Generation of catalase overexpression transgenic mice and production of anthrax lethal toxin
Catalase overexpressing transgenic mouse generation was described in detail previously . In brief, an 8-kb CAT driven by α-myosin heavy chain (α-MHC) promoter containing the entire coding sequences of the catalase cDNA was purified on a matrix of diatomaceous earth (Prepagene, Bio-Rad Hercules, CA, USA) and filtered through a 0.22-μm filter. Approximately, 100 copies of the purified transgene insert were microinjected into one pronucleus of each one-cell mouse embryo of the inbred strain FVB. The transgene transcription of catalase was controlled by the mouse α-MHC gene. To identify transgenic founder mice, genomic DNA was isolated from 1-cm tail clips from four-week-old mice. DNA was subjected to Southern and dot blot analyses, which were probed with a 550-bp SmaI/NotI fragment derived from the rat insulin II portion of the CAT. This probe hybridized to an 8,000-bpEcoRI fragment of the transgene, consistent with the presence of a unique EcoRI site in the CAT (600-bp upstream of the MHC transcription initiation site). Founder mice were bred with mice of the same strain and transgenic offspring were routinely identified by a polymerase chain reaction (PCR) using a primer pair derived from the MHC promoter and rat catalase cDNA with the reverse sequence of AAT ATC GTG GGT GAC CTC AA and the forward sequence of CAG ATG AAG CAG TGG AAG GA. These transgenic mice have approximately 60-fold catalase overexpression.
Recombinant PA and LF were produced and purified as previously described [15, 24, 25]. In brief, plasmid pSJ115 encoding LF from SCS110 E. coli stocks was transformed into BH445 crippled B. anthracis strain by electroporation . Single colonies from LB kanamycin agar plates were grown overnight and 20 mL culture media were inoculated into 6 L modified FA media in the presence of 1 mL polypropylene glycol P200 and kanamycin at 37°C at 100 to 300 rpm agitation rate and 2 L air/min constant sparging for 14 h. Cultures were centrifuged, and 2 mM EDTA and 0.1 mM PMSF added to supernatant. Supernatants were sterilized using 0.22-μm filters and concentrated to 300 mL with 10,000 Da Mr cutoff cellulose membranes in a Pellicon system. A total of 40% ammonium sulfate was added, and the sample was loaded, washed and eluted from Fast Flow PhenylSepharose six columns with a 1.5 to 0 M ammonium sulfate gradient in 20 mM Hepes/5 mM EDTA pH 7.5. LF containing fractions were concentrated in Centricon Plus-80, dialyzed into 20 mM Hepes/0.1 mM EDTA pH 8, and eluted from a Q-Sepharose Fast Flow column with a 0 to 0.5 M NaCl in 10 mM Tris/0.5 mM EDTA pH 8 gradient. Eluted LF fractions were pooled, concentrated again with Centricon Plus-80 filters, diluted in 1x PBS, filter sterilized and frozen at -80°C. pYS5-wild-type PA vector was transformed into B. anthracis as described above. Colonies were expanded in overnight cultures with kanamycin and were inoculated into 6 L modified FA medium with polypropylene glycol and kanamycin for LF and PA fermentation similar to LF . After 14 h, cultures were centrifuged prior to the addition of EDTA and PMSF. Filtration and concentration were conducted using a Pellicon system. A total of 20% ammonium sulfate was added, and PA loaded, washed and eluted from Phenyl Sepharose Fast Flow with a 1.5 to 0 M ammonium sulfate gradient in 10 mM HEPES/5 mM EDTA pH 7. PA containing fractions were dialyzed against 20 mM Tris/5 mM EDTA pH 8.9 and loaded and eluted from a Q Sepharose Fast Flow column with a 0 to 0.5 M NaCl in 20 mM Tris/5 mM EDTA pH 8.9 gradient. Pooled PA fractions were purified using the Superdex 75 chromatography in PBS/5 mM EDTA pH 7.4 and were pooled, concentrated with Centricon Plus-80 filters, diluted in 1x PBS, filter sterilized and frozen at -80°C until experimentation. Immediately before in vivo injection, toxin components were thawed and mixed in PBS.
