Use of a Perfused Rat Heart Model to Investigate Anthrax Lethal and Edema Toxins

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Abstract

Both LeTx and ETx toxins, contribute to shock with anthrax. Common to each toxin is a component called protective antigen (PA), which is necessary for cellular uptake of the toxic components; lethal factor (LF) for LeTx and edema factor (EF) for ETx. Both factors are capable of disrupting key steps in essential but very different intracellular signaling pathways. Lethal factor is a protease, which inhibits stress kinase pathways and has been shown to inhibit host defense and other responses necessary for the survival of the host. Emerging data also suggests that LF may have a direct depressant effect on mitochondrial function. Edema factor has adenyl cyclase activity and increases intracellular cAMP levels to very high levels. While edema factor is capable of depressing host defense, it may also have direct effects on myocardial and vascular function via its adenyl cyclase activity. Findings from studies we have now conducted in toxin challenged rat and canine models have raised several questions regarding the effects of LeTx and ETx. First, does LeTx decrease heart rate or the strength with which the heart pumps. Second, does LeTx initiate reactions that lead to later reductions in heart strength despite cessation of toxin administration? Third, does ETx increase heart rate directly? Fourth, does ETx produce reductions in the strength of the heart that the canine model was insensitive to detecting? The present study is designed to further investigate the effects of LeTx and ETx on myocardial function employing a re-circulating Langendorff perfused rat heart model. This model directly measures myocardial function in excised hearts that are allowed to continue to beat in an ex vivo system. The present study is being conducted in three parts. In the initial part procedures were developed to establish and determine the stability of the perfused rat heart model. The second part has assessed the direct effects of LeTx and ETx added alone or together to the perfusion circuit on the heart rate and contractility of hearts taken from normal animals. In the third part, now underway, we are assessing the heart rates and contractility of hearts taken from animals previously challenged with either LeTx or ETx alone or together. In this final part, prior to removal of hearts for perfusion, animals are undergoing in vivo cardiopulmonary measures performed including blood pressure, heart rate, cardiac echo and arterial blood gas measures. During perfused heart studies, in addition to cardiac functional measures, perfusate is collected at timed intervals to measure markers of cardiac injury (creatinine phosphokinase, myocardial troponin I, cardiac myosin light chain-1), nitric oxide, and cytokines. At the completion of perfusion, in some studies, hearts are fixed with gluteraldehyde for EM studies