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Acute vincristine pretreatment protects adult mouse cardiac myocytes from oxidative stress
Journal of Molecular and Cellular Cardiology 43 (2007) 327 – 336 www.elsevier.com/locate/yjmcc
Original article
Acute vincristine pretreatment protects adult mouse cardiac myocytes from oxidative stress Kanu Chatterjee a,c,e,1 , Jianqing Zhang b,1 , Norman Honbo b , Uschi Simonis f , Robin Shaw a,d , Joel S. Karliner b,c,d,⁎ a
e
Cardiology Division, University of California, San Francisco, USA b Cardiology Section, VA Medical Center, San Francisco, USA c Department of Medicine, University of California, San Francisco, USA d Cardiovascular Research Institute, University of California, San Francisco, USA Chatterjee Center for Cardiac Research, University of California, San Francisco, USA f Department of Chemistry and Biochemistry, San Francisco State University, USA Received 24 May 2007; accepted 12 June 2007 Available online 21 June 2007
Abstract Vincristine is a chemotherapeutic agent that disrupts microtubules. We noted that paclitaxel (Taxol), which stabilizes microtubules, protected cultured adult mouse cardiac myocytes from oxidative stress induced by H2O2. We hypothesized that vincristine, which disrupts microtubules, should have the opposite effect. To our surprise, we found that pretreatment with concentrations of vincristine ranging from 30 to 120 μmol/L for 60 min preserved myocyte viability and morphology after incubation with 30 μmol/L of H2O2 for 35 min as measured by trypan blue exclusion. The cardioprotective effects of vincristine were also observed during prolonged hypoxia. With continuous exposure to vincristine, survival lasted for as long as 24 h, but longer periods of exposure up to 42 h resulted in extensive cell death. Despite microtubule disruption evidenced on deconvolution microscopy, vincristine activated a prosurvival pathway resulting in increased phosphorylation of Akt, ERK and GSK-3β and in reduced cytochrome C release into the cytosol. Pharmacological inhibitors of Akt and Erk attenuated the cardioprotective effect of vincristine. We conclude that short-term pretreatment with vincristine exerts dramatic protective effects in cultured adult mouse myocytes subjected to acute oxidative stress. Despite causing microtubule disruption, vincristine initiates a prosurvival signaling pathway. As vincristine and doxorubicin are often used in conjunction to treat patients, it is possible that vincristine could be used to modify the cardiotoxicity of doxorubicin. © 2007 Elsevier Inc. All rights reserved. Keywords: Vincristine; Cardiac myocytes; Cell culture; Hydrogen peroxide; Oxidative stress; Hypoxia; Cardioprotection; Signal transduction
1. Introduction Vincristine is a member of the vinca alkaloid family and is widely used in chemotherapy for various malignancies [1,2]. The cytotoxicity of vinca alkaloids is based on well-established pharmacologic properties that include microtubule depolymerization and arrest of cell cycle division during metaphase [2]. As microtubules are integral components of the mitotic spindle, interference with microtubule dynamics by vinca alkaloids such ⁎ Corresponding author. Cardiology Section (111C5), VA Medical Center, 4150 Clement St., San Francisco, CA 94121, USA. Fax: +1 415 750 6959. E-mail address: [email protected] (J.S. Karliner). 1 Both authors contributed equally to this work. 0022-2828/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.yjmcc.2007.06.005
as vincristine promotes not only cell division arrest but also apoptosis [3]. However microtubule-interfering agents like vincristine may also affect non-neoplastic cells because microtubules are necessary to maintain other cellular functions such as membrane and cellular scaffolding, intracellular transport of organelles and proteins, and transmission of signals initiated at cell surface receptors [3]. Such effects on nonmalignant cells constitute mechanisms for potential cardiotoxicity of these agents. Among chemotherapeutic agents, anthracyclines have been well documented to produce cardiotoxicity and cardiomyopathy in patients receiving these drugs for treatment of various malignancies, but the use of vinca alkaloids in isolation is usually not associated with cardiotoxicity although neurotoxi-
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city is not infrequent [4]. However, one report noted that the vinca alkaloid vinblastine was cardiotoxic to isolated hearts subjected to ischemia/reperfusion injury [5]. Paclitaxel (Taxol) is also widely used for the chemotherapy of malignancies, but in contrast to vincristine it stabilizes microtubules, and in the same study paclitaxel exhibited a cardioprotective effect in isolated perfused rat hearts [5]. As Taxol is a microtubule-stabilizing agent and vincristine is a microtubule-destabilizing agent similar to vinblastine, we hypothesized that vincristine would not exert a cardioprotective effect and might in fact cause cardiac toxicity as noted above. To test this hypothesis, we asked the following questions: (1) Is vincristine toxic to adult mouse cardiac myocytes, and if so, at what level? (2) What are the effects of vincristine on adult mouse cardiac myocytes subjected to chemical and hypoxic oxidative stress? (3) What is the mechanism of these effects? 2. Experimental procedures 2.1. Materials Vincristine (VCR) and paclitaxel were purchased from Sigma-Aldrich (St. Louis, MO). Antibodies against cytochrome C were purchased from BD Biosciences (Franklin Lakes, NJ), and antibodies against GAPDH were obtained from Abcam (Cambridge, MA). Antibodies against phosphorylated and total Akt, ERK and GSK-3β were purchased from Cell Signaling Technology (Danvers, MA). LY294002 and PD98059 were obtained from Biomol (Plymouth Meeting, PA). Mouse laminin and pertussis toxin were purchased from Invitrogen (Carlsbad, CA). 2.2. Cell culture Male C57B1/6 mice (19–25 g) from Charles River Laboratories (Hollister, CA) were used for the preparation of myocytes. Mice received standard rodent chow and water ad libitum. The study was approved by the Institutional Animal Care and Use Committee of the San Francisco Veterans Affairs Medical Center. Cardiac myocytes were cultured according to the method described by Zhou et al. with modifications [6]. Following anticoagulation with heparin (50 units, i.p.), mice were euthanized with sodium pentobarbital (200 mg/kg, i.p.). After excision, hearts were cannulated via the aorta for retrograde perfusion for 2 min with Ca2+-free isolation buffer containing (mmol/L) NaCl 120, KCl 5.4, MgSO4 1.2, NaH2PO4 1.2, glucose 5.6, NaHCO3 4.6, HEPES 10, taurine 5, 2,3-butanedione monoxine (BDM) 10. The hearts were then perfused for approximately 9 min with the same isolation buffer which in addition contained 50 μmol/L CaCl2 and 1.5 mg/mL collagenase II (Worthington, Lakewood, NJ). Ventricles were then separated by trimming the other cardiac and major vascular structures. Ventricular fibers were teased apart with forceps, pipetted in collagenase II buffer, filtered through a cell dissociation sieve, and centrifuged at 40×g for 1 min. Cardiac myocyte pellets were resuspended in isolation buffer serially supplemented with 100, 250, 500 and 1200 μmol/L CaCl2.
