another method using KCl. When rapamycin was added to the cardiomyocytes following depolarization with 30 mM KCl, which increases [Ca 2+]i (Eble et al., 1998), it decreased [Ca 2+]i (Fig. 8). This result further indicates stimulation of the Na +/Ca 2+ exchanger. As expected, there was no change of [Ca 2+]i after the addition of rapamycin in Tyrode–choline with 30 mM KCl (data not shown). Figs. 6, 7, and 8 indicate that rapamycin activated the “forward” mode of the Na+/Ca2+ exchanger, since rapamycin decreased [Ca 2+]i in Thyrode buffer after its elevation with extracellular Ca2+, thapsigargin, and KCl (Figs. 6A, 7A, 8), a phenomenon that was not detected in Tyrode–choline buffer that inhibited the Na+/Ca2+ exchanger. Cardioprotective effect of KB-R7943 against hypoxia In order to show that rapamycin specifically activates the “forward” mode of the Na +/Ca 2+ exchanger and not the “reverse” mode, KB-R7943 (10 μM), an inhibitor of the “reverse” mode of the Na +/Ca 2+ exchanger (Iwamoto et al., 1996, 2007), was applied. Heart cultures were treated with 10 μM rapamycin and 10 μM KB-R7943 for 20 min before being subjected to hypoxia/reoxygenation. Under H/R conditions, LDH and CK levels in the medium were reduced by 40 and 70% by rapamycin and KB-R7943 (Fig. 9A, B). Although KB-R7943 did
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Fig. 5. Effect of rapamycin on Ca2+ accumulation by the SR. Ca2+ uptake into the SR was decreased by rapamycin in a dose-dependent (A) and time-dependent (B) manner. Data represent the mean ± SD (*p b 0.05, compared to control). Representative results from 6 experiments are shown.
Fig. 6. Rapamycin reversed [Ca2+]i elevation by increased extracellular [Ca2+]. A) Increase of extracellular calcium from 1.8 to 3.8 mM (arrow) caused an increase in diastolic [Ca2+]i level. An application of 10 μM rapamycin (arrow) reduced [Ca2+]i to baseline. The experiment was performed in Tyrode buffer containing 140 mM Na+ and 1.8 mM CaCl2. B) This phenomenon was not detected in Na+-free buffer (Tyrode–choline). The experiment was performed as in Fig. 6A but using Tyrode–choline buffer, in which rapamycin failed to decrease [Ca2+]i following extracellular calcium elevation. Representative results from 6 experiments are shown.
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Time (sec) Fig. 7. Rapamycin reversed [Ca2+]i elevation following thapsigargin treatment. A) Five μM thapsigargin increased diastolic [Ca2+]i and then the application of10 μM rapamycin (arrow) reduced [Ca2+]i in 140 mM Na+ Tyrode buffer (A), but not in Na+-free Tyrode– choline buffer (B). Representative results from 6 experiments are shown.
not inhibit the cardioprotective effect of rapamycin against hypoxic damage, KB-R7943 itself exhibited cardioprotection against hypoxic damage (Fig. 9A, B). This implies that either activation of the “forward” mode of the Na +/Ca 2+ exchanger or inhibition of the “reverse” mode of the Na +/Ca 2+ exchanger brings about protection of cardiac cells from hypoxia. Discussion
[Ca2+]i (410/490 nm)
We have demonstrated here that rapamycin protects rat heart cultures from hypoxic reoxygenation damage, as revealed by assays of LDH and CK leakage to the medium (Fig. 1A, B), by MTT absorbance (Fig. 1C) and by desmin immunostaining (Fig. 2). In addition, treatment of heart cultures with rapamycin following hypoxia attenuated the gradual increase of [Ca 2+]i level (Fig. 4A). Our results support the findings of Khan et al. (2006) in a Langendorff model of isolated adult mouse heart, in which rapamycin induced a potent preconditioning-like effect against myocardial infarction. However, whereas Khan et al. (2006) suggested that the protection is via
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Time (sec) Fig. 8. Rapamycin reversed [Ca2+]i elevation following depolarization. Heart cultures in Tyrode buffer were treated with 30 mM KCl, which causes depolarization and [Ca2+]i elevation (arrow). Treatment with rapamycin following depolarization with 30 mM KCl (arrow) decreased [Ca2+]i level. Representative results from 6 experiments are shown.
