Download 1 B. anthracis Edema but not Lethal Toxin Challenge in Rats Is

Articles in PresS. Am J Physiol Heart Circ Physiol (April 10, 2015). doi:10.1152/ajpheart.00851.2014
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B. anthracis Edema but not Lethal Toxin Challenge in Rats Is Associated with Depressed
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Myocardial Function in Hearts Isolated and Tested in a Langendorff System
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Yan Li, MD1, Mones Abu-Asab, PhD2, Junwu Su, MD, PhD3, Ping Qiu, MD, PhD4, Jing Feng1,
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Lernik Ohanjanian, MD, MPH1, Hanish Sampath Kumar, MBBS1, Yvonne Fitz, BS1,
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Peter Q. Eichacker, MD1*, Xizhong Cui MD, PhD1*
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Critical Care Medicine Department, Clinical Center, National Institutes of Health, Bethesda,
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MD 20892; 2National Eye Institute, National Institutes of Health, Bethesda, MD 20892;
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MD 20852
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*These authors contributed equally to this study and manuscript
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Running Head: Cardiac effects of anthrax lethal and edema toxins
Anzhen Hospital, Capital Medical University, Beijing, China; 4OncoImmune Inc., Rockville,
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Abstract word count: 249
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Body word count: 4078
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Corresponding Author:
Peter Q. Eichacker, MD
Critical Care Medicine Department
National Institutes of Health
Building 10, Room 2C145
9000 Rockville Pike
Bethesda, MD 20892
(301) 496-9320
Fax : (301) 402-1213
Email : [email protected]
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Copyright © 2015 by the American Physiological Society.
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Abstract
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Background: Although direct myocardial depression is implicated in the lethal effects of B.
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anthracis lethal toxin (LT), in hearts isolated from healthy rats and perfused under constant
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pressure, neither LT or edema toxin (ET) in typically lethal concentrations depressed myocardial
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function. Here we challenged rats with LT and ET and performed in and ex vivo heart measures.
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Methods and Results: Sprague-Dawley rats infused over 24h with LT (n=94), ET (n=99) or
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diluent (controls, n=50), were studied at 8, 24 or 48h. Compared to controls (all survived),
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survival rates with LT (56.1%) and ET (37.3%) were reduced (p<0.0001) similarly (p=0.66 for
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LT vs. ET). LT decreased mean arterial blood pressure from 12 to 20h (p≤0.05) while ET
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decreased it progressively throughout (p<0.05). On echocardiography, LT decreased left
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ventricular ejection fraction (LVEF) at 8 and 48h but increased it at 24h and decreased cardiac
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output (p≤0.05 for the time interaction or averaged over time). ET decreased systolic and
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diastolic volumes and increased LVEF at 24h (p≤0.05). In isolated hearts perfused for 120min
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under constant pressure, LT did not significantly alter LV systolic or developed pressures at any
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time point, while ET decreased both of these at 24h (p<0.0001 initially). ET but not LT
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progressively increased plasma creatine phosphokinase and cardiac troponin levels (p<0.05).
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Conclusion: Despite echocardiographic changes, in vivo lethal LT challenge did not produce
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evidence of myocardial depression in isolated rat hearts. While lethal ET challenge did depress
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isolated heart function, this may have resulted from prior hypotension and ischemia.
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Key Words: anthrax; edema toxin; lethal toxin; heart function
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Introduction
B. anthracis (anthrax) produces two binary toxins, lethal (LT) and edema (ET) toxin,
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which are both thought to play a role in hypotension and lethality during infection. Each toxin
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includes protective antigen (PA) which mediates cellular uptake of the toxins’ toxigenic
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moieties; lethal factor (LF), a zinc metalloprotease, for LT and edema factor (EF), a molecule
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with calmodulin-dependent adenyl cyclase activity, for ET. The intracellular actions of LF and
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EF differ. Lethal factor inhibits mitogen-activated protein kinase kinases and stimulates
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inflammasome formation (6, 26). Edema factor increases intracellular cAMP levels (14, 15).
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Evidence from in vivo models has raised the possibility that LT but not ET produces
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hypotension in part by depressing myocardial function (22). In canines, lethality and hypotension
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with a 24h LT challenge was associated with gradual tachycardia and reductions in left
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ventricular ejection fraction (LVEF) on echocardiography, changes consistent with the effects of
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LT some small animal models (21, 25, 28, 29). Different from LT, lethality and hypotension with
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24h lethal ET challenge in this canine model was associated with early and striking increases in
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heart rate (HR) and cardiac index but no significant changes in LVEF, alterations also noted by
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other groups (28, 29). These findings and others have suggested that although LT and ET both
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alter myocardial function, LT but not ET depresses it (22). However, since both toxins also
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decreased central venous pressure (CVP) and systemic vascular resistance (SVR) in the canine
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model, it was unclear whether their myocardial effects measured in vivo were direct ones or
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instead related to changes in preload or afterload.
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We previously employed a constant pressure Langendorff isolated perfused rat heart
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model to examine the effects of LT and ET on heart function independent of the toxins’ potential
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preload and afterload effects (11). In hearts isolated from healthy Sprague-Dawley rats, typically
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lethal LT concentrations had little effect on heart function. In contrast, lethal ET concentrations
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produced increases in HR and actually augmented myocardial function and coronary blood flow,
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changes consistent with increased intracellular cAMP (11). In order to further investigate the
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myocardial effects of LT and ET, 8, 24 or 48h after the initiation of 24h toxin or PA only
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(control) infusions in Sprague-Dawley rats, hearts were isolated and myocardial function
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investigated in the constant pressure perfusion system. Immediately before heart isolation,
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animals had blood pressure and HR measures followed by in vivo echocardiography and blood
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sampling for cardiac markers. In other experiments, animals were sacrificed at these same time
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points for cardiac electron microscopy.
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Materials and Methods
Animal care
The protocol (ASP CCM09-03) used in the present study was approved by the Animal
Care and Use Committee of the Clinical Center, National Institutes of Health.