The final myocyte pellets were resuspended in minimal essential medium (MEM) containing Hanks' buffered salt solution (HBSS), 10 μg/mL penicillin, 1.5 μmol/L vitamin B12, 2.5% bovine calf serum (BCS), and 10 μmol/L BDM. Isolated myocytes were then plated on dishes coated with 10 μg/L laminin and in media containing 2.5% serum and 10 mM BDM at a density of 50 rod-shaped cells/mm2 for 2 h. Culture media were changed to 1 mM BDM without serum for overnight incubation at 37 °C in a humidified atmosphere of 1% CO2 and air. Cells were viable at pH 7.2 for 72 h. Experimental protocols were performed the day following myocyte isolation and plating. 2.3. Measurement of cell survival After overnight incubation, media were changed to remove BDM. Myocytes were treated with vincristine or vehicle for a minimum of 60 min or for as long as 42 h. H2O2 was routinely prepared as a 10 mM stock solution. In each experiment, 3 μL of this solution were added to 1 mL of buffer in each dish to yield a concentration of 30 μmol/L. Cell numbers were kept constant for each experiment at ∼ 50,000 cells/35 mm culture dish. To assess the effects of chemical oxidative stress, myocytes were exposed or not to 30 μmol/L H2O2 for 35 min in the presence or absence of vincristine. In some experiments, pretreatment with pharmacological inhibitors of signaling molecules was for 30 min in the presence or absence of vincristine. In these experiments, myocytes were treated with vincristine (30 μmol/L) or vehicle for 60 min. To induce hypoxic stress, media were changed twice with glucose-free MEM/HBSS without BDM or bovine fetal serum that had been equilibrated in either normoxia in a 37 °C humidified CO2 incubator (1% CO2, Forma Scientific, Marietta, OH) or in hypoxia in a Bactron I Anaerobic Chamber (99% N2/1% CO2, Sheldon Manufacturing, Inc., Cornelius, OR). The pretreated myocytes were then incubated in the same MEM medium in normoxia or hypoxia for approximately 4 to 5 h prior to induction of hypoxia. Survival of the cultured myocytes was determined by staining cells in tissue culture dishes for 10 min at room temperature with trypan blue solution (Gibco, Grand Island, NY) diluted to a final concentration of 0.04% (w/v). When cell membranes are irreversibly damaged, trypan blue, which is an anionic dye, is taken up by dead cells and binds to nuclei [7,9]. The trypan blue exclusion (TBE) assay is a widely accepted and validated method to determine cell survival and changes in cell morphology in experimental models, and has previously been used in our laboratory [8,9]. For the calculation of viability and morphologic changes, myocytes were visualized at 100× magnification by microscopy. Cells that excluded trypan blue (TBE) were considered viable. Healthy rod-shaped myocytes (rods) were identified when the length/width ratio was N3:1 as previously described [10]. Contracted cells were defined when the length/width ratio was b 3:1. Trypan blue-positive cells were identified when the trypan blue was present intracellularly irrespective of whether the cells were rod-shaped or contracted [10]. Morphologic
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changes were measured by determining the number of rods relative to all TBEs (rods and contracted cells) in ten fields/ dish. The percent survival and viability were calculated as follows: Percent survival Total number of H2 O2 TBE cells in 10 fields=dish ¼ Total number of rods and contracted cells in the same 10 fields 100
Percent viability Total number of rod−shaped TBEs in 10 fields=dish ¼ Total number of TBE rods and contracted cells in the same 10 fields 100
The second technique for assay of cell viability and death was employed using the Live/Dead Viability/Cytotoxicity Assay kit (Molecular Probes, Carlsbad, CA). This method is also well established and validated and has previously been used in our laboratory [11]. It is based on determination of intracellular esterase activity and plasma membrane integrity. The polyanionic dye calcein, which fluoresces green, is retained exclusively by live cells. Ethidium homodimer enters the cells only when the plasma membrane is damaged, and after binding to nucleic acids, it exhibits red fluorescence. Thus this method also allows assessment of apoptotic cell death [12,13]. For these experiments, myocytes were grown on chamber slides. The culture medium was replaced with 2 μmol/L calcein acetoxymethyl ester and 4 μmol/L ethidium homodimer-1. Live cells were recognized by the intense uniform green fluorescence of calcein and the dead cells by red fluorescence of the ethidium homodimer [11]. 2.4. Western blot analysis Lysates were prepared from cultured myocytes and were subjected to SDS–PAGE. Selective primary antibodies, appropriate secondary antibodies conjugated with horseradish peroxidase, and enhanced chemiluminescence (ECL, Amersham Biosciences, Piscataway, NJ) were used for detection of the proteins of interest. Densitometry was used for quantitative assessment of the signals. 2.5. Immunostaining and deconvolution imaging Myocytes were fixed at − 20 °C with methanol for 5 min, permeabilized with 0.2% Triton X-100 in PBS, and blocked in PBS + 0.1% Triton X-100 + 5% goat serum or donkey serum (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). Mouse anti-tubulin primary antibodies were diluted into PBS + 0.1 % Triton X-100 + 2% serum (1:1000, Sigma-Aldrich, St. Louis, MO) and incubated at room temperature for 1 h. Coverslips were washed in PBS, followed by incubation with goat or donkey anti-mouse secondary antibodies conjugated to Alexa 488 or Cy3 (Jackson ImmunoResearch Laboratories, 1:1000 dilution in PBS + 0.1% Triton X-100 + 1% serum) with 1 g/mL Hoechst 33342 (Invitrogen, Carlsbad, CA) for 1 h at
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room temperature. Cells were mounted on microscope slides with GelMount aqueous mounting medium (Biomeda Corp., Foster City, CA). Fixed cells were imaged with a Nikon TE2000-U inverted fluorescence microscope equipped with a Photometrics Coolsnap HQ CCD camera and MetaVue software (Molecular Devices, Foster City, CA). For deconvolution processing, whole cell stacks with 100 nm vertical spacing were acquired. For tubulin, the stacks were deconvoluted using 3D blind deconvolution using Autoquant software. 2.6. Creatine kinase (CK) CK depletion from the cells was measured with a commercially available kit. (Stanbio CK-NAC, Boerne, TX). 2.7. NMR experiments The 1H NMR spectra were acquired at 310 K (36 °C) on an Avance DRX 300-MHz spectrometer from Bruker BioSpin operating at 299.9197 MHz and were referenced to D2O resonating at 4.800 ppm. The spectra were acquired using the one-pulse sequence zg available in the BRUKER pulse sequence library. Typically, the FIDs were acquired using a relaxation delay of 1 s, a spectral width of 7500 Hz, a data size of 32 K data points, and a pulse width of 50° (90° pulse = 11.0 μs) and 512 acquisitions depending on sample concentration. The free induction decays (FIDs) were apodized with an exponential multiplication using a line-broadening factor of 0.10 Hz and phase-corrected to yield the spectra. 2.8. Statistical analysis All results are reported as mean ± S.E.M. Comparisons were made by one-way analysis of variance (ANOVA). Post hoc analysis was performed using the Student–Newman–Keuls test. P b 0.05 was considered significant. 3. Results 3.1. Viability assays In initial experiments, we found that pretreatment with the microtubule-stabilizing chemotherapeutic agent paclitaxel (Taxol) enhanced survival of non-dividing cultured adult mouse cardiac myocytes in response to chemical oxidative stress (H2O2) as measured by the percentage of live cells determined by trypan blue exclusion (Fig. 1). Based on these observations, we wondered whether the microtubule-disrupting agent vincristine, which is commonly used in cancer chemotherapy, would have the opposite effect. Results of a trypan blue exclusion assay in myocytes treated or not with vincristine before H2O2 exposure in a representative experiment are illustrated in Fig. 2A. Rod-shaped and contracted cells that excluded trypan blue were considered as live, while cells that failed to exclude trypan blue were regarded as dead regardless of morphology. In the control dish, the majority of the cells were rod-shaped and there were only a few contracted and trypan
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Fig. 1. Effects of Taxol pretreatment on survival as measured by the trypan blue assay in cultured adult mouse cardiac myocytes in response to chemical oxidative stress. Cells were pretreated with 30 μmol/L of H2O2 for 35 min or not. Taxol was dissolved in 0.1% DMSO (final concentration) and pretreatment was for 60 min. Numbers in bars show the number of experiments for each condition. *P b 0.05 vs. vehicle alone and Taxol. Veh = vehicle.