Experimental animals and anthrax lethal toxin challenge
All animal procedures used in this study were approved by the Animal Care and Use Committee at the University of Wyoming (Laramie, WY, USA). Animals were housed under well-controlled conditions of temperature (22 ± 2°C), humidity (55 ± 5%) and circadian cycle (12 h/12 h light-dark) with access to food and water ad libitum. Four month-old adult male cardiac-specific catalase overexpressing (CAT) mice and their wild-type (WT) littermates were used. For lethal toxin challenge, mice were injected intraperitoneally with PBS or anthrax lethal toxin (2 μg/g) (LF + PA 1:2 ratio) and were sacrificed 18 h following in vivo lethal toxin injection .
Murine cardiomyocyte isolation and drug treatments
After ketamine/xylazine (80 mg/kg:12 mg/kg, i.p.) sedation, hearts were removed and perfused with Ca2+ -free Tyrode's solution containing (in mM): NaCl 135, KCl 4.0, MgCl2 1.0, HEPES 10, NaH2PO4 0.33, glucose 10, butanedione monoxime 10 and the solution was gassed with 5% CO2/95% O2. Hearts were digested with Liberase Blendzyme 4 (Hoffmann-La Roche Inc., Indianapolis, IN, USA) for 20 minutes. Left ventricles were removed and minced before being filtered. Extracellular Ca2+ was added incrementally back to 1.20 mM over a period of 30 minutes. Myocyte yield was approximately 75%, which was not affected by either lethal toxin challenge or catalase overexpression. Only rod-shaped myocytes with clear edges were selected for mechanical study . To assess the cause-effect relationship of autophagy in lethal toxin-induced cardiomyocyte anomalies, in vitro studies were performed using cardiomyocytes isolated from WT and catalase transgenic mice treated with or without lethal toxin (100 ng/ml) at 37°C for 3 h in the presence or absence of the autophagy inhibitor 3-methyladenine (3-MA, 10 mM) or the autophagy inducer rapamycin (5 μM) prior to mechanical and biochemical evaluation .
Cell shortening and relengthening
Mechanical properties of cardiomyocytes were evaluated using a MyoCam system (IonOptix Corporation, Milton, MA, USA) . Briefly, cardiomyocytes were visualized under an inverted microscope (IX-70, Olympus Optical Co., Tokyo, Japan) and were stimulated at a frequency of 0.5 Hz. The myocyte being observed was displayed on a computer monitor using an IonOptix MyoCam camera (IonOptix Corporation, Milton, MA, USA). IonOptix SoftEdge software was utilized to capture cell shortening and re-lengthening. The indices measured included peak shortening (PS), time-to-peak shortening (TPS), time-to-90% re-lengthening (TR90), and maximal velocity of shortening/re-lengthening (± dL/dt).
Intracellular Ca2+ transients
Isolated cardiomyocytes were loaded with fura-2/AM (0.5 μM) for 10 minutes. Fluorescence intensity was recorded with a dual-excitation fluorescence photomultiplier system (IonOptix). Myocytes were placed onto an Olympus IX-70 inverted microscope and imaged through a Fluor 40x objective. Cells were exposed to light emitted by a 75 W lamp and passed through either a 360 or a 380 nm filter while being stimulated to contract at 0.5 Hz. Fluorescence emissions were detected between 480 and 520 nm, and qualitative change in fura-2 fluorescence intensity was inferred from the fura-2 fluorescence intensity ratio at 360 and 380 nm. Fluorescence decay time (both single and bi-exponential curve fits) was calculated as an indicator of intracellular Ca2+ clearing .