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Study Design
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Twenty-six weekly experiments were performed. Each week, Sprague-Dawley rats (200
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to 230g) with carotid arterial and jugular venous catheters were randomized to receive challenge
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with doses of B. anthracis ET (400 ug/kg EF + 800 ug/kg PA) or LT (50 ug/kg LF + 100 ug/kg
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PA) previously shown to produce approximate 50% lethality rates (4) or with PA alone (control)
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as continuous 24h infusions. A total of 243 animals were studied. Each experiment included at
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least one animal from each group. In one set of experiments (Study 1) animals randomly selected
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at 8, 24 or 48h, had blood pressure and heart rate (HR) measured while awake via their
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indwelling carotid arterial catheters, after which they were lightly anesthetized with isoflurane
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(1-3%) and had echocardiography performed. After echocardiography, animals were more
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deeply anesthetized (isoflurane 3-5%) and their hearts were excised and perfused in a constant
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pressure perfusion system. Immediately before heart excision, arterial blood was drawn for
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creatine phosphokinase (CPK), cardiac troponin I (cTnI), alanine and aspartate amino-
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transferases (ALT and AST), blood urea nitrogen (BUN), creatinine (Cr), sodium (Na+),
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potassium (K+), arterial blood gas, and lactate measures at 8, 24 and 48h. A subgroup of animals
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(n=28) in Study 1 had blood pressure and HR measured for 24h, immediately after which their
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hearts were excised and perfused and perfusion effluent was collected for CPK, cTnI and cAMP
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measures. The number of animals randomized and then studied in each experiment for Study 1 at
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the 3 time points is shown in Table 1. In a second set of experiments (Study 2), animals were
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randomly selected at 8, 24 or 48h and anesthetized and sacrificed for cardiac electron
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microscopy studies (Table 1). Prior to sacrifice, animals had blood pressure and HR measured
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and blood sampled as in Study 1.
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Echocardiography
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Animals were lightly anesthetized with isoflurane (1 to 3%). M-mode echocardiography
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was performed using a Vevo 770 (Visualsonics Inc, Toronto, Canada) with a frame rate of 300–
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500 frames/s and a 12-MHz linear transducer as previously described (23). Data representing the
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average of nine cardiac cycles from at least two separate scans was analyzed. Heart rate was
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averaged over the number of beats measured. After tracing end diastolic and systolic dimensions,
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manufacturer software computed end diastolic and systolic volumes (EDV and ESV respectively,
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µl), stroke volume (SV, µl), cardiac output (CO, ml/min), and percent ejection fraction (EF %).
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Studies were interpreted without knowledge of study group.
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Langendorff Model
Rats were anesthetized with 3-5% isoflurane and anticoagulated with heparin (500 IU/kg)
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through the inferior vena cava as previously described (11). Hearts were rapidly excised and
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arrested in cold (4o C) Krebs–Henseleit (KH) buffer (10, 11, 24, 27). A cannula was inserted in
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the aorta and the heart was perfused retrograde at constant pressure (95 cmH2O) with KH buffer
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gassed with 95% O2 and 5% CO2 at 37°C and filtered through a 5 µm filter. A water-filled latex
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balloon connected to a water column and pressure transducer was introduced into the left
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ventricle. Left ventricular end-diastolic pressure (LVEDP) was adjusted to between 4 and 8
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mmHg. The hearts were allowed to equilibrate for 10 min and then serial readings of left
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ventricular systolic pressure (LVSP, mmHg), LV end diastolic pressure (LVEDP, mmHg), and
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heart rate (HR, bpm) were measured continuously over a 120 min period (ML880P PowerLab
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16/30 with LabChart Pro, ADInstruments, Colorado Springs, CO). Following equilibration and
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at 30min intervals thereafter, measures obtained over the 5min period immediately preceding
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each designated time point (i.e. 0, 30, 60, 90 and 120min) were averaged for analysis. Coronary
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flow (CF, ml/min) was calculated based on coronary effluent measures every 30min. Hearts
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were perfused with fresh KH buffer throughout. Two to four hearts including both control and
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toxin subjects were studied daily. Calculated data included left ventricular developed pressure
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(LVDP = LVSP – LVEDP, mmHg), rate pressure product (RPP = LVDP x HR, mmHg∙bpm),
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dP/dt max (the maximal rate of change in left ventricular pressure during contraction,
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mmHg/sec), and dP/dt min (the minimal rate of change in left ventricular pressure during
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relaxation, mmHg/sec). In a subgroup of animals at 24h (see above), perfusion effluent was
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collected at 0, 30, 60, and 90min for cAMP, CPK and cTnI measures.
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Electron Microscopy
A section (2x2 mm) of the left ventricular anterior wall was double-fixed in PBS-
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buffered glutaraldehyde (2.5%) and osmium tetroxide (0.5%), dehydrated, and embedded into
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Spurr’s epoxy resin. Ultrathin sections (90 nm) were made and double-stained with uranyl
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acetate and lead citrate, and viewed in a JEOL JEM 1010 transmission electron microscope.
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Myocardial injury was assessed on the ultrastructural level with a numerical scale (0= no injury
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to 5 = most severe injury) by a pathologist blinded to the study groups. The ultrastructural
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assessment included changes in the cardiac muscle’s endothelial cells’ membranes and nuclei,
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evidence of myocyte mitochondrial degeneration and swelling in the outer and inner membranes,
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as well as damage to myocyte sarcomeres and nuclei.
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Hemodynamic and Other Laboratory Measures
Indwelling arterial and central venous catheters from each animal were connected via
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protected extension tubing to pressure transducers for mean blood pressure (MBP) and HR
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measures or to syringe pumps for toxin delivery respectively (5, 16). Arterial blood gases were
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measured with Critical Care Xpress (Nova Biomedical, Waltham, MA). Plasma or effluent
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chemistries (CPK, ALT, AST, BUN and creatinine) were measured with Alfa Wassermann
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(Diagnostic Technologies, West Caldwell, NJ). Plasma and effluent cTnI was measured using
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ELISA (Life Diagnostics, West Chester, PA). Effluent cAMP was measured with a cAMP
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Chemiluminescent Immunoassay Kit (Arbor Assays, Ann Arbor, MI).
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Toxin and Treatments
All toxin components [protective antigen (PA), lethal factor (LF), and edema factor (EF)]
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were recombinant proteins prepared from Escherichia coli and provided by Human Genome
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Sciences (formerly of Rockville, MD) (3, 9, 12) Edema and lethal toxins were comprised of EF
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or LF respectively with PA in ratios of 1:2 on the basis of weight (4). The control solution
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contained diluent and PA alone.
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Statistical Analysis
A Wilcoxon Rank test compared survival between control and either LT or ET
challenged animals and between LT and ET challenged ones, accounting for survival time,
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sacrifice of animals at 8, 24 or 48 h and death within the 48 h experimental period. Electron
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microscopy scores for myocardial cell mitochondrial and endothelial injury were not normally
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distributed and were ranked using PROC RANK for analysis. The cTnI was log-transformed due
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to its abnormal distribution. Two way ANOVA and 2-way repeated measures ANOVA, where
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applicable, accounting for toxin (LT, ET or control) and time of measurement, and one way
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ANOVA comparing different time points in controls and toxin vs control at each time point,
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were performed on all continuous or ranked data using PROC MIXED in SAS Version 9.2
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software (SAS Institute, Inc., Cary, NC). Two-sided p-values of 0.05 or less were considered
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significant. Multiple comparisons were not controlled. For echocardiography, electron
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microscopy, and blood chemistry and blood gas measures, values for control animals are shown
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in Table 2, while figures show the effect of each toxin compared to control, calculated by
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subtracting the mean control values from the mean toxin values. For blood pressure, HR and
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perfused heart experiments, figures compare the values for controls and LT and ET challenged
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animals.