blue-positive cells. Following exposure to H2O2, there was a dramatic decrease in the number of rods and a substantial increase in the trypan blue-positive cells. After pretreatment with vincristine and following exposure to H2O2, we were surprised to find that the majority of the cells were rod-shaped and only a few cells were contracted and trypan blue-positive. These results, which were replicated numerous times (see Figs. 4 and 5), suggested that pretreatment with vincristine improved survival of cultured adult mouse cardiac myocytes exposed to chemical oxidative stress. An additional assessment of viability, the “Live/Dead” assay, illustrated in Fig. 2B, confirmed these observations. Without pretreatment with vincristine or H2O2, (control), the proportion of viable cells (green fluorescent) was much higher than the dead cells (red fluorescent). Following exposure to H2O2 and without pretreatment with vincristine the proportion of viable cells markedly decreased and that of dead cells markedly increased. Following vincristine pretreatment, however, cell survival was largely preserved as there were many more green than red fluorescent cells. Fig. 3A depicts the effects of increasing concentrations of vincristine pretreatment for 60 min on myocyte survival as measured by the trypan blue exclusion assay following exposure to H2O2. Cell counts are expressed as the percent change relative to control values which were set at 100%. Following H2O2 exposure and without vincristine pretreatment, the proportion of cells that excluded trypan blue decreased to an average of 53%. After pretreatment with 30 to 120 μmol/L of vincristine, the proportion of cells that excluded trypan blue remained at 90% to 96%, consistent with almost complete protection from chemical oxidative stress. Pretreatment with vincristine also preserved adult mouse cardiac myocyte morphology against oxidative stress. As shown in Fig. 3B, the percentage of rod-shaped myocytes relative to the total number of cells that excluded trypan blue in the control cultures was N 80%. Vehicle pretreatment followed by H2O2 exposure decreased the proportion to ≈ 25%. With
vincristine pretreatment, the percentage of rod-shaped myocytes was reduced to about 60% after H2O2 exposure, suggesting that pretreatment with vincristine improved myocyte viability by more than 2-fold after exposure to chemical oxidative stress. To determine whether vincristine also is protective during oxygen deprivation, a key component of ischemia, cultured cardiomyocytes were exposed to hypoxia after the pretreatment with vincristine. As shown in Fig. 4, preincubation of adult mouse cardiac myocytes with 30 μmol/L vincristine for 1 h reduced hypoxia-induced cell death. After 5 h of hypoxia, the survival of vincristine-pretreated myocytes was significantly improved in comparison to vehicle controls (Fig. 4). To determine the effects of treatment duration with vincristine on survival and morphologic changes, myocytes were incubated for 6 to 42 h. Even with the highest dose used (1 mmol/L), there was no decrease in survival after 6 or 24 h. However, as shown in Fig. 5A, after prolonged exposure to vincristine for an average of 42 h survival decreased progressively with increasing concentrations to a maximum of 100 μmol/L. The magnitude of CK activity in the myocytes progressively declined with increasing concentrations of vin-
Fig. 2. (A) Typical results of a trypan blue exclusion assay in a representative experiment. H2O2 at 30 μmol/L for 35 min was associated with an increased number of trypan blue positive cells and a decrease in rod-shaped cells compared to the vehicle control panel. Pretreatment for 60 min with 60 μM vincristine dissolved in H2O was associated with fewer trypan blue-positive cells and persistence of rod-shaped cells, indicating increased survival and viability of the cultured adult mouse cardiac myocytes. These results were replicated numerous times (see text and Figs. 3 and 4). VCR = vincristine; Veh = vehicle. (B) Treatments were the same as in panel A. Live/Dead assay showing that pretreatment with 60 μmol/L vincristine for 60 min increased survival (calcein ester-positive/green fluorescence) and decreased the proportion of dead cells (ethidium-positive/red nuclei) after exposure to 30 μmol/L H2O2 for 35 min. This experiment was replicated four times. VCR = vincristine; Veh = vehicle; EthD-1 = ethidium homodimer-1.
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Fig. 4. Pretreatment with 30 mmol/L vincristine for 60 min significantly increased the total survival of cardiomyocytes during hypoxia. Numbers in bars show the number of experiments for each condition. VCR = vincristine; Veh = vehicle; Nx = normoxia; Hx = hypoxia.
pretreated with vincristine in the presence of specific inhibitors of PI3-K/Akt and MEK/ERK activation: LY294002 and PD98059, respectively. Cells were exposed to H2O2 after washout of vincristine and the inhibitors. Survival of cardiomyocytes was determined by trypan blue exclusion. As shown in Fig. 6, inhibition of PI3-K/Akt and ERK each significantly reduced the percentage of myocytes protected by vincristine treatment. These findings suggest that more than one prosurvival
Fig. 3. (A) Effect of increasing concentrations of vincristine pretreatment for 60 min on the proportion of surviving myocytes following exposure to 30 μmol/L H2O2 for 35 min. n = number of experiments per data point. *P b 0.05 vs. vehicle control. Con = control; Veh = vehicle; VCR = vincristine. (B) Effect of increasing concentration of vincristine pretreatment for 60 min on the proportion of rodshaped myocytes following exposure to 30 μmol/L H2O2 for 35 min. n = number of experiments per data point. *P b 0.05 vs. vehicle control.
cristine given over 42 h and paralleled changes in survival (Fig. 5B). Next we asked how long cardioprotection persisted after 1 h of pretreatment with vincristine. Cultured myocytes were exposed to H2O2 after washout of vincristine and survival was measured at 1, 2, 4, and 6 h. At 1 and 2 h, survival, as determined by trypan blue exclusion, was maintained; but at 4 and 6 h, the cardioprotective effects of vincristine were no longer observed (data not shown). 3.2. Mechanism of vincristine-induced resistance to oxidative stress To identify possible components of a protective pathway stimulated by vincristine, we examined the survival of myocytes
Fig. 5. Effects of vincristine pretreatment for an average of 42 h on survival (A) and creatine kinase depletion (B) with increasing concentrations of vincristine. n = number of experiments per data point. *P b 0.05 vs. control. Con = control.
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signaling pathway is involved in cardioprotection by vincristine. Overnight preincubation with 100 μg/mL pertussis toxin had no significant effect on the prosurvival effect of vincristine (n = 6, data not shown), indicating that a receptor-mediated Gi-coupled mechanism was not involved in vincristine signaling. To verify that vincristine activated a prosurvival signal transduction cascade, we initially performed time and concentration–response experiments to identify the optimal conditions for measuring signaling responses. The magnitude of Akt, ERK and GSK-3β phosphorylation was proportional to the concentration of vincristine used and reached a plateau at 30 to 100 μmol/L. Figs. 7A–C illustrate typical effects of vincristine pretreatment on phosphorylation of Akt (S473), ERK/MAPK and GSK-3β. Compared to vehicle controls, phosphorylation of each of these molecules increased several-fold. The increased phosphorylation of Akt and ERK suggests an activation of prosurvival signals resulting from vincristine pretreatment, and is consistent with improved survival conferred by vincristine in the cultured cardiac myocytes (Fig. 6). The increased phosphorylation of GSK-3β is consistent with inhibition of its adverse effects on cell demise, as previously reported [14]. In parallel with the effects of LY294002 and PD98059 on myocyte viability shown in Fig. 6, we observed that phosphorylation of Akt and GSK-3β by vincristine was abrogated by LY294002 pretreatment (Figs. 7A and B). These data indicate that PI3-K is required for Akt S473 phosphorylation. ERK activation was inhibited completely by PD98059 but not by LY294002 as expected (Fig. 7C).
western analysis. Myocyte cytosolic extracts were prepared after exposure to H2O2 with and without pretreatment with vincristine and blots were probed with a cytochrome C specific antibody. Compared to H2O2 alone, vincristine pretreatment resulted in a significant decrease of mitochondrial cytochrome C release, suggesting reduction of oxidative stress and inhibition of mitochondrial permeability transition (Fig. 6D). 3.4. Vincristine and H2O2: is there a direct interaction? Using 1H NMR spectroscopy, we observed no alteration in the spectra of vincristine when it was incubated with H2O2 in the presence or absence of cardiomyocytes. Thus our experiments do not support a direct interaction of vincristine and H2O2. 3.5. Microtubular architecture Fixed cardiomyocyte immunostaining with deconvolution imaging provides a planar transverse view of microtubule organization (Fig. 8). In control cells, microtubules are organized along the longitudinal axis in keeping with expected myocardial anisotropy. In contrast, following pretreatment with vincristine the microtubules are highly disrupted with clumping and little discernable organization. Thus it appears that vincristine significantly disrupts the microtubules and yet still exerts cardioprotective effects in cultured adult mouse cardiac myocytes.
3.3. Cytochrome C release
4. Discussion
To determine the effects of vincristine on mitochondrial transition pore function, we measured cytochrome C release by
The main and unexpected finding of the present study is that despite microtubule disruption short-term pretreatment with vincristine exerts dramatic protective effects in cultured adult mouse cardiac myocytes subjected to acute oxidative stress. The mechanisms involved include activation of prosurvival signaling pathways. 4.1. Myocyte survival
Fig. 6. Pharmacological inhibitors of PI3K and Erk pretreatment abolish the vincristine protective effect on myocytes survival following exposure to 30 μmol/L H2O2 for 35 min. Myocytes were pretreated with 10 μmol/L of LY294002 (LY) or 30 μmol/L of PD98059 (PD) for 30 min before followed by vincristine treatment or vehicle. Both the control value (asterisk) and the vincristine-treated cells (cross) subjected to hydrogen peroxide show significantly greater survival than all other sets of cells; none of the other values [H2O2 + either vehicle, LY or PD], or [vincristine + H2O2 and either LY or PD], are significantly different from each other.
We determined cell survival by two different methods, the trypan blue exclusion assay and the Live/Dead assay. Initially we used the trypan blue assay to determine the effects of vincristine pretreatment on survival and viability of the cultured adult mouse myocytes exposed to H2O2. The results demonstrate that vincristine pretreatment for only 1 h exerts substantial protection of cardiac myocytes exposed to this chemical oxidative stress. With 30 to 120 μmol/L of vincristine pretreatment, myocyte salvage doubled and approximated control values (Fig. 3A). Furthermore, cell viability as measured by preserved morphology also improved 2-fold with vincristine pretreatment (Fig. 3B). The two-color fluorescence assay technique (Live/Dead assay), which was performed in a series of separate experiments, yielded virtually identical results. Thus, two independent methods of measuring myocyte viability, the TBE method and the Live/Dead approach, verified the prosurvival effects of vincristine.
