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. 2009 May;5(5):e1000456.
doi: 10.1371/journal.ppat.1000456. Epub 2009 May 29.

The heart is an early target of anthrax lethal toxin in mice: a protective role for neuronal nitric oxide synthase (nNOS)

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The heart is an early target of anthrax lethal toxin in mice: a protective role for neuronal nitric oxide synthase (nNOS)

Mahtab Moayeri et al. PLoS Pathog. 2009 May.

Abstract

Anthrax lethal toxin (LT) induces vascular insufficiency in experimental animals through unknown mechanisms. In this study, we show that neuronal nitric oxide synthase (nNOS) deficiency in mice causes strikingly increased sensitivity to LT, while deficiencies in the two other NOS enzymes (iNOS and eNOS) have no effect on LT-mediated mortality. The increased sensitivity of nNOS-/- mice was independent of macrophage sensitivity to toxin, or cytokine responses, and could be replicated in nNOS-sufficient wild-type (WT) mice through pharmacological inhibition of the enzyme with 7-nitroindazole. Histopathological analyses showed that LT induced architectural changes in heart morphology of nNOS-/- mice, with rapid appearance of novel inter-fiber spaces but no associated apoptosis of cardiomyocytes. LT-treated WT mice had no histopathology observed at the light microscopy level. Electron microscopic analyses of LT-treated mice, however, revealed striking pathological changes in the hearts of both nNOS-/- and WT mice, varying only in severity and timing. Endothelial/capillary necrosis and degeneration, inter-myocyte edema, myofilament and mitochondrial degeneration, and altered sarcoplasmic reticulum cisternae were observed in both LT-treated WT and nNOS-/- mice. Furthermore, multiple biomarkers of cardiac injury (myoglobin, cardiac troponin-I, and heart fatty acid binding protein) were elevated in LT-treated mice very rapidly (by 6 h after LT injection) and reached concentrations rarely reported in mice. Cardiac protective nitrite therapy and allopurinol therapy did not have beneficial effects in LT-treated mice. Surprisingly, the potent nitric oxide scavenger, carboxy-PTIO, showed some protective effect against LT. Echocardiography on LT-treated mice indicated an average reduction in ejection fraction following LT treatment in both nNOS-/- and WT mice, indicative of decreased contractile function in the heart. We report the heart as an early target of LT in mice and discuss a protective role for nNOS against LT-mediated cardiac damage.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. nNOS−/− mice are more susceptible to LT.
nNOS−/−, eNOS−/−, iNOS−/−, and C57BL6/J mice were treated with LT ((A) 100 µg IP, or (B) 40 µg IP) and monitored for survival. The experiment shown in (A) represents data from four independent toxin challenge experiments that included all four mouse strains. Eleven additional independent toxin challenge experiments that included controls and nNOS−/− mice also yielded the same results. The n values for panel (A) are as follows: C57BL/6J (n =  36), nNOS−/− (n = 33), eNOS−/− (n =  16), iNOS−/− (n = 21). Logrank test P-values for comparison of nNOS−/− mouse survival curves with each of the eNOS−/−, iNOS−/−, and C57BL/6J mouse survival curves are <0.0001 for panel A. The experiment in (B) (n = 10 for each strain) is a single experiment representative of two independent experiments.
Figure 2
Figure 2. nNOS−/− mouse macrophages are not sensitive to LT and nNOS−/− mice do not release IL-1β following LT treatment.
(A) BMDMs derived from C57BL/6J, nNOS−/− and Balb/cJ mice were treated with varying doses of LT for 2 h and viability assessed by MTT staining. Percent viability is expressed relative to untreated controls. (B) Balb/cJ, C57BL/6J, and nNOS−/− mice were treated with LT (100 µg IP) and bled at four time points after LT injection (n = 2 mice/time point). IL-1β level in serum was assessed by ELISA.
Figure 3
Figure 3. 7-NI sensitizes nNOS-sufficient WT mice to LT.
C57BL/6J or nNOS−/− mice were treated with 7-NI (50 mg/kg or 25 mg/kg), PBS, or drug vehicle (10% DMSO) according to the schedule shown in the top panel. LT was administered on day 3 and animals were followed for survival (n = 5 for each group). Data is representative of two similar experiments. Logrank test P-values were <0.005 when comparing survival curves for C57BL/6J /25 mg/kg+LT, C57BL/6J /50 mg/kg+LT, nNOS−/−/PBS+LT, and nNOS−/−/vehicle+LT mouse groups to either the C57BL/6J/PBS or C57BL/6J/vehicle mouse groups.
Figure 4
Figure 4. Histopathological analysis of LT-treated hearts in nNOS−/− mice.