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Results
Survival
All animals challenged with PA alone (controls, n=50) survived to the time of sacrifice
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(8, 24 or 48h) (Figure 1). Compared to controls, in animals challenged with LT (n=94) or ET
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(n=99) and after accounting for those sacrificed for studies, survival rates were significantly
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reduced (56.1% and 37.3% respectively) (p<0.0001 for each) but not different when comparing
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the toxins (p=0.66).
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Mean Arterial Blood Pressure and Heart Rate Measures
In controls, compared to baseline MBP decreased over time (p<0.0001 for the time
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interaction), but stayed between 100-120 mmHg. Compared to controls, LT decreased MBP
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from 12 to 20h (p≤0.05) and HR from 14 to 22 h and at 44h (p≤0.05) (Figure 2). Edema toxin
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reduced MBP significantly at each time point (p≤0.05) and in a pattern that increased over time
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(p<0.0001 for the time interaction) and increased HR significantly from 2 to 18 h, but decreased
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it at 30 and 32 h (p≤0.05).
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Echocardiography Measures
On echocardiography in controls, compared to 8h, at 48h HR was lower (0.008) and end-
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diastolic volume was increased in a trend approaching significance (p=0.08) (Table 2).
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Compared to controls, LT challenge decreased left ventricular ejection fraction (LVEF) at 8 and
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48h but increased it at 24h in patterns that differed over time (p=0.049 for the time interaction)
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(Figure 3). Lethal toxin also decreased cardiac output (CO) across the three time points in an
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overall pattern that was significant (p=0.04 averaged over time). At 24h, ET decreased both end-
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systolic and end-diastolic volumes but increased LVEF (p≤0.05). However stroke volume,
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possibly more informative than LVEF in the setting of reduced end-diastolic volume, was
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decreased at 24h with ET, but not significantly. ET also increased HR at 48h (p=0.048).
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Blood Cardiac Markers
Plasma creatine phosphokinase (CPK) and cardiac troponin I (cTnI) measures did not
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change significantly over time in controls (Table 2). Compared to controls, LT challenge did not
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significantly alter blood CPK or cTnI levels at any of the three time points measured (8, 24 or
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48h). ET challenge increased both measures significantly compared to control at 24 and 48h
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(p≤0.05).
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Isolated Perfused Cardiac Measures
Compared to controls and despite its lethal and hypotensive effects, prior in vivo LT
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challenge did not significantly alter at any of the three time points (8, 24 or 48h) LVEDP, LVSP,
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LVDP, RPP, HR or dP/dt min (Figures 5 and 6). At 24h, LT produced a small decrease in dP/dt
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max (p=0.054) at a single time point (60min) and decreased CF from 30 to 120min (p≤0.05).
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However, even when the effects of LT on LVSP, LVDP, RPP, dP/dt max and dP/dt min were
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analyzed across all perfusion time points (0, 30, 60, 90 and 120min) at 24h, its effects did not
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differ over time (p=0.19 to 0.49) and when averaged, none of its overall effects were
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significantly different from controls (p=0.33 to 0.46). In perfusion effluent at 24h, LT did not
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significantly alter CPK levels but did increase cTnI at 60min (p=0.02)(Table 3). In contrast to
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LT, prior in vivo ET challenge had marked effects in isolated hearts. Compared to controls, at 8h
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ET increased HR at 30 and 60min of perfusion (p≤0.05). Most notably however, at 24h ET
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caused highly significant reductions in LVSP, LVDP, dP/dt max and RPP and increases in dP/dt
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min evident immediately following equilibration at 24h (p<0.0001 for all except RPP which was
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p=0.0002). Decreases in LVSP and LVDP persisted and continued to be significant (p≤0.05) for
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up to 90 and 60min respectively. Edema toxin also then increased RPP at 120min (p<0.05) at
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24h and increased it from 30 to 120min at 48h (p≤0.001), possibly related to increases in HR
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with ET that were evident throughout perfusion at both 24 and 48h (p≤0.001). At 24 and 48h, ET
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also increased CF throughout the perfusion period (p≤0.001). Finally, at 48h ET increased dP/dt
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max and decreased dP/dt min from 60 to 120min (p≤0.05). ET was not associated with
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significant changes in CPK or cTnI in perfusion effluent. Also, while ET at 24h was associated
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with increases in cAMP levels in perfusion effluent at 0, 30 and 120min, these changes were not
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significant (Table 3).
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Cardiac Electron Microscopy
Electron microscopy revealed minimal myocardial injury from either LT or ET
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challenge. Compared to controls (Table 2), hearts from LT challenged animals demonstrated
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myocyte sarcomere injury at 48h (p=0.05) (Figure 7). Hearts from ET challenged animals
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demonstrated myocyte mitochondrial inner membrane and myocyte sarcomere injury at 24h and
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sarcomere injury when averaged over the three time points (p≤0.05).
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Other Blood Measures
Compared to controls (Table 2), LT had no significant effects on chemistry and arterial
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blood gas measures (Figures 8). In contrast ET increased ALT and AST at 24 and 48h, and BUN
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at 8, 24 and 48h, and first decreased Cr at 8h and then increased it at 24 and 48h (p≤0.05 for
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each). Edema toxin also decreased pH at 24h, increased lactate at 24 and 48h and decreased ABE
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and Na+ at all time points, K+ at 8h and AaO2 at 24h (p≤0.05 for each, AaO2 not shown).
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Changes with ET in ALT, BUN, Cr, pH, ABE, lactate, Na+ and K+ were greater at later time
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points (p≤0.05 for the time interactions).
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Discussion
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In the present study in rats, a 24h B. anthracis LT challenge in doses causing significant
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reductions in survival and blood pressure, did not produce evidence of myocardial depression in
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isolated perfused hearts at 8, 24 or 48h as reflected by measures of LVSP, LVDP, or RPP. None
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of these measures in LT challenged hearts differed from control hearts over 120min of
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observation at any of the three time points. Consistent with this, blood CPK and cTnI levels were
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also not altered in LT challenged animals. Although a decrease in dP/dt max was noted at 24h,
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this change was small and only evident at a single time point (60min). In fact when data was
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analyzed over all perfusion time points at 24h, LTs effects on dP/dt max, LVSP, LVDP, RPP and
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dP/dt min were not significantly different compared to controls (p=0.33 to 0.46).