Measurement of O2
- levels in myocardium
In situ production of myocardial O2
- was assessed using the oxidative fluorescent dye dihydroethidium (DHE) . The cell membrane permeable DHE is oxidized to fluorescent hydroxyethidine by O2
- and then intercalated into DNA. In brief, myocardial tissues were placed in ice-cold PBS, rinsed and embedded in the optimal cutting temperature (OCT) compound medium (Sakura Finetek USA, Inc., Torrance, CA, USA) for cryosectioning. The cryomoulds were stored at -80°C. Sections (30 μm) from the cryomoulds were thawed to room temperature and were incubated with DHE (10 μM) for 45 minutes at room temperature in a dark chamber. After being washed three times with PBS, sections were fixed with aqueous mounting medium (VectaMount AQ, Vector Laboratories, Burlingame, CA, USA) and images were obtained using a Zeiss LSM 710 confocal microscope (Carl Zeiss MicroImaging GmbH, Jena, Germany). Nuclei with red fluorescence were measured using the ImageJ analysis software (version 1.34S, developed by NIH).
Measurement of ROS production
Production of cellular reactive oxygen species (ROS) was evaluated by analyzing the changes in fluorescence intensity resulting from oxidation of the intracellular fluoroprobe 5-(6)-chloromethyl-2, 7-dichlorodihydrofluorescein diacetate (CM-H2DCFDA). In brief, cardiomyocytes were isolated from WT and catalase transgenic mice challenged with or without lethal toxin (2 μg/g, i.p., for 18 h) and were loaded with 10 μM of the nonfluorescent dye CM-H2DCFDA (Molecular Probes, Eugene, OR, USA) at 37°C for 30 minutes. The myocytes were rinsed with KRH buffer (125 mM NaCl, 5 mM KCl, 1.8 mM CaCl2, 2.6 mM MgSO4, 5 mM HEPES, pH 7.4) and the fluorescence intensity was then measured with a SpectraMax XS fluorescence microplate spectrophotometer (Spectra MaxGeminiXS, spectra Max, Atlanta, GA, USA) at an excitation wavelength of 480 nm and an emission wavelength of 530 nm. Sections (30 μm) of frozen tissues were thawed to room temperature and were incubated with CM-H2DCFDA (10 μM) for 1 h at room temperature in a dark chamber. After being washed three times with PBS, sections were fixed with aqueous mounting medium (VectaMount AQ, Vector Laboratories) and images were obtained using a Zeiss LSM 710 confocal microscope (Carl Zeiss MicroImaging GmbH, Jena, Germany) .
H9C2 cells, a clonal cell line derived from fetal rat heart, purchased from ATCC (Manassas, VA, USA). Cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Gibco, Grand Island, NY, USA) and 1% penicillin and streptomycin and maintained in 95% air and 5% CO2 at 37°C. Cells were grown to 80% confluence before treating with anthrax lethal-toxin .
Measurement of mitochondrial membrane potential (ΔΨm)
Murine cardiomyocytes from lethal toxin-treated mice or H9C2 cells following in vitro lethal-toxin treatment (100 ng/ml for 1 h) were incubated with JC-1 (5 μM) for 15 minutes at 37°C. Fluorescence was analyzed with a Texas red-FITC filter cube (Spectra MaxGeminiXS, spectra Max, Atlanta, GA, USA). Red emission represents a potential-dependent aggregation in the mitochondria, reflecting ΔΨm. Green fluorescence represented the monomeric form of JC-1, appearing in the cytosol after mitochondrial membrane depolarization. Fluorescence of each sample was read at an excitation wavelength of 490 nm and emission wavelength of 530 (Green) and 590 (Red) nm using a spectrofluorimeter (Spectra MaxGeminiXS, spectra Max, Atlanta, GA, USA) at an interval of 10 sec. Results in fluorescence intensity were expressed as 590-to-530 nm emission ratio. The mitochondrial uncoupler carbonyl cyanide m-cholorophenylhydrazone (CCCP, 50 μmol/L) was used as a positive control for ΔΨm measurement .