opening of mitochondrial KATP channels, we propose that rapamycin also activates the sodium calcium exchanger (NCX) in the “forward” mode, thereby reducing calcium overload. It has been generally accepted that Ca 2+ influx through the L-type 2+ Ca channel induces Ca2+ release from the SR via ryanodine channels, which leads to contraction (Eisner et al., 2009). Subsequently, Ca2+ is taken up by the SR Ca2+ATPase, and is also extruded from the cardiomyocyte by the NCX operating in the “forward” mode. Hence, NCX competes with the SR Ca 2+-ATPase in lowering [Ca 2+]i and thereby causing relaxation (Inserte et al., 2009). The NCX can also operate in a “reverse” mode, causing an influx of 1 Ca 2+ ion in exchange for 3 Na + ions during excitation. The “reverse” function of the NCX has been proposed to increase the content of [Ca2+]i in the cardiomyocytes. Preclinical studies have shown that pharmacological interventions targeted against sarcolemmal sodium ion transporters, such as inhibition of the "reverse" mode or activation of the “forward” mode of NCX, are effective in ameliorating heart failure (Baartscheer and van Borren, 2008). Rapamycin caused an immediate [Ca 2+]i decrease (Fig. 3). This reduction of [Ca 2+]i lasted as long as rapamycin remained in the dish. Upon washing, the cells resumed their previous [Ca 2+]i levels (Fig. 3). A similar effect of rapamycin was obtained in the membrane of smooth muscle cells (Weidelt and Isenberg, 2000). Whereas rapamycin reduced the amplitude of the Ca 2+ transients in rabbit heart, it increased their amplitude in mice (Raphael et al., 2008; Su et al., 2003). 45 Rapamycin also decreased ATP-dependent Ca 2+ uptake into the sarcoplasmic reticulum of cardiomyocytes via SERCA2a in a dose- and time-dependent manner (Fig. 5A, B). A similar inhibition of the SR Ca 2+ ATPase (SERCA1a) by rapamycin was observed in skeletal muscle sarcoplasmic reticulum (Bilmen et al., 2002) and in smooth muscle cells (Bultynck et al., 2000). Ischemia and reperfusion are associated with oxidative stress and intracellular Ca 2+ overload (Dhalla et al., 2007). Previous studies have shown different protective mechanisms against hypoxia/ reoxygenation-induced damage, and the involvement of SERCA2a in these mechanisms (El-Ani et al., 2007; Rickover et al., 2008; Shmist et al., 2005; Shneyvays et al., 2005; Zinman et al., 2006). An increase in diastolic [Ca 2+]i following elevation of extracellular calcium could be reversed by the subsequent application of rapamycin in Tyrode solution (Fig. 6A). Likewise, the application of thapsigargin, a SERCA2a inhibitor, increased diastolic [Ca 2+]i in heart cultures, and this increase was reversed by the immediate application of rapamycin (Fig. 7A). Similarly, depolarization caused [Ca 2+]i elevation, which was reduced by rapamycin (Fig. 8). In Na +-free Tyrode–choline buffer, rapamycin did not reduce [Ca 2+]i following exogenic Ca 2+ elevation, thapsigargin application, or following depolarization (Figs. 6B, 7B). Because Ca 2+ uptake into the SR was blocked by thapsigargin, we attribute the extrusion of cytosolic Ca 2+ to the possibility of stimulation of the sarcolemmal NCX by rapamycin. We further hypothesize that this reduction in [Ca 2+]i following exogenic Ca 2+ elevation (Fig. 6A) is probably via stimulation of the “forward” mode of NCX by rapamycin to extrude Ca 2+ out of the cell. Stimulation of NCX is one of the mechanisms for protecting the heart from ischemic insults (Chen et al., 2006; Yeung et al., 2007). The NCX in cardiac myocytes usually operates in the “forward” mode. During the action potential, the NCX can also operate in the “reverse” mode, in which Ca 2+ enters the