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These findings differ from other studies in mouse, rat, rabbit and canine models in which
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LT challenge was noted on echocardiography to depress in vivo myocardial function as reflected
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by changes in left ventricular volumes, velocity of propagation, circumferential fiber shortening,
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or LVEF (13, 21, 25, 28). In these models LT was also associated with increased cardiac
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enzymes in blood and myocardial injury on electron microscopy (13, 21). In studies inhibiting
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LT, PA directed-monoclonal antibody treatment in LT challenged canines or selective deletion
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of anthrax toxin receptor-2 from cardiomyocytes in LT challenged mice both increased LVEF
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measured with echocardiography (1, 19). Possibly consistent with these other studies, in the
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present one on echocardiography LT decreased LVEF at 8 and 48h but not 24h and reduced
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cardiac output when averaged over all time points. LT was also associated with modest changes
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on electron microscopy at 48h but not in circulating CPK or cTnI levels.
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Differing effects of LT on myocardial function in the present study versus studies like
those noted above (13, 21, 24, 27) may reflect the techniques employed to measure that function.
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Growing data suggests that LT decreases endothelial barrier and vascular smooth muscle
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function (8, 19). These changes could alter preload or afterload and secondarily affect
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myocardial performance measured in vivo with techniques like echocardiography (13, 21, 24,
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27). Of note in the prior canine study we conducted, although pulmonary artery occlusion
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pressure (PAOP) was not altered by LT, CVP was reduced and volume loading increased LVEF
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in some animals (24). Both of these latter findings suggest that reductions in preload related to
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LT may have influenced LVEF. Assessment of the effects of LT on myocardial function
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employing measures less likely influenced by preload and afterload have been limited. On the
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one hand, an abstract reported that pressure-volume measures in four canines demonstrated
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reductions in stroke volume, end systolic pressure and LVEF and increased LV end-diastolic
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pressures in patterns consistent with heart failure (2). However, we showed previously that
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perfusion with LT in concentrations typically producing high lethality rates in vivo did not alter
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the function of hearts isolated from healthy rats and tested for up to 240 min under constant
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pressure in a Langendorff system (11). In that study, LT only altered myocardial function when
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its concentration was increased to10 times those producing lethality in vivo. Lack of an effect of
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LT (unless administered at very high concentrations) may have been because myocardial
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changes with ex vivo toxin administration required more time to develop than the model
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permitted. However, the present findings demonstrate that even when rat hearts are exposed in
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vivo to lethal LT doses over a 24h period, once isolated they do not demonstrate evidence of
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myocardial depression in a constant pressure perfusion system. Consistent with these findings in
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isolated hearts, LT was also not associated with increases in CPK or cTnI in blood and its effects
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on cardiac electron microscopic findings were minimal. The basis for the reduction in CF with
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LT at 24h is unclear. Since CF in this model is dependent on the perfusion pressure the system is
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exposed to, which was constant throughout, and the resistance of the coronary arteries, an effect
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of LT on the latter must be considered. Importantly however, these reductions in CF were not
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associated with significant alterations in the pressure hearts were capable of generating.
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It is possible that ex vivo measurement of heart function at later time points (e.g. 72 or
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96h) after the start of LT infusion might have demonstrated myocardial depression. However,
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lethality with LT appeared complete by 48h in this model, and the relevance of later myocardial
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changes for lethality would be unclear. It’s also possible that the myocardial effects of LT differ
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in the rat compared to other species. Notably, time to lethality with LT and the mechanisms
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underlying this lethality appear to differ in some rat and mouse models (18). However, we have
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found that the effects of either LT or ET on systemic hemodynamics have been comparable in
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species as different as the rat and canine (4, 25). Finally, LT challenge in doses more lethal or
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which had greater hypotensive effects than the ones employed here may have resulted in
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myocardial depression in isolated hearts. Yet LT in the present study produced highly significant
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lethality (p<0.0001) approaching 40% and blood pressure reductions close to when hearts were
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isolated and measured. The reason why cTnI was increased in perfusion effluents at 24h with LT
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challenge while these measures were unchanged in blood immediately before hearts were
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isolated is unclear. However these cTnI levels are inconsistent with the absence of alterations in
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developed pressures or on electron microscopy in hearts exposed to LT.
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Different from LT in the present study, hearts from rats exposed in vivo to a lethal ET
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challenge demonstrated both depressed and stimulated function. Most notably, hearts isolated
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24h after the start of ET, and when systemic blood pressure had reached its lowest point, had
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marked reductions in LVDP, LVSP, RPP and dP/dt max. There are several potential explanations
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for these changes. First, it is possible that ET directly depressed myocardial function. On the one
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hand this appears unlikely though, since acute increases in myocardial cAMP levels with ET
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would be expected to increase contractility (2, 11). As noted above, exposure of hearts isolated
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from healthy animals to ET in concentrations comparable to ones producing lethality in vivo,
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increased LVDP and dP/dt max as well as myocardial tissue and effluent cAMP levels. It is
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possible though that longer exposure to ET in vivo in the present study elicited mechanisms
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capable of producing myocardial depression. We demonstrated that ET induced arterial
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relaxation in an aortic ring model is partially endothelium dependent (15). These findings
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implicate endothelial derived relaxant factors such as nitric oxide (NO) in the cardiovascular
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effects of ET. Studies have shown that cAMP can induce NO production (17, 30), while others
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have found an association between NO and cardiac dysfunction (7, 20). A second possibility
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however is that hypotension and ischemia resulting from ETs systemic vasodilatory effects
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produced myocardial depression secondarily. Consistent with this, ET increased lactate and
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worsened liver and renal function (manifested by increased AST, ALT, BUN and Cr). In this
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regard, increases in CPK and cTnI in blood and changes on cardiac electron microscopy noted
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with ET may reflect myocardial ischemia as opposed to direct myocardial injury. Arguing
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against this possibility though is that while reductions in blood pressure with ET persisted until
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48h, evidence of myocardial depression had resolved by this later time point.
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The depression in LV function in isolated hearts following ET challenge at 24h contrasts
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with the increase in LVEF noted on echocardiography at this time point. However, reductions in
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afterload caused by hypotension with ET, as well as relative differences in end systolic and
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diastolic volumes may have increased LVEF on echocardiography.
