The Heart Is an Early Target of Anthrax Lethal Toxin in Mice: A Protective Role for Neuronal Nitric Oxide Synthase (nNOS)

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. Injection of pure anthrax lethal toxin (LT) at the levels found in terminally-ill, anthrax-infected animals induces an atypical vascular collapse in several experimental animals without the associated hemorrhagic manifestations and disseminated intravascular coagulopathies seen in bacteria-induced shock states. Nevertheless, LT alone produces the pleural edema that is a classic manifestation of anthrax. All the histopathological changes previously observed in LT-treated mice can be explained as predictable sequelae of vascular insufficiency. In this report, electron microscopic analysis was used to identify rapid and striking pathological changes in endothelial cells, myocytes, mitochondria, and the sarcoplasmic reticulum cisternae in the hearts of LT-challenged mice. These morphological changes were paralleled by release of very high levels of multiple cardiac injury biomarkers. LT-induced pathological changes occurred earlier and with markedly higher severity in nNOS knockout mice compared to wild-type or iNOS or eNOS knockout mice, reflecting the well-established role of nNOS in maintenance of cardiac function, and suggesting that nitric oxide produced by nNOS counteracts the damage induced by LT.