cells. Conditions such as ischemia/reperfusion-induced elevation of cytosolic calcium can cause heart cell damage. Recently, it has been reported by Yeung et al. (2007) that chronic intermittent hypoxia alters Ca 2+ handling in rat cardiomyocytes by augmenting NCX and ryanodine receptor activity in ischemia-reperfusion by protein kinase A (PKA)- and protein kinase C (PKC)-dependent protection. Despite the impressive inhibition by rapamycin of Ca 2+ uptake into the SR, rapamycin attenuated the amplitude of calcium transients (Fig. 3) and reduced the increase in [Ca 2+]i as a result of hypoxia (Fig. 4A), suggesting a mechanism to prevent intracellular Ca 2+
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Fig. 9. Effect of rapamycin (Rp) and KB-R7943 on LDH and CK release to the medium following hypoxia and reoxygenation (H/R). Heart cultures were treated with 10 μM rapamycin (Rp) and 10 μM KB-R7943 (KBR) 20 min before being subjected to H/R. Rapamycin and KB-R7943 decreased LDH and CK release after H/R. (A, B) [* p b 0.05 compared with H/R, ** p b 0.05 compared with control]. Data represent the mean ± SD. Representative results from 6 experiments are shown.
overload. According to our results, this prevention of Ca 2+ overload is not via SERCA2a, but probably via activation of NCX. Our results suggest that rapamycin induces cardioprotection against hypoxic damage by removing Ca2+ from the cytosol through NCX activation. In addition, KB-R7943 – an inhibitor of NCX in the reverse mode (Iwamoto et al., 1996; Yoshitomi et al., 2005) – on the one hand protected the cells from hypoxia, but on the other hand, did not inhibit the cardioprotective effect of rapamycin following hypoxia (Fig. 9A, B). Thus, both treatments – rapamycin and KB-R7943 – prevent intracellular Ca2+ accumulation under hypoxic conditions that leads to activation of proteolytic enzymes and cell damage. Based on the current findings, we suggest that rapamycin induces cardioprotection against hypoxic/reoxygenation damage in heart cultures by stimulating NCX to extrude Ca 2+ from the cardiomyocytes. Thus, rapamycin preserves Ca 2+ homeostasis and prevents Ca 2+ overload via extrusion of surplus Ca 2+ across the sarcolemma, thereby protecting the cells from hypoxic reoxygenation stress. Conflict of interest statement The authors declare that there are no conflicts of interest related to this manuscript.
Acknowledgments We thank A. Isak for technical support. We also thank Sharon Victor for helping to prepare this manuscript for publication. This work was supported in part by an ISF grant no. 876/05 and through the generous support of the Adar Program for the Advancement of Research in Heart Function at Bar-Ilan University. The sponsors were not involved in any way in the making of this paper or the decision to submit it for publication. References Baartscheer A, van Borren MM. Sodium ion transporters as new therapeutic targets in heart failure. Cardiovasc Hematol Agents Med Chem 2008;6:229–36. Bilmen JG, Wootton LL, Michelangeli F. The inhibition of the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase by macrocyclic lactones and cyclosporin A. Biochem J 2002;366:255–63. Bose AK, Mocanu MM, Carr RD, Yellon DM. Myocardial ischaemia-reperfusion injury is attenuated by intact glucagon like peptide-1 (GLP-1) in the in vitro rat heart and may involve the p70s6K pathway. Cardiovasc Drug Ther 2007;21 253–25. Bultynck G, De Smet P, Weidema AF, Ver Heyen M, Maes K, Callewaert G, et al. Effects of the immunosuppressant FK506 on intracellular Ca2+ release and Ca2+ accumulation mechanisms. J Physiol 2000;525(Pt 3):681–93.
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