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While ET reduced LVDP, LVSP and dP/dt max at 24h, it increased HR in the isolated
heart at all three time points, dP/dt max and RPP at 48h and coronary flow at 24 and 48h. These
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changes are very consistent with increased myocardial cAMP levels and they are similar to ones
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we observed when healthy hearts were exposed to ET in the Langendorff system (11). Notably
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though, while ET increased HR during perfusion in isolated hearts at all three time points, HR
400
increases were only clearly evident in vivo on arterial tracings at 8h and on echocardiography at
401
8 and 48h. Thus, ongoing hypoperfusion or acidosis may have inhibited the chronotropic effects
402
of increased cAMP in vivo. Also, although CF was increased when measured under constant
403
pressure ex vivo, this increase may have been insufficient to maintain myocardial perfusion in the
404
face of systemic hypotension with ET in vivo. Of note, increased HR and CF were still evident at
405
48h in isolated hearts (i.e. 24h following the cessation of ET challenge) and may have reflected
406
the residual effects of toxin or were compensatory ones related to the prolonged systemic
407
hypotension caused by ET.
408
In conclusion, the findings from this study in a Langendorff constant pressure perfused
409
rat heart model do not support a direct myocardial depressant effect of B. anthracis LT. While
410
ET challenge did depress myocardial function at 24h, whether this was a direct effect of the toxin
411
or was related to systemic hypotension and secondary ischemia is not clear. Further defining the
412
mechanisms underlying the cardiovascular and lethal effects of these toxins will help improve
413
the management of B. anthracis infection in the future.
414
415
416
417
418
419
18
420
Acknowledgements
421
422
423
424
425
426
427
428
429
430
431
432
433
434
435
436
437
438
439
440
441
442
19
443
444
445
Figures Legends
446
447
Figure 1. The proportion of animals surviving over time following the initiation of 24h infusions
448
of edema toxin (ET) or lethal toxin (LT) or diluent with protective antigen only (controls).
449
Animals sacrificed for studies were considered to be survivors up until the time of sacrifice, at
450
which time they were censored from further analysis. Both ET and LT resulted in progressive
451
reductions in survival in patterns that were significantly different from controls (p<0.0001), but
452
which did not differ when comparing the two toxins (p=0.66).
453
454
Figure 2. Serial mean (±sem) mean arterial blood pressures (Panel A) and heart rates (Panel B)
455
measured for up to 48h following the initiation of 24h infusions of lethal or edema toxin or
456
control .
457
458
Figure 3. Mean (±sem) effects of lethal toxin (LT) or edema toxin (ET), compared to controls
459
on echocardiography determined left ventricular end-systolic (Panel A) and end-diastolic (Panel
460
B) volumes, left ventricular ejection fraction (Panel C), heart rate (Panel D), stroke volume
461
(Panel E), and cardiac output (Panel F) at either 8, 24 or 48h following the initiation of 24h
462
infusions of toxin or diluent. Effects were calculated by subtracting control values from toxin
463
values (see methods). Data for control animals is presented in Table 2.
464
20
465
Figure 4. Mean (±sem) effects of lethal toxin (LT) or edema toxin (ET), compared to controls on
466
blood creatine phosphokinase (CPK; Panel A) and cardiac troponin I (cTnI; Panel B) levels at
467
either 8, 24 or 48h following the initiation of 24h infusions of toxin or diluent. Effects were
468
calculated by subtracting control values from toxin values (see methods). Data for control
469
animals is presented in Table 2.
470
471
Figure 5. Mean (±sem) left ventricular end diastolic pressures (LVEDP), LV developed pressures
472
(LVDP), LV systolic pressures (LVSP) and dP/dt max in hearts excised from animals and
473
perfused under constant pressure at either 8, 24 or 48h following the initiation of 24h infusions
474
of lethal toxin (LT), edema toxin (ET) or diluent with protective antigen only (controls).
475
476
Figure 6. Mean (±sem) dP/dt min, heart rates (HR), rate pressure products (RPP) and coronary
477
flows (CF) in hearts excised from animals and perfused under constant pressure at either 8, 24 or
478
48h following the initiation of 24h infusions of lethal toxin (LT), edema toxin (ET) or diluent
479
with protective antigen only (controls).
480
481
Figure 7. Mean (±sem) effects of lethal toxin (LT) or edema toxin (ET), compared to controls
482
on the ranked tissue injury scores determined with electron microscopy in sections taken from
483
the hearts of animals at either 8, 24 or 48h following the initiation of 24h infusions of toxin or
484
diluent. The ranked injury scores were determined for endothelial membranes (Panel A) and
485
nuclei (Panel B), and for myocyte mitochondrial outer (Panel C) and inner (Panel D) membranes
486
and myocyte sarcomeres (Panel E) and nuclei (Panel F) Effects were calculated by subtracting
487
control values from toxin values (see methods). Data for control animals is presented in Table 2.
21
488
489
Figure 8. Mean (±sem) effects of lethal toxin (LT) or edema toxin (ET), compared to controls on
490
plasma or blood alanine aminotransferase (ALT; Panel A), aspartate aminotransferase (AST;
491
Panel B), blood urea nitrogen (BUN; Panel C), creatinine (Panel D), arterial blood pH (Panel E),
492
lactate (Panel F), arterial base excess (ABE; Panel G), Sodium (Na+; Panel H) and potassium
493
(K+; Panel I) from animals at either 8, 24 or 48h following the initiation of 24h infusions of toxin
494
or diluent. Effects were calculated by subtracting control values from toxin values (see methods).
495
Data for control animals is presented in Table 2.
496
497
498
22
499
References
500
1. Barochia AV, Cui X, Sun J, Li Y, Solomon SB, Migone TS, Subramanian GM, Bolmer
501
SD, and Eichacker PQ. Protective antigen antibody augments hemodynamic support in
502
anthrax lethal toxin shock in canines. J Infect Dis 205: 818-829, 2012.
503
2. Cheng CP, Masutani S, Cheng HJ, Cross M, Zhang CX, Zhou P, Cann J, Cline JM,
504
Little WC, Kuo SR, and Frankel AE. Progressive left ventricle, myocyte dysfunction, and
505
heart failure in the lethality of anthrax toxin in conscious dogs. Circulation 116: 758-758,
506
2007.
507
3. Cooksey BA, Sampey GC, Pierre JL, Zhang X, Karwoski JD, Choi GH, and Laird MW.
508
Production of biologically active Bacillus anthracis edema factor in Escherichia coli.
509
Biotechnol Prog 20: 1651-1659, 2004.
510
4. Cui X, Li Y, Li X, Laird MW, Subramanian M, Moayeri M, Leppla SH, Fitz Y, Su J,
511
Sherer K, and Eichacker PQ. Bacillus anthracis edema and lethal toxin have different
512
hemodynamic effects but function together to worsen shock and outcome in a rat model. J
513
Infect Dis 195: 572-580, 2007.