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Fig. 7. Panels A–C show changes in phosphorylation of Akt, ERK and GSK-3β in cultured adult mouse myocytes after 30 min of pretreatment with 60 μmol/L vincristine with or without pretreatment with the pharmacological inhibitors. The upper panels show representative western blots and the lower panels show the foldincrease in signal intensity produced by vincristine. Numbers in bars show the number of independent experiments for each of the signals depicted. *P b 0.05 vs. all other values; **Pb0.05 vs. Con and PD. Con = vehicle; VCR = vincristine. Panel D shows western blot analysis of cytochrome C release in response to 30 μmol/L H2O2 for 35 min. Pretreatment with 60 μmol/L vincristine for 60 min decreased cytochrome C release into the cytosol. Con = control; VCR = vincristine. Equal loading was verified with anti-GAPDH antibody. *P b 0.05 vs. control.
Most of the experiments were done with quiescent myocyte cultures. After harvest and plating, myocytes were incubated for 24 h before vincristine treatment followed by H2O2 exposure. However, the same protective effect of vincristine was observed with freshly isolated cardiomyocytes (data not shown). In this
study, pretreatment with vincristine also provided protective effects in cultured adult mouse cardiac myocytes subjected to hypoxia (Fig. 4). The magnitude of protection from hypoxia was similar to that found in cells subjected to chemical oxidative stress. These findings suggest that pretreatment with
Fig. 8. Deconvolution microscopic studies showing effects of pretreatment with 60 μmol/L vincristine for 60 min on microtubular structure. Vincristine pretreatment is associated with microtubular disruption. Nuclei are shown in blue. For details see text.
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vincristine has the potential to provide cardioprotection irrespective of type of oxidative stress imposed. Our culture protocol includes overnight incubation in medium containing BDM at 1 mM final concentration which substantially increases the number of healthy and functional ventricular myocytes available for experimentation on the morning after isolation. For methods requiring homogeneous populations of adult mouse ventricular myocytes to be functional for at least 24 h, other laboratories have published protocols in which BDM is present throughout heart tissue digestion and Ca2+ restoration. Zhou et al. [15] demonstrated the utility of this cell culture paradigm for cellular genetic physiology. Hilal-Dandan et al. [16] confirmed that adult mouse cardiac myocytes remain excitable and useful for contractile and calcium measurements [17]. Sollott et al. [18] demonstrated that treatment of adult rat ventricular myocytes with 1 mM BDM had no significant effect on myocyte length and [Ca2+]i at rest and during electrical stimulation. It should be emphasized that we routinely incubate cardiac myocytes in a normoxic medium that does not contain BDM on the morning following isolation for 2 h prior to the addition of pharmacological reagents. Under the conditions studied, cells incubated with vincristine were viable for at least for 24 h even when myocytes were exposed to a high concentration of the drug (100 μmol/L). However, very prolonged exposure, for example for up to 42 h, induced cardiotoxicity. When vincristine was washed out after 1 h of pretreatment, the cardioprotective effects in response to oxidative stress lasted only 2 h. Thus it appears that, in order to maintain cardioprotection, continuous vincristine treatment is necessary. In contrast to our findings, Skobel and Kammermeier reported that vinca alkaloids can enhance cardiac myocardial damage [5]. In isolated perfused rat hearts subjected to ischemia/reperfusion injury, vinblastine, which has similar therapeutic and biologic properties, increased rat cardiac myocyte death as determined by creatine kinase release and TBE assays in paraffin sections [5]. The reason for this discrepant result remains unclear. In their study, Taxol, a microtubule-stabilizing agent and cytochalasin D, an actin-destabilizing agent, decreased the proportion of irreversible cell injury, suggesting that microtubule-destabilizing effects of vinblastine may be the mechanism of its observed adverse effects. Both vinblastine and vincristine disrupt microtubular architecture by binding to tubulin dimers at a site located on the beta subunits at the plus ends of microtubules [19,20]. Our deconvolution microscopic studies demonstrated that vincristine pretreatment indeed is associated with microtubular disruption. Despite this adverse response, we noted that vincristine pretreatment produced dramatic cardioprotective effects. Thus microtubule disruption in these nondividing cells does not appear to be the explanation for the discrepant results between our study and that of Skobel and Kammermeier [5]. It has been reported that the oxidative breakdown of vincristine by myeloperoxidase is H2O2-dependent [21]. Degradation of vincristine was not seen when vincristine was incubated with H2O2 in the absence of enzyme or when vincristine was incubated with the enzyme in the absence of H2O2 [21]. Using
1
H NMR spectroscopy, we observed no alteration in the spectra of vincristine when it was incubated with H2O2 in the presence or absence of cardiomyocytes. Thus our experiments do not support a direct interaction of vincristine and H2O2. During oxidative stress, microtubules may have a protective structural effect. Therefore, in intact beating hearts subjected to ischemia, the intensity of any microtubular structural disruption may be one determinant of the extent of myocardial injury. However, in isolated quiescent myocytes, our study has revealed that disruption of microtubules is associated with a surprising and marked viability advantage concurrent with activation of prosurvival biochemical changes. Determining how this biochemical advantage might be linked to microtubule disruption or if it is independent of this response, and whether it occurs in isolated hearts and intact animals is an important topic of future investigation. 4.2. Signal transduction Akt activation is mediated mainly through phosphorylation of S473, and is regulated through a PI3-K-dependent mechanism [22]. PI3-K/Akt signaling is involved in cell survival in many cellular systems as well as in adult cardiomyocytes [23]. Our results with LY294002 show that Akt activation is PI3-Kdependent in mouse cardiomyocytes (Fig. 7A). It has been reported that the beneficial effects of Akt activation on cardiac myocyte survival during oxidative stress is at least partly due to phosphorylation of the downstream signaling molecule GSK3β [14]. Our Western blot experiments are consistent with these observations and indicate that pretreatment of cultured adult mouse myocytes with vincristine is associated with increased phosphorylation of GSK-3β (Fig. 7B), thereby leading to its inactivation and enhancing cell survival [14]. As noted earlier, there was increased phosphorylation of Akt and ERK which may also promote cell survival by preventing mitochondrial dysfunction [23]. Conversely, inhibition of Akt and ERK activation attenuated their cardioprotective effect. Thus activation of these survival pathways appears to be necessary in mediating the cardioprotective effect of vincristine. 4.3. Cytochrome C release It is well-recognized that cytochrome C release from mitochondria and impairment of respiratory complex function by activation of mitochondrial permeability transition causes cell injury [24]. Reactive oxygen species (ROS) generated by respiratory complex III during ischemia-induced oxidative stress depletes mitochondrial cardiolipin, thereby permitting cytochrome C release into cytosolic compartments [25]. In the present study, pretreatment with vincristine decreased cytochrome C release induced by chemical oxidative stress by exposure to H2O2. Although we did not measure ROS in this study, H2O2 is known to result in ROS generation [26]. Therefore our findings suggest that one additional potential mechanism of cardioprotection activated by short-term vincristine treatment is diminished ROS production resulting in decreased mitochondrial permeability transition.
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4.4. Study limitations Many prior studies reporting signaling responses initiated by vincristine have been performed. Almost all of these are in tumor cell lines and have described the apoptosis-inducing effects of vincristine. Examples of reported pro-apoptotic signals induced by vincristine in vitro include JNK/SAP, ras and ASK1 [27], Bcl2 phosphorylation [28], p53 and p21 [29], ERK and p38 [30], NF-κB/IκB [31], and RhoA [32]. All of these responses occur in conjunction with disruption of the dynamics of microtubule assembly, but how microtubular disarray activates these pathways has yet to be defined. Our results are novel as they unequivocally demonstrate that in the experimental model employed, short term pretreatment with vincristine provides survival and viability benefit in primary cultures of adult cardiac myocytes for at least 24 h following exposure to chemical oxidative stress. To the best of our knowledge, the prosurvival effect of vincristine in these cells has not been previously reported, and represents a new model of cardioprotection. In this connection, recent evidence is consistent with our observations that vincristine can stimulate non-apoptotic signals. For example, in a nontransformed murine fetal liver cell line it was reported that vincristine activated mTOR, which facilitates Akt activation [33]. Thus, further studies to elucidate the mechanisms by which vincristine triggers myocyte survival are warranted. The clinical implications of this study suggest the possibility that vincristine, which is often used in conjunction with doxorubicin, could abrogate the cardiotoxicity of the latter drug. Studies are currently underway in our laboratory to test this hypothesis. Acknowledgments Supported by the Foundation for Cardiac Research and PO1 HL 68738 from the National Heart, Lung, and Blood Institute, National Institutes of Health.