Proteasome activity was measured as described . Briefly, heart tissues were homogenized in HEPES buffer (in mM: NaCl 137, KCl 4.6, KH2PO4 1.1, MgSO4 0.6, EDTA 1, and DTT 1) without protease inhibitor at 4°C and then centrifuged at 16,168 g to obtain the soluble fraction. Protein (50 μg in 50 μl) was incubated in 50 μl of 50 mM Tris-HCl buffer, pH 7.5, containing 20 mM KCl, 0.5 mM MgCl2 and 1 mM DTT for 1 h with 200 μM fluorogenic substrates Suc-LLVY-AMC/Z-LLE-AMC (Enzo Life Sciences, Plymouth Meeting, PA, USA). Fluorescence products were measured using a spectrofluorimeter (Spectra MaxGeminiXS, spectra Max, Atlanta, GA, USA) using a 355-nm excitation and 460-nm emission filter.
LC3B-GFP-adenovirus production and infection
Adenovirus containing LC3-GFP construct (GFP tagged on the N-terminal of LC3) was kindly provided by Dr. Cindy Miranti (Van Andel Institute, Grand Rapids, MI, USA) and was propagated using HEK293 cell line. Briefly, cells were infected with LC3-GFP adenovirus and collected upon plaque formation. Cell debris was collected by centrifugation, and aliquots of supernatant with viral particles were stored at -80°C. Adenovirus was purified using an Adeno-X Maxi purification kit from Clontech (Clontech Laboratories, Inc., Mountain View, CA, USA). H9C2 cells were grown to confluence on Lab-Tek chamber slides. Cells were then infected at an MOI of 2 with adenoviruses expressing LC3-GFP fusion protein. Medium was replaced with fresh DMEM after 6 h. Twenty four hours later, cells were observed for autophagy using confocal microscopy .
Quantification of GFP-LC3
H9C2 cells transfected with GFP-LC3 adenovirus were treated with or without anthrax lethal toxin (100 ng/ml). Cells were fixed with 4% paraformaldehyde in PBS for 20 minutes at room temperature. Cells were then washed with PBS three times. These fixed cells were treated with DAPI for five minutes followed by three washes with PBS. Cover slips were mounted on slides using Vecta mount™ AQ-aqueous mounting medium (Vector Laboratories, Inc.,). For analysis of autophagy, cells were visualized at 40x magnification using a Zeiss LSM 710 confocal microscope (Carl Zeiss MicroImaging GmbH, Jena, Germany) and the percentage of cells showing numerous GFP-LC3 puncta (>10 dots/cells) were scored as described previously . A minimum of 75 to 100 cells were scored for each condition in at least three independent experiments.
Western blot analysis
Protein samples for Western blot analysis were prepared as described . Briefly, ventricular tissues or H9C2 cells were homogenized and sonicated in a lysis buffer containing 20 mM Tris (pH 7.4), 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM ethyleneglycoltetraacetic acid (EGTA), 1% Triton, 0.1% SDS and 1% protease inhibitor cocktail. Equal amounts (50 μg) of proteins were separated on 7% to 15% SDS-polyacrylamide gels in a minigel apparatus (Mini-PROTEAN II, Bio-Rad Laboratories Inc.,) and were transferred electrophoretically to nitrocellulose membranes. The membranes were blocked with 5% milk in Tris-buffered saline before overnight incubation at 4°C with anti-Beclin-1 (1:1,000), anti-Atg-7 (1:1,000), anti-LC3-II (1:1,000), anti-phospholamban (1:1,000), anti-phosphorylated phospholamban (1:1,000), anti-SERCA2a (1:1,000), anti-Na+-Ca2+ exchanger (NCX, 1:1,000), anti-ubiquitin (1:1000), and anti-GAPDH (loading control, 1:1,000) antibodies. Proteins were visualized after subsequent incubation with a 1:5,000 dilution of anti-mouse or anti-rabbit IgG conjugated to horseradish peroxidase and a LumiGLO® Chemiluminescence detection procedure (Cell Signaling Technologies, Beverly, MA, USA).
Data were expressed as mean ± SEM. Statistical significance (P <0.05) for each variable was estimated by analysis of variance (ANOVA) followed by the Tukey's test for post-hoc analysis.