514
5. Cui X, Moayeri M, Li Y, Li X, Haley M, Fitz Y, Correa-Araujo R, Banks SM, Leppla
515
SH, and Eichacker PQ. Lethality during continuous anthrax lethal toxin infusion is
516
associated with circulatory shock but not inflammatory cytokine or nitric oxide release in
517
rats. Am J Physiol Regul Integr Comp Physiol 286: R699-709, 2004.
518
6. Duesbery NS, Webb CP, Leppla SH, Gordon VM, Klimpel KR, Copeland TD, Ahn NG,
519
Oskarsson MK, Fukasawa K, Paull KD, and Vande Woude GF. Proteolytic inactivation
520
of MAP-kinase-kinase by anthrax lethal factor. Science 280: 734-737, 1998.
23
521
522
523
524
7. Garcia-Estan J, Ortiz MC, and Lee SS. Nitric oxide and renal and cardiac dysfunction in
cirrhosis. Clin Sci (Lond) 102: 213-222, 2002.
8. Guichard A, Nizet V, and Bier E. New insights into the biological effects of anthrax toxins:
linking cellular to organismal responses. Microbes Infect 14: 97-118, 2012.
525
9. Gwinn W, Zhang M, Mon S, Sampey D, Zukauskas D, Kassebaum C, Zmuda JF, Tsai
526
A, and Laird MW. Scalable purification of Bacillus anthracis protective antigen from
527
Escherichia coli. Protein Expr Purif 45: 30-36, 2006.
528
529
530
10. Hearse DJ and Sutherland FJ. Catecholamines and preconditioning: studies of contraction
and function in isolated rat hearts. Am J Physiol 277: H136-143, 1999.
11. Hicks CW, Li Y, Okugawa S, Solomon SB, Moayeri M, Leppla SH, Mohanty A,
531
Subramanian GM, Mignone TS, Fitz Y, Cui X, and Eichacker PQ. Anthrax edema toxin
532
has cAMP-mediated stimulatory effects and high-dose lethal toxin has depressant effects in
533
an isolated perfused rat heart model. Am J Physiol Heart Circ Physiol 300: H1108-1118,
534
2011.
535
12. Laird MW, Zukauskas D, Johnson K, Sampey GC, Olsen H, Garcia A, Karwoski JD,
536
Cooksey BA, Choi GH, Askins J, Tsai A, Pierre J, and Gwinn W. Production and
537
purification of Bacillus anthracis protective antigen from Escherichia coli. Protein Expr
538
Purif 38: 145-152, 2004.
539
13. Lawrence WS, Marshall JR, Zavala DL, Weaver LE, Baze WB, Moen ST, Whorton EB,
540
Gourley RL, and Peterson JW. Hemodynamic effects of anthrax toxins in the rabbit model
541
and the cardiac pathology induced by lethal toxin. Toxins (Basel) 3: 721-736, 2011.
542
543
14. Leppla SH. Anthrax toxin edema factor: a bacterial adenylate cyclase that increases cyclic
AMP concentrations of eukaryotic cells. Proc Natl Acad Sci U S A 79: 3162-3166, 1982.
24
544
15. Li Y, Cui X, Solomon SB, Remy K, Fitz Y, and Eichacker PQ. B. anthracis edema toxin
545
increases cAMP levels and inhibits phenylephrine-stimulated contraction in a rat aortic ring
546
model. Am J Physiol Heart Circ Physiol 305: H238-250, 2013.
547
16. Li Y, Cui X, Su J, Haley M, Macarthur H, Sherer K, Moayeri M, Leppla SH, Fitz Y,
548
and Eichacker PQ. Norepinephrine increases blood pressure but not survival with anthrax
549
lethal toxin in rats. Crit Care Med 37: 1348-1354, 2009.
550
17. Liu D, Homan LL, and Dillon JS. Genistein acutely stimulates nitric oxide synthesis in
551
vascular endothelial cells by a cyclic adenosine 5'-monophosphate-dependent mechanism.
552
Endocrinology 145: 5532-5539, 2004.
553
554
18. Liu S, Moayeri M, and Leppla SH. Anthrax lethal and edema toxins in anthrax
pathogenesis. Trends Microbiol 22: 317-325, 2014.
555
19. Liu S, Zhang Y, Moayeri M, Liu J, Crown D, Fattah RJ, Wein AN, Yu ZX, Finkel T,
556
and Leppla SH. Key tissue targets responsible for anthrax-toxin-induced lethality. Nature
557
501: 63-68, 2013.
558
559
20. Lv M, Liu K, Fu S, Li Z, and Yu X. Pterostilbene attenuates the inflammatory reaction
induced by ischemia/reperfusion in rat heart. Mol Med Rep, 2014.
560
21. Moayeri M, Crown D, Dorward DW, Gardner D, Ward JM, Li Y, Cui X, Eichacker P,
561
and Leppla SH. The heart is an early target of anthrax lethal toxin in mice: a protective role
562
for neuronal nitric oxide synthase (nNOS). PLoS Pathog 5: e1000456, 2009.
563
22. Remy KE, Qiu P, Li Y, Cui X, and Eichacker PQ. B. anthracis associated cardiovascular
564
dysfunction and shock: the potential contribution of both non-toxin and toxin components.
565
BMC Med 11: 217, 2013.
25
566
23. Su J, Cui X, Li Y, Mani H, Ferreyra GA, Danner RL, Hsu LL, Fitz Y, and Eichacker
567
PQ. SB203580, a p38 inhibitor, improved cardiac function but worsened lung injury and
568
survival during Escherichia coli pneumonia in mice. J Trauma 68: 1317-1327, 2010.
569
570
571
24. Sutherland FJ and Hearse DJ. The isolated blood and perfusion fluid perfused heart.
Pharmacol Res 41: 613-627, 2000.
25. Sweeney DA, Cui X, Solomon SB, Vitberg DA, Migone TS, Scher D, Danner RL,
572
Natanson C, Subramanian GM, and Eichacker PQ. Anthrax lethal and edema toxins
573
produce different patterns of cardiovascular and renal dysfunction and synergistically
574
decrease survival in canines. J Infect Dis 202: 1885-1896, 2010.
575
26. Vitale G, Pellizzari R, Recchi C, Napolitani G, Mock M, and Montecucco C. Anthrax
576
lethal factor cleaves the N-terminus of MAPKKs and induces tyrosine/threonine
577
phosphorylation of MAPKs in cultured macrophages. Biochem Biophys Res Commun 248:
578
706-711, 1998.