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Original article
Acute vincristine pretreatment protects adult mouse cardiac myocytes from oxidative stress Kanu Chatterjee a,c,e,1 , Jianqing Zhang b,1 , Norman Honbo b , Uschi Simonis f , Robin Shaw a,d , Joel S. Karliner b,c,d,⁎ a
e
Cardiology Division, University of California, San Francisco, USA b Cardiology Section, VA Medical Center, San Francisco, USA c Department of Medicine, University of California, San Francisco, USA d Cardiovascular Research Institute, University of California, San Francisco, USA Chatterjee Center for Cardiac Research, University of California, San Francisco, USA f Department of Chemistry and Biochemistry, San Francisco State University, USA Received 24 May 2007; accepted 12 June 2007 Available online 21 June 2007
Abstract Vincristine is a chemotherapeutic agent that disrupts microtubules. We noted that paclitaxel (Taxol), which stabilizes microtubules, protected cultured adult mouse cardiac myocytes from oxidative stress induced by H2O2. We hypothesized that vincristine, which disrupts microtubules, should have the opposite effect. To our surprise, we found that pretreatment with concentrations of vincristine ranging from 30 to 120 μmol/L for 60 min preserved myocyte viability and morphology after incubation with 30 μmol/L of H2O2 for 35 min as measured by trypan blue exclusion. The cardioprotective effects of vincristine were also observed during prolonged hypoxia. With continuous exposure to vincristine, survival lasted for as long as 24 h, but longer periods of exposure up to 42 h resulted in extensive cell death. Despite microtubule disruption evidenced on deconvolution microscopy, vincristine activated a prosurvival pathway resulting in increased phosphorylation of Akt, ERK and GSK-3β and in reduced cytochrome C release into the cytosol. Pharmacological inhibitors of Akt and Erk attenuated the cardioprotective effect of vincristine. We conclude that short-term pretreatment with vincristine exerts dramatic protective effects in cultured adult mouse myocytes subjected to acute oxidative stress. Despite causing microtubule disruption, vincristine initiates a prosurvival signaling pathway. As vincristine and doxorubicin are often used in conjunction to treat patients, it is possible that vincristine could be used to modify the cardiotoxicity of doxorubicin. © 2007 Elsevier Inc. All rights reserved. Keywords: Vincristine; Cardiac myocytes; Cell culture; Hydrogen peroxide; Oxidative stress; Hypoxia; Cardioprotection; Signal transduction
1. Introduction Vincristine is a member of the vinca alkaloid family and is widely used in chemotherapy for various malignancies [1,2]. The cytotoxicity of vinca alkaloids is based on well-established pharmacologic properties that include microtubule depolymerization and arrest of cell cycle division during metaphase [2]. As microtubules are integral components of the mitotic spindle, interference with microtubule dynamics by vinca alkaloids such ⁎ Corresponding author. Cardiology Section (111C5), VA Medical Center, 4150 Clement St., San Francisco, CA 94121, USA. Fax: +1 415 750 6959. E-mail address: [email protected] (J.S. Karliner). 1 Both authors contributed equally to this work. 0022-2828/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.yjmcc.2007.06.005
as vincristine promotes not only cell division arrest but also apoptosis [3]. However microtubule-interfering agents like vincristine may also affect non-neoplastic cells because microtubules are necessary to maintain other cellular functions such as membrane and cellular scaffolding, intracellular transport of organelles and proteins, and transmission of signals initiated at cell surface receptors [3]. Such effects on nonmalignant cells constitute mechanisms for potential cardiotoxicity of these agents. Among chemotherapeutic agents, anthracyclines have been well documented to produce cardiotoxicity and cardiomyopathy in patients receiving these drugs for treatment of various malignancies, but the use of vinca alkaloids in isolation is usually not associated with cardiotoxicity although neurotoxi-
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city is not infrequent [4]. However, one report noted that the vinca alkaloid vinblastine was cardiotoxic to isolated hearts subjected to ischemia/reperfusion injury [5]. Paclitaxel (Taxol) is also widely used for the chemotherapy of malignancies, but in contrast to vincristine it stabilizes microtubules, and in the same study paclitaxel exhibited a cardioprotective effect in isolated perfused rat hearts [5]. As Taxol is a microtubule-stabilizing agent and vincristine is a microtubule-destabilizing agent similar to vinblastine, we hypothesized that vincristine would not exert a cardioprotective effect and might in fact cause cardiac toxicity as noted above. To test this hypothesis, we asked the following questions: (1) Is vincristine toxic to adult mouse cardiac myocytes, and if so, at what level? (2) What are the effects of vincristine on adult mouse cardiac myocytes subjected to chemical and hypoxic oxidative stress? (3) What is the mechanism of these effects? 2. Experimental procedures 2.1. Materials Vincristine (VCR) and paclitaxel were purchased from Sigma-Aldrich (St. Louis, MO). Antibodies against cytochrome C were purchased from BD Biosciences (Franklin Lakes, NJ), and antibodies against GAPDH were obtained from Abcam (Cambridge, MA). Antibodies against phosphorylated and total Akt, ERK and GSK-3β were purchased from Cell Signaling Technology (Danvers, MA). LY294002 and PD98059 were obtained from Biomol (Plymouth Meeting, PA). Mouse laminin and pertussis toxin were purchased from Invitrogen (Carlsbad, CA). 2.2. Cell culture Male C57B1/6 mice (19–25 g) from Charles River Laboratories (Hollister, CA) were used for the preparation of myocytes. Mice received standard rodent chow and water ad libitum. The study was approved by the Institutional Animal Care and Use Committee of the San Francisco Veterans Affairs Medical Center. Cardiac myocytes were cultured according to the method described by Zhou et al. with modifications [6]. Following anticoagulation with heparin (50 units, i.p.), mice were euthanized with sodium pentobarbital (200 mg/kg, i.p.). After excision, hearts were cannulated via the aorta for retrograde perfusion for 2 min with Ca2+-free isolation buffer containing (mmol/L) NaCl 120, KCl 5.4, MgSO4 1.2, NaH2PO4 1.2, glucose 5.6, NaHCO3 4.6, HEPES 10, taurine 5, 2,3-butanedione monoxine (BDM) 10. The hearts were then perfused for approximately 9 min with the same isolation buffer which in addition contained 50 μmol/L CaCl2 and 1.5 mg/mL collagenase II (Worthington, Lakewood, NJ). Ventricles were then separated by trimming the other cardiac and major vascular structures. Ventricular fibers were teased apart with forceps, pipetted in collagenase II buffer, filtered through a cell dissociation sieve, and centrifuged at 40×g for 1 min. Cardiac myocyte pellets were resuspended in isolation buffer serially supplemented with 100, 250, 500 and 1200 μmol/L CaCl2.