Left and central panels shown H&E analysis (10× and 100×) of heart sections from mice treated with PBS (top) or 25 h after LT (100 µg IP) treatment (bottom). Arrows point to the swollen pale cardiomyocytes seen only in toxin-treated mice. Right panels are TUNEL stain (20×) of mouse cardiac tissue from the same untreated (top) and LT-treated (bottom) mice. Single arrow points to a single TUNEL-positive cell in the untreated control.
Figure 5
Figure 5. Cardiac injury biomarkers myoglobin, cTroponin-I (cTnI), and heart-type fatty acid binding protein (H-FABP) are elevated after LT treatment of mice.
Groups of C57BL/6J (WT) (n = 49) and nNOS−/− mice (n = 36) were injected with a single bolus of LT (100 µg IP) and groups of mice were bled to obtain serum at various time points after injection. Three control animals were treated with PA (100 µg IP) or left untreated (NT). Levels of myoglobin (A), cTroponin-I (B) and H-FABP (C) were measured for all samples by ELISA. Each symbol represents an individual mouse; mean measurements for each group are shown.
Figure 6
Figure 6. Electron microscopy of LT-treated nNOS−/− and WT mouse hearts.
Groups of C57BL/6J (WT) (n = 10) and nNOS−/− mice (n = 10) were injected with a single bolus of LT (100 µg IP) and hearts harvested at 12 h (when both strains show no malaise symptoms) or after moderate to marked malaise symptoms (28–30 h for nNOS−/− and 55–60 h for C57BL/6J mice). Three control mice were treated with PA (100 µg IP) or left untreated (NT). Images representative of each group are shown at differing magnifications as shown by the scale markers. (A) Untreated C57BL/6J with intact myocytes and endothelium (E = erythrocyte, N = myocyte nucleus). (B–C) Untreated nNOS−/− mice with swollen sarcoplasmic reticulum (SR) cisternae (*) and general mitochondrial swelling. (D–I) nNOS−/− mice, +LT, 28 h: (D) Capillary necrosis (e = endothelial cell) and inter-fiber edema (*). (E) necrotic capillary with expansion of lumen (c), inter-fiber edema (*), and debris (E =  erythrocyte). (F) endothelial necrosis (pyknotic endothelial nucleus, arrowhead); increased density of endothelial intercellular junction (arrow); fragmented myofilament (*)(e = endothelial cell). (G) Capillary necrosis (arrow); inter-fiber edema and cell debris (*). (H) shows detail of (G): myofilament degeneration with separation of myofibrils from mitochondria (**), swollen SR cisternae and transverse (T) tubules (arrow); arrowhead points to lipid. (I) Further detail of (G): endothelial and capillary necrosis with intraluminal erythrocyte (E) and platelet (P); inter-fiber edema and cell debris (*); (e = endothelial cell). (J–L) C57BL/6J mice+LT, 55 h: (J) Endothelial cell swelling, mitochondrial degeneration (arrows), multiple swollen SR cisternae (*); M = mitochondria and e = endothelial cell. (K) Detail of degenerate mitochondrion (arrowhead) and swollen SR (*). (L) Endothelial degeneration; endothelial cytoplasmic swelling with increased vacuolation; irregular endothelial plasma membrane (e = endothelial cell, arrow marks plasma membrane, arrowhead points to representative abnormally enlarged cytoplasmic vacuole).
Figure 7
Figure 7. Effects of allopurinol, nitrite therapy, and carboxy-PTIO on LT-induced mortality.
(A) Allopurinol treatment: C57BL/6J mice were gavaged with allopurinol (300 mg/kg, daily, for 5 days) or PBS. One drug-treated group and the PBS-treated controls were injected with LT (100 µg IP) 2 h after drug/PBS administration on day 2 while a second drug-only control group did not receive toxin. Animals were monitored for survival (n = 5/group). P-value for survival curve of allpurinol/LT vs. PBS/LT is 0.0431. (B) Nitrite Therapy: C57BL/6J and nNOS−/− mice were injected with sodium nitrite (1 mg/kg), IP, daily, 7 days) or PBS. A single bolus of LT was injected into mice 2 h after sodium nitrite administration on day 2 and animals were monitored for survival (n = 7 for all groups, except n = 10 for the C57BL/6J+LT group). Logrank-test based P-value for nNOS−/− PBS/LT vs. nNOS−/− Nitrite/LT is 0.2928 and P-value for C57BL/6J/PBS/LT vs. C57BL/6J/Nitrite/LT is 0.3907. (C) Carboxy-PTIO treatment. C57BL/6J mice were injected daily with carboxy-PTIO (100 µg/200 µl/mouse IP) and LT (100 µg) was injected 2 h after drug injection on the second day (n = 10 for both LT-treated groups and n = 3 for drug only control group). Logrank-test based P-value comparing the C57BL/6J PTIO/LT survival curve to the LT alone survival curve is 0.0248.

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