579
27. Wang QD, Tokuno S, Valen G, Sjoquist PO, and Thoren P. Cyclic fluctuations in the
580
cardiac performance of the isolated Langendorff-perfused mouse heart: pyruvate abolishes
581
the fluctuations and has an anti-ischaemic effect. Acta Physiol Scand 175: 279-287, 2002.
582
28. Watson LE, Kuo SR, Katki K, Dang T, Park SK, Dostal DE, Tang WJ, Leppla SH, and
583
Frankel AE. Anthrax toxins induce shock in rats by depressed cardiac ventricular function.
584
PLoS One 2: e466, 2007.
585
29. Watson LE, Mock J, Lal H, Lu G, Bourdeau RW, Tang WJ, Leppla SH, Dostal DE,
586
and Frankel AE. Lethal and edema toxins of anthrax induce distinct hemodynamic
587
dysfunction. Front Biosci 12: 4670-4675, 2007.
26
588
589
30. Zhang XP and Hintze TH. cAMP signal transduction induces eNOS activation by
promoting PKB phosphorylation. Am J Physiol Heart Circ Physiol 290: H2376-2384, 2006.
590
591
27
Table 1. The Number of Animals Randomized to 24h Infusions with Diluent and PA alone (Control), Lethal Toxin
(LT) or Edema Toxin (ET) and then Sampled at 8, 24 or 48h (T8, T24 and T48 respectively) after the Start of
Infusion in Each of 12 Experiments (Exp.) in Study 1 for Echocardiography and Perfused Heart Measures and 10
Experiments in Study 2 for Electron Microscopy Measures
Study 1
Exp.
Number of Animals
Control
Randomized
LT
Sampled
Randomized
T8 T24 T48
1
2
3
4
5
6
7
8
9
10
11
12
11*
12*
13*
14*
2
2
2
2
2
2
2
2
2
2
2
1
1
2
2
3
1
1
1
1
1
1
1
1
1
1
1
2
2
3
ET
1
1
1
1
1
1
1
1
1
1
1
Sampled
Randomized
T8 T24 T48
4
3
4
3
4
3
4
5
4
5
4
3
3
3
4
3
1
1
1
1
1
2
1
1
1
1
1
1
2
2
2
1
1
1
1
1
1
1
1
2
1
2
2
Sampled
T8 T24 T48
3
3
4
3
4
3
3
5
4
5
4
3
2
2
2
2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
1
2
1
2
1
1
2
2
2
2
Study 2
1
2
1
1
2
1
3
1
1
2
1
1
2
1
3
1
3
1
1
1
1
3
1
4
2
1
1
2
1
1
2
1
1
5
2
1
2
1
1
4
1
1
6
1
1
2
1
1
7
1
1
3
1
1
2
1
8
5
1
1
3
9
1
1
2
10
1
1
9
4
1
1
2
9
1
1
2
11
1
1
2
10
1
1
4
11
3
* These experiments tested hemodynamics, perfused isolated heart function and effluent myocardial biomarker and
cAMP only
Table 2. Mean (±SEM) Values for Hemodynamic, Echocardiography, Electron Microscopy, Chemistry and Arterial
Blood Gas Measures for Control Animals at 8, 24 and 48h after the Start of an Infusion of Diluent with Protective
Antigen Only
Echocardiography
End-Systolic
End-Diastolic
Ejection Fraction
Heart Rate
Stroke Volume Cardiac Output
Volume (µl)
Volume (µl)
(%)
(bpm)
(µl)
(ml/min)
8
59.2±12.6
257.8±24.8
77.9±3.5
393±22
198±20
77.4±7.5
24
81.5±12.6
309.7±24.8
74.5±3.5
356±25
228±20
83.2±8.4
48
77.1±8.5
309.9±16.7
75.1±2.3
320±15
233±13
74.7±5.1
8
24
48
8
24
48
Endothelial
Membrane
Endothelial
Nucleus
8.3±1.9
6.6±2.2
7.7±1.6
7.8±1.7
6.8±2.5
7.2±1.1
CPK
(U/L)
431±143
235±120
299±115
Electron Microscopy
Myocyte
Myocyte
Mitochondria Outer Mitochondria
Membrane
Inner Membrane
8.8±2.0
6.8±2.0
9.4±2.4
8.9±2.2
6.5±1.2
8.1±1.9
cTnI
[Ln(ng/ml)]
0.01±0.07
0.10±0.05
0.00±0.06
Plasma Chemistry
ALT
(U/L)
52.0±34.3
31.7±28.7
44.6±26.2
AST
(U/L)
160±101
128±84
213±77
Myocyte
Sarcomere
Myocyte
Nucleus
8.9±2.1
9.1±2.3
5.81.5
7.9±1.6
8.6±1.8
7.6±1.5
BUN
(mg/dL)
16.6±9.3
17.6±7.8
21.8±7.1
Creatinine
(mg/dL)
0.34±0.09
0.31±0.07
0.25±0.07
Arterial Blood Gas
pH
Lactate
(mmol/l)
ABE
(mmol/l)
Na+
(mmol/l)
K+
(mmol/l)
8
7.47±0.01
1.25±0.27
2.52±0.69
141.6±0.6
3.41±0.14
24
7.47±0.01
1.01±0.30
1.05±0.78
141.8±0.7
3.43±0.17
48
7.47±0.01
1.05±0.40
1.98±1.05
141.6±0.8
3.28±0.18
CPK-creatine phosphokinase; cTnI-cardiac troponin I; ALT- alanine amino-transferases; AST-aspartate aminotransferases; BUN-blood urea nitrogen; ABE-arterial base excess
Table 3. Mean (±SEM) Values for Isolated Perfused Heart Effluents Creatine Phosphokinases
(CPK), Cardiac Troponin I (cTnI) and cyclic AMP (cAMP) Levels at the Time (0), and 30, 60 and
90 min After Perfusion
Time
PA Control
Lethal Toxin
Edema Toxin
CPK
(U/L)
0
30
60
90
5.3±0.5
3.6±0.5
2.9±0.5
0.9±0.5
5.9±0.6
3.2±0.6
2.1±0.6
2.1±0.6
4.1±0.5
2.3±0.6
2.3±0.6
2.1±0.5
CTNI
[Ln(ng/ml)]
0
30
60
90
0.21±0.02
0.16±0.02
0.16±0.02
0.15±0.02
0.27±0.02
0.24±0.02*
0.20±0.02
0.19±0.02
0.23±0.02
0.17±0.02
0.17±0.02
0.19±0.02
8.0±3.8
7.2±3.8
7.1±3.8
10.3±3.8
8.4±4.1
5.8±4.1
4.3±4.1
10.9±4.1
12.3±3.8
14.9±3.8
6.8±4.1
15.6±3.8
0
cAMP
30
(pmol/ml)
60
90
* p=0.02 compared to PA Control
Fig. 1