The final myocyte pellets were resuspended in minimal essential medium (MEM) containing Hanks' buffered salt solution (HBSS), 10 μg/mL penicillin, 1.5 μmol/L vitamin B12, 2.5% bovine calf serum (BCS), and 10 μmol/L BDM. Isolated myocytes were then plated on dishes coated with 10 μg/L laminin and in media containing 2.5% serum and 10 mM BDM at a density of 50 rod-shaped cells/mm2 for 2 h. Culture media were changed to 1 mM BDM without serum for overnight incubation at 37 °C in a humidified atmosphere of 1% CO2 and air. Cells were viable at pH 7.2 for 72 h. Experimental protocols were performed the day following myocyte isolation and plating. 2.3. Measurement of cell survival After overnight incubation, media were changed to remove BDM. Myocytes were treated with vincristine or vehicle for a minimum of 60 min or for as long as 42 h. H2O2 was routinely prepared as a 10 mM stock solution. In each experiment, 3 μL of this solution were added to 1 mL of buffer in each dish to yield a concentration of 30 μmol/L. Cell numbers were kept constant for each experiment at ∼ 50,000 cells/35 mm culture dish. To assess the effects of chemical oxidative stress, myocytes were exposed or not to 30 μmol/L H2O2 for 35 min in the presence or absence of vincristine. In some experiments, pretreatment with pharmacological inhibitors of signaling molecules was for 30 min in the presence or absence of vincristine. In these experiments, myocytes were treated with vincristine (30 μmol/L) or vehicle for 60 min. To induce hypoxic stress, media were changed twice with glucose-free MEM/HBSS without BDM or bovine fetal serum that had been equilibrated in either normoxia in a 37 °C humidified CO2 incubator (1% CO2, Forma Scientific, Marietta, OH) or in hypoxia in a Bactron I Anaerobic Chamber (99% N2/1% CO2, Sheldon Manufacturing, Inc., Cornelius, OR). The pretreated myocytes were then incubated in the same MEM medium in normoxia or hypoxia for approximately 4 to 5 h prior to induction of hypoxia. Survival of the cultured myocytes was determined by staining cells in tissue culture dishes for 10 min at room temperature with trypan blue solution (Gibco, Grand Island, NY) diluted to a final concentration of 0.04% (w/v). When cell membranes are irreversibly damaged, trypan blue, which is an anionic dye, is taken up by dead cells and binds to nuclei [7,9]. The trypan blue exclusion (TBE) assay is a widely accepted and validated method to determine cell survival and changes in cell morphology in experimental models, and has previously been used in our laboratory [8,9]. For the calculation of viability and morphologic changes, myocytes were visualized at 100× magnification by microscopy. Cells that excluded trypan blue (TBE) were considered viable. Healthy rod-shaped myocytes (rods) were identified when the length/width ratio was N3:1 as previously described [10]. Contracted cells were defined when the length/width ratio was b 3:1. Trypan blue-positive cells were identified when the trypan blue was present intracellularly irrespective of whether the cells were rod-shaped or contracted [10]. Morphologic
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changes were measured by determining the number of rods relative to all TBEs (rods and contracted cells) in ten fields/ dish. The percent survival and viability were calculated as follows: Percent survival Total number of H2 O2 TBE cells in 10 fields=dish ¼ Total number of rods and contracted cells in the same 10 fields 100
Percent viability Total number of rod−shaped TBEs in 10 fields=dish ¼ Total number of TBE rods and contracted cells in the same 10 fields 100
The second technique for assay of cell viability and death was employed using the Live/Dead Viability/Cytotoxicity Assay kit (Molecular Probes, Carlsbad, CA). This method is also well established and validated and has previously been used in our laboratory [11]. It is based on determination of intracellular esterase activity and plasma membrane integrity. The polyanionic dye calcein, which fluoresces green, is retained exclusively by live cells. Ethidium homodimer enters the cells only when the plasma membrane is damaged, and after binding to nucleic acids, it exhibits red fluorescence. Thus this method also allows assessment of apoptotic cell death [12,13]. For these experiments, myocytes were grown on chamber slides. The culture medium was replaced with 2 μmol/L calcein acetoxymethyl ester and 4 μmol/L ethidium homodimer-1. Live cells were recognized by the intense uniform green fluorescence of calcein and the dead cells by red fluorescence of the ethidium homodimer [11]. 2.4. Western blot analysis Lysates were prepared from cultured myocytes and were subjected to SDS–PAGE. Selective primary antibodies, appropriate secondary antibodies conjugated with horseradish peroxidase, and enhanced chemiluminescence (ECL, Amersham Biosciences, Piscataway, NJ) were used for detection of the proteins of interest. Densitometry was used for quantitative assessment of the signals. 2.5. Immunostaining and deconvolution imaging Myocytes were fixed at − 20 °C with methanol for 5 min, permeabilized with 0.2% Triton X-100 in PBS, and blocked in PBS + 0.1% Triton X-100 + 5% goat serum or donkey serum (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). Mouse anti-tubulin primary antibodies were diluted into PBS + 0.1 % Triton X-100 + 2% serum (1:1000, Sigma-Aldrich, St. Louis, MO) and incubated at room temperature for 1 h. Coverslips were washed in PBS, followed by incubation with goat or donkey anti-mouse secondary antibodies conjugated to Alexa 488 or Cy3 (Jackson ImmunoResearch Laboratories, 1:1000 dilution in PBS + 0.1% Triton X-100 + 1% serum) with 1 g/mL Hoechst 33342 (Invitrogen, Carlsbad, CA) for 1 h at
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room temperature. Cells were mounted on microscope slides with GelMount aqueous mounting medium (Biomeda Corp., Foster City, CA). Fixed cells were imaged with a Nikon TE2000-U inverted fluorescence microscope equipped with a Photometrics Coolsnap HQ CCD camera and MetaVue software (Molecular Devices, Foster City, CA). For deconvolution processing, whole cell stacks with 100 nm vertical spacing were acquired. For tubulin, the stacks were deconvoluted using 3D blind deconvolution using Autoquant software. 2.6. Creatine kinase (CK) CK depletion from the cells was measured with a commercially available kit. (Stanbio CK-NAC, Boerne, TX). 2.7. NMR experiments The 1H NMR spectra were acquired at 310 K (36 °C) on an Avance DRX 300-MHz spectrometer from Bruker BioSpin operating at 299.9197 MHz and were referenced to D2O resonating at 4.800 ppm. The spectra were acquired using the one-pulse sequence zg available in the BRUKER pulse sequence library. Typically, the FIDs were acquired using a relaxation delay of 1 s, a spectral width of 7500 Hz, a data size of 32 K data points, and a pulse width of 50° (90° pulse = 11.0 μs) and 512 acquisitions depending on sample concentration. The free induction decays (FIDs) were apodized with an exponential multiplication using a line-broadening factor of 0.10 Hz and phase-corrected to yield the spectra. 2.8. Statistical analysis All results are reported as mean ± S.E.M. Comparisons were made by one-way analysis of variance (ANOVA). Post hoc analysis was performed using the Student–Newman–Keuls test. P b 0.05 was considered significant. 3. Results 3.1. Viability assays In initial experiments, we found that pretreatment with the microtubule-stabilizing chemotherapeutic agent paclitaxel (Taxol) enhanced survival of non-dividing cultured adult mouse cardiac myocytes in response to chemical oxidative stress (H2O2) as measured by the percentage of live cells determined by trypan blue exclusion (Fig. 1). Based on these observations, we wondered whether the microtubule-disrupting agent vincristine, which is commonly used in cancer chemotherapy, would have the opposite effect. Results of a trypan blue exclusion assay in myocytes treated or not with vincristine before H2O2 exposure in a representative experiment are illustrated in Fig. 2A. Rod-shaped and contracted cells that excluded trypan blue were considered as live, while cells that failed to exclude trypan blue were regarded as dead regardless of morphology. In the control dish, the majority of the cells were rod-shaped and there were only a few contracted and trypan
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Fig. 1. Effects of Taxol pretreatment on survival as measured by the trypan blue assay in cultured adult mouse cardiac myocytes in response to chemical oxidative stress. Cells were pretreated with 30 μmol/L of H2O2 for 35 min or not. Taxol was dissolved in 0.1% DMSO (final concentration) and pretreatment was for 60 min. Numbers in bars show the number of experiments for each condition. *P b 0.05 vs. vehicle alone and Taxol. Veh = vehicle.
blue-positive cells. Following exposure to H2O2, there was a dramatic decrease in the number of rods and a substantial increase in the trypan blue-positive cells. After pretreatment with vincristine and following exposure to H2O2, we were surprised to find that the majority of the cells were rod-shaped and only a few cells were contracted and trypan blue-positive. These results, which were replicated numerous times (see Figs. 4 and 5), suggested that pretreatment with vincristine improved survival of cultured adult mouse cardiac myocytes exposed to chemical oxidative stress. An additional assessment of viability, the “Live/Dead” assay, illustrated in Fig. 2B, confirmed these observations. Without pretreatment with vincristine or H2O2, (control), the proportion of viable cells (green fluorescent) was much higher than the dead cells (red fluorescent). Following exposure to H2O2 and without pretreatment with vincristine the proportion of viable cells markedly decreased and that of dead cells markedly increased. Following vincristine pretreatment, however, cell survival was largely preserved as there were many more green than red fluorescent cells. Fig. 3A depicts the effects of increasing concentrations of vincristine pretreatment for 60 min on myocyte survival as measured by the trypan blue exclusion assay following exposure to H2O2. Cell counts are expressed as the percent change relative to control values which were set at 100%. Following H2O2 exposure and without vincristine pretreatment, the proportion of cells that excluded trypan blue decreased to an average of 53%. After pretreatment with 30 to 120 μmol/L of vincristine, the proportion of cells that excluded trypan blue remained at 90% to 96%, consistent with almost complete protection from chemical oxidative stress. Pretreatment with vincristine also preserved adult mouse cardiac myocyte morphology against oxidative stress. As shown in Fig. 3B, the percentage of rod-shaped myocytes relative to the total number of cells that excluded trypan blue in the control cultures was N 80%. Vehicle pretreatment followed by H2O2 exposure decreased the proportion to ≈ 25%. With
vincristine pretreatment, the percentage of rod-shaped myocytes was reduced to about 60% after H2O2 exposure, suggesting that pretreatment with vincristine improved myocyte viability by more than 2-fold after exposure to chemical oxidative stress. To determine whether vincristine also is protective during oxygen deprivation, a key component of ischemia, cultured cardiomyocytes were exposed to hypoxia after the pretreatment with vincristine. As shown in Fig. 4, preincubation of adult mouse cardiac myocytes with 30 μmol/L vincristine for 1 h reduced hypoxia-induced cell death. After 5 h of hypoxia, the survival of vincristine-pretreated myocytes was significantly improved in comparison to vehicle controls (Fig. 4). To determine the effects of treatment duration with vincristine on survival and morphologic changes, myocytes were incubated for 6 to 42 h. Even with the highest dose used (1 mmol/L), there was no decrease in survival after 6 or 24 h. However, as shown in Fig. 5A, after prolonged exposure to vincristine for an average of 42 h survival decreased progressively with increasing concentrations to a maximum of 100 μmol/L. The magnitude of CK activity in the myocytes progressively declined with increasing concentrations of vin-
Fig. 2. (A) Typical results of a trypan blue exclusion assay in a representative experiment. H2O2 at 30 μmol/L for 35 min was associated with an increased number of trypan blue positive cells and a decrease in rod-shaped cells compared to the vehicle control panel. Pretreatment for 60 min with 60 μM vincristine dissolved in H2O was associated with fewer trypan blue-positive cells and persistence of rod-shaped cells, indicating increased survival and viability of the cultured adult mouse cardiac myocytes. These results were replicated numerous times (see text and Figs. 3 and 4). VCR = vincristine; Veh = vehicle. (B) Treatments were the same as in panel A. Live/Dead assay showing that pretreatment with 60 μmol/L vincristine for 60 min increased survival (calcein ester-positive/green fluorescence) and decreased the proportion of dead cells (ethidium-positive/red nuclei) after exposure to 30 μmol/L H2O2 for 35 min. This experiment was replicated four times. VCR = vincristine; Veh = vehicle; EthD-1 = ethidium homodimer-1.