Proportion of Animals Surviving
1.0
0.8
0.6
0.4
0.2
Control (n=50)
Edema Toxin (n=99)
Lethal Toxin (n=94)
Toxin or Diluent with
Protective Antigen (PA) Infusion
0.0
0
12
24
36
48
Hours after Initiation of Toxin or Diluent with PA (Control) Infusion
Fig. 2
A. Mean Arterial Blood Pressure (MBP, mmHg)
B. Heart Rate (HR, BPM)
Serial Mean (+sem) of Hemodynamics
140
550
120
PA Control (n=50)
Edema Toxin (n=91)
Lethal Toxin T (n=90)
500
#
*
#
100
#
#
#
#
#
#
*
#
#
#
* *
#
#
#
80
#
450
#
#
#
#
#
400
#
#
#
60
350
#
#
#
#
#
Toxin or PA Infusion
#
#
#
#
#
#
#
#
40
4
8
12
16
20
24
28
32
36
40
44
48
#
*
*
Toxin or PA Infusion
300
0
#
0
4
8
12
16
20
Hours after Initiation of Toxin or PA (Control) Infusion
* p<0.05, # p<0.001 for the effect of toxin versus control
24
28
*
*
32
36
40
44
48
Fig. 3
Increase
No Effect
Decrease
LT
150
ET
60
*
10
50
20
X
0
0
-20
0
-50
-40
-10
-100
-60
*
*
-150
D. Heart Rate (BPM)
Increase
ET
LT
#
40
80
No Effect
20
ET
LT
100
-80
Decrease
Mean (+sem) Effect of ET or LT Compared to Control
80
C. Ejection Fraction (%)
B. End-Diastolic Volume (Pl)
A. End-Systolic Volume (Pl)
-20
E. Stroke Volume (Pl)
60
ET
LT
60
*
40
F. Cardiac Output (ml/min)
LT
40
ET
LT
40
20
20
20
X
0
†
0
-20
ET
0
-20
-40
-20
-40
-60
-60
-80
8
24
48
8
24
48
-40
8
24
48
8
24
48
8
24
48
Hours after Initiation of Toxin or Diluent with Protective Antigen (Control) Infusion
* p<0.05 for the effect of toxin versus control
p=0.049 for the time interaction between toxin versus control
†p=0.04 for the overall effect of toxin versus control over time
#
8
24
48
Fig. 4
B. cTnI [Ln(ng/ml)]
0.4
1000
*
Increase
*
No Effect
X
*
0.2
500
Decrease
Mean (+sem) Effect of ET or LT Compared to Control
A. CPK (U/L)
*
0.0
0
-500
-0.2
-1000
-0.4
8
24
48
8
24
48
8
24
48
8
24
Hours after Initiation of Toxin or Diluent with Protective Antigen (Control) Infusion
* p<0.05 for the effect of toxin versus control
48
LVEDP (mmHg)
Fig. 5
8h
15
48h
24h
Control (n=12)
ET (n=14)
LT (n=14)
Control (n=5)
ET (n=6)
LT (n=6)
20
Control (n=11)
ET (n=13)
LT (n=14)
10
5
*
0
LVDP (mmHg)
120
100
80
60
#
*
*
*
*
40
LVSP (mmHg)
140
120
100
80
#
60
*
dP/dtmax (mmHg/sec)
40
4000
*
3500
*
*
90
120
3000
2500
*
#
2000
1500
0
30
60
90
120
0
30
60
90
120
0
Time of Perfusion (min) Following Equilibration
* p<0.05, # p<0.001 for the comparison of toxin versus control
30
60
dP/dtmin (mmHg/Sec)
Fig. 6
8h
-500
Control (n=5)
ET (n=6)
LT (n=6)
-1000
Control (n=11)
ET (n=13)
LT (n=14)
Control (n=12)
ET (n=14)
LT (n=14)
#
-1500
-2000
450
#
400
HR (BPM)
48h
24h
*
350
#
#
#
#
#
*
*
*
#
#
#
#
#
#
*
300
250
Coronary Flow (ml/min)
RPP (mmHg.bpm)
200
50000
40000
#
#
*
30000
20000
#
10000
24
#
20
#
#
#
16
#
#
#
#
#
#
12
8
4
0
30
60
90
120
0
*
*
*
*
30
60
90
120
0
Time of Perfusion (min) Following Equilibration
* p<0.05, # p<0.001 for the comparison of toxin versus control
30
60
90
120
Fig. 7
Increase
No Effect
Decrease
C. Myocyte Mitochondrial Outer Membrane
B. Endothelial Nucleus
LT
ET
LT
ET
LT
ET
8
4
X
0
-4
-8
-12
D. Myocyte Mitochondrial Inner Membrane
LT
F. Myocyte Nucleus
E. Myocyte Sarcomere
ET
LT
*
LT
ET
ET
*
8
*
4
No Effect
Increase
12
0
X
Decrease
Mean (+sem) Effect of ET or LT on the Ranked Injury Score Compared to Control
A. Endothelial Membrane
12
-4
-8
-12
8
24
48
8
24
48
8
24
48
8
24
48
8
24
48
Hours after Initiation of Toxin or Diluent with Protective Antigen (Control) Infusion
* p<0.05 for the effect of toxin versus control
8
24
48
Fig. 8
Decrease No Effect Increase
A. ALT (U/L)
**
200
X
Decrease No Effect Increase
B. AST (U/L)
150
*
**
50
*
0
#
-100
C. BUN (mg/dL)
100
**
0
0
**
#
-50
-600
-200
-100
-300
0.6
1200
600
*
100
-150
-1200
D. Creatinine (mg/dL)
*
0.4
0.15
8 F. Lactate (mmol/L)
E. pH
6
0.10
0.2
0.05
*
0
-0.2
-0.05
-2
*
-4
-0.10
#
**
-0.15
-0.6
-6
-8
H. Na+ (mmol/L)
G. ABE (mmol/L)
10
5
*
2
0.00
-0.4
**
4
#
X
0.0
Decrease No Effect Increase
Mean (+sem) Effect of ET or LT Compared to Control
300
I. K+ (mmol/L)
10
1.0
5
0.5
0
0.0
#
0
X
-5
**
-10
**
8
24
48
8
**
-5
24
*
48
#
*
24
48
-10
8
24
48
8
-0.5
*
*
#
-1.0
8
24
48
Hours after Initiation of Toxin or Diluent with Protective Antigen (Control) Infusion
* p<0.05 for the effect of toxin versus control; ** p<0.001 for the effect of toxin versus control
#
p<0.05 for the time interaction between toxin versus control;
8
24
48