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Fig. 4. Pretreatment with 30 mmol/L vincristine for 60 min significantly increased the total survival of cardiomyocytes during hypoxia. Numbers in bars show the number of experiments for each condition. VCR = vincristine; Veh = vehicle; Nx = normoxia; Hx = hypoxia.
pretreated with vincristine in the presence of specific inhibitors of PI3-K/Akt and MEK/ERK activation: LY294002 and PD98059, respectively. Cells were exposed to H2O2 after washout of vincristine and the inhibitors. Survival of cardiomyocytes was determined by trypan blue exclusion. As shown in Fig. 6, inhibition of PI3-K/Akt and ERK each significantly reduced the percentage of myocytes protected by vincristine treatment. These findings suggest that more than one prosurvival
Fig. 3. (A) Effect of increasing concentrations of vincristine pretreatment for 60 min on the proportion of surviving myocytes following exposure to 30 μmol/L H2O2 for 35 min. n = number of experiments per data point. *P b 0.05 vs. vehicle control. Con = control; Veh = vehicle; VCR = vincristine. (B) Effect of increasing concentration of vincristine pretreatment for 60 min on the proportion of rodshaped myocytes following exposure to 30 μmol/L H2O2 for 35 min. n = number of experiments per data point. *P b 0.05 vs. vehicle control.
cristine given over 42 h and paralleled changes in survival (Fig. 5B). Next we asked how long cardioprotection persisted after 1 h of pretreatment with vincristine. Cultured myocytes were exposed to H2O2 after washout of vincristine and survival was measured at 1, 2, 4, and 6 h. At 1 and 2 h, survival, as determined by trypan blue exclusion, was maintained; but at 4 and 6 h, the cardioprotective effects of vincristine were no longer observed (data not shown). 3.2. Mechanism of vincristine-induced resistance to oxidative stress To identify possible components of a protective pathway stimulated by vincristine, we examined the survival of myocytes
Fig. 5. Effects of vincristine pretreatment for an average of 42 h on survival (A) and creatine kinase depletion (B) with increasing concentrations of vincristine. n = number of experiments per data point. *P b 0.05 vs. control. Con = control.
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signaling pathway is involved in cardioprotection by vincristine. Overnight preincubation with 100 μg/mL pertussis toxin had no significant effect on the prosurvival effect of vincristine (n = 6, data not shown), indicating that a receptor-mediated Gi-coupled mechanism was not involved in vincristine signaling. To verify that vincristine activated a prosurvival signal transduction cascade, we initially performed time and concentration–response experiments to identify the optimal conditions for measuring signaling responses. The magnitude of Akt, ERK and GSK-3β phosphorylation was proportional to the concentration of vincristine used and reached a plateau at 30 to 100 μmol/L. Figs. 7A–C illustrate typical effects of vincristine pretreatment on phosphorylation of Akt (S473), ERK/MAPK and GSK-3β. Compared to vehicle controls, phosphorylation of each of these molecules increased several-fold. The increased phosphorylation of Akt and ERK suggests an activation of prosurvival signals resulting from vincristine pretreatment, and is consistent with improved survival conferred by vincristine in the cultured cardiac myocytes (Fig. 6). The increased phosphorylation of GSK-3β is consistent with inhibition of its adverse effects on cell demise, as previously reported [14]. In parallel with the effects of LY294002 and PD98059 on myocyte viability shown in Fig. 6, we observed that phosphorylation of Akt and GSK-3β by vincristine was abrogated by LY294002 pretreatment (Figs. 7A and B). These data indicate that PI3-K is required for Akt S473 phosphorylation. ERK activation was inhibited completely by PD98059 but not by LY294002 as expected (Fig. 7C).
western analysis. Myocyte cytosolic extracts were prepared after exposure to H2O2 with and without pretreatment with vincristine and blots were probed with a cytochrome C specific antibody. Compared to H2O2 alone, vincristine pretreatment resulted in a significant decrease of mitochondrial cytochrome C release, suggesting reduction of oxidative stress and inhibition of mitochondrial permeability transition (Fig. 6D). 3.4. Vincristine and H2O2: is there a direct interaction? Using 1H NMR spectroscopy, we observed no alteration in the spectra of vincristine when it was incubated with H2O2 in the presence or absence of cardiomyocytes. Thus our experiments do not support a direct interaction of vincristine and H2O2. 3.5. Microtubular architecture Fixed cardiomyocyte immunostaining with deconvolution imaging provides a planar transverse view of microtubule organization (Fig. 8). In control cells, microtubules are organized along the longitudinal axis in keeping with expected myocardial anisotropy. In contrast, following pretreatment with vincristine the microtubules are highly disrupted with clumping and little discernable organization. Thus it appears that vincristine significantly disrupts the microtubules and yet still exerts cardioprotective effects in cultured adult mouse cardiac myocytes.
3.3. Cytochrome C release
4. Discussion
To determine the effects of vincristine on mitochondrial transition pore function, we measured cytochrome C release by
The main and unexpected finding of the present study is that despite microtubule disruption short-term pretreatment with vincristine exerts dramatic protective effects in cultured adult mouse cardiac myocytes subjected to acute oxidative stress. The mechanisms involved include activation of prosurvival signaling pathways. 4.1. Myocyte survival
Fig. 6. Pharmacological inhibitors of PI3K and Erk pretreatment abolish the vincristine protective effect on myocytes survival following exposure to 30 μmol/L H2O2 for 35 min. Myocytes were pretreated with 10 μmol/L of LY294002 (LY) or 30 μmol/L of PD98059 (PD) for 30 min before followed by vincristine treatment or vehicle. Both the control value (asterisk) and the vincristine-treated cells (cross) subjected to hydrogen peroxide show significantly greater survival than all other sets of cells; none of the other values [H2O2 + either vehicle, LY or PD], or [vincristine + H2O2 and either LY or PD], are significantly different from each other.
We determined cell survival by two different methods, the trypan blue exclusion assay and the Live/Dead assay. Initially we used the trypan blue assay to determine the effects of vincristine pretreatment on survival and viability of the cultured adult mouse myocytes exposed to H2O2. The results demonstrate that vincristine pretreatment for only 1 h exerts substantial protection of cardiac myocytes exposed to this chemical oxidative stress. With 30 to 120 μmol/L of vincristine pretreatment, myocyte salvage doubled and approximated control values (Fig. 3A). Furthermore, cell viability as measured by preserved morphology also improved 2-fold with vincristine pretreatment (Fig. 3B). The two-color fluorescence assay technique (Live/Dead assay), which was performed in a series of separate experiments, yielded virtually identical results. Thus, two independent methods of measuring myocyte viability, the TBE method and the Live/Dead approach, verified the prosurvival effects of vincristine.
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Fig. 7. Panels A–C show changes in phosphorylation of Akt, ERK and GSK-3β in cultured adult mouse myocytes after 30 min of pretreatment with 60 μmol/L vincristine with or without pretreatment with the pharmacological inhibitors. The upper panels show representative western blots and the lower panels show the foldincrease in signal intensity produced by vincristine. Numbers in bars show the number of independent experiments for each of the signals depicted. *P b 0.05 vs. all other values; **Pb0.05 vs. Con and PD. Con = vehicle; VCR = vincristine. Panel D shows western blot analysis of cytochrome C release in response to 30 μmol/L H2O2 for 35 min. Pretreatment with 60 μmol/L vincristine for 60 min decreased cytochrome C release into the cytosol. Con = control; VCR = vincristine. Equal loading was verified with anti-GAPDH antibody. *P b 0.05 vs. control.
Most of the experiments were done with quiescent myocyte cultures. After harvest and plating, myocytes were incubated for 24 h before vincristine treatment followed by H2O2 exposure. However, the same protective effect of vincristine was observed with freshly isolated cardiomyocytes (data not shown). In this
study, pretreatment with vincristine also provided protective effects in cultured adult mouse cardiac myocytes subjected to hypoxia (Fig. 4). The magnitude of protection from hypoxia was similar to that found in cells subjected to chemical oxidative stress. These findings suggest that pretreatment with
Fig. 8. Deconvolution microscopic studies showing effects of pretreatment with 60 μmol/L vincristine for 60 min on microtubular structure. Vincristine pretreatment is associated with microtubular disruption. Nuclei are shown in blue. For details see text.
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vincristine has the potential to provide cardioprotection irrespective of type of oxidative stress imposed. Our culture protocol includes overnight incubation in medium containing BDM at 1 mM final concentration which substantially increases the number of healthy and functional ventricular myocytes available for experimentation on the morning after isolation. For methods requiring homogeneous populations of adult mouse ventricular myocytes to be functional for at least 24 h, other laboratories have published protocols in which BDM is present throughout heart tissue digestion and Ca2+ restoration. Zhou et al. [15] demonstrated the utility of this cell culture paradigm for cellular genetic physiology. Hilal-Dandan et al. [16] confirmed that adult mouse cardiac myocytes remain excitable and useful for contractile and calcium measurements [17]. Sollott et al. [18] demonstrated that treatment of adult rat ventricular myocytes with 1 mM BDM had no significant effect on myocyte length and [Ca2+]i at rest and during electrical stimulation. It should be emphasized that we routinely incubate cardiac myocytes in a normoxic medium that does not contain BDM on the morning following isolation for 2 h prior to the addition of pharmacological reagents. Under the conditions studied, cells incubated with vincristine were viable for at least for 24 h even when myocytes were exposed to a high concentration of the drug (100 μmol/L). However, very prolonged exposure, for example for up to 42 h, induced cardiotoxicity. When vincristine was washed out after 1 h of pretreatment, the cardioprotective effects in response to oxidative stress lasted only 2 h. Thus it appears that, in order to maintain cardioprotection, continuous vincristine treatment is necessary. In contrast to our findings, Skobel and Kammermeier reported that vinca alkaloids can enhance cardiac myocardial damage [5]. In isolated perfused rat hearts subjected to ischemia/reperfusion injury, vinblastine, which has similar therapeutic and biologic properties, increased rat cardiac myocyte death as determined by creatine kinase release and TBE assays in paraffin sections [5]. The reason for this discrepant result remains unclear. In their study, Taxol, a microtubule-stabilizing agent and cytochalasin D, an actin-destabilizing agent, decreased the proportion of irreversible cell injury, suggesting that microtubule-destabilizing effects of vinblastine may be the mechanism of its observed adverse effects. Both vinblastine and vincristine disrupt microtubular architecture by binding to tubulin dimers at a site located on the beta subunits at the plus ends of microtubules [19,20]. Our deconvolution microscopic studies demonstrated that vincristine pretreatment indeed is associated with microtubular disruption. Despite this adverse response, we noted that vincristine pretreatment produced dramatic cardioprotective effects. Thus microtubule disruption in these nondividing cells does not appear to be the explanation for the discrepant results between our study and that of Skobel and Kammermeier [5]. It has been reported that the oxidative breakdown of vincristine by myeloperoxidase is H2O2-dependent [21]. Degradation of vincristine was not seen when vincristine was incubated with H2O2 in the absence of enzyme or when vincristine was incubated with the enzyme in the absence of H2O2 [21]. Using
1
H NMR spectroscopy, we observed no alteration in the spectra of vincristine when it was incubated with H2O2 in the presence or absence of cardiomyocytes. Thus our experiments do not support a direct interaction of vincristine and H2O2. During oxidative stress, microtubules may have a protective structural effect. Therefore, in intact beating hearts subjected to ischemia, the intensity of any microtubular structural disruption may be one determinant of the extent of myocardial injury. However, in isolated quiescent myocytes, our study has revealed that disruption of microtubules is associated with a surprising and marked viability advantage concurrent with activation of prosurvival biochemical changes. Determining how this biochemical advantage might be linked to microtubule disruption or if it is independent of this response, and whether it occurs in isolated hearts and intact animals is an important topic of future investigation. 4.2. Signal transduction Akt activation is mediated mainly through phosphorylation of S473, and is regulated through a PI3-K-dependent mechanism [22]. PI3-K/Akt signaling is involved in cell survival in many cellular systems as well as in adult cardiomyocytes [23]. Our results with LY294002 show that Akt activation is PI3-Kdependent in mouse cardiomyocytes (Fig. 7A). It has been reported that the beneficial effects of Akt activation on cardiac myocyte survival during oxidative stress is at least partly due to phosphorylation of the downstream signaling molecule GSK3β [14]. Our Western blot experiments are consistent with these observations and indicate that pretreatment of cultured adult mouse myocytes with vincristine is associated with increased phosphorylation of GSK-3β (Fig. 7B), thereby leading to its inactivation and enhancing cell survival [14]. As noted earlier, there was increased phosphorylation of Akt and ERK which may also promote cell survival by preventing mitochondrial dysfunction [23]. Conversely, inhibition of Akt and ERK activation attenuated their cardioprotective effect. Thus activation of these survival pathways appears to be necessary in mediating the cardioprotective effect of vincristine. 4.3. Cytochrome C release It is well-recognized that cytochrome C release from mitochondria and impairment of respiratory complex function by activation of mitochondrial permeability transition causes cell injury [24]. Reactive oxygen species (ROS) generated by respiratory complex III during ischemia-induced oxidative stress depletes mitochondrial cardiolipin, thereby permitting cytochrome C release into cytosolic compartments [25]. In the present study, pretreatment with vincristine decreased cytochrome C release induced by chemical oxidative stress by exposure to H2O2. Although we did not measure ROS in this study, H2O2 is known to result in ROS generation [26]. Therefore our findings suggest that one additional potential mechanism of cardioprotection activated by short-term vincristine treatment is diminished ROS production resulting in decreased mitochondrial permeability transition.
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4.4. Study limitations Many prior studies reporting signaling responses initiated by vincristine have been performed. Almost all of these are in tumor cell lines and have described the apoptosis-inducing effects of vincristine. Examples of reported pro-apoptotic signals induced by vincristine in vitro include JNK/SAP, ras and ASK1 [27], Bcl2 phosphorylation [28], p53 and p21 [29], ERK and p38 [30], NF-κB/IκB [31], and RhoA [32]. All of these responses occur in conjunction with disruption of the dynamics of microtubule assembly, but how microtubular disarray activates these pathways has yet to be defined. Our results are novel as they unequivocally demonstrate that in the experimental model employed, short term pretreatment with vincristine provides survival and viability benefit in primary cultures of adult cardiac myocytes for at least 24 h following exposure to chemical oxidative stress. To the best of our knowledge, the prosurvival effect of vincristine in these cells has not been previously reported, and represents a new model of cardioprotection. In this connection, recent evidence is consistent with our observations that vincristine can stimulate non-apoptotic signals. For example, in a nontransformed murine fetal liver cell line it was reported that vincristine activated mTOR, which facilitates Akt activation [33]. Thus, further studies to elucidate the mechanisms by which vincristine triggers myocyte survival are warranted. The clinical implications of this study suggest the possibility that vincristine, which is often used in conjunction with doxorubicin, could abrogate the cardiotoxicity of the latter drug. Studies are currently underway in our laboratory to test this hypothesis. Acknowledgments Supported by the Foundation for Cardiac Research and PO1 HL 68738 from the National Heart, Lung, and Blood Institute, National Institutes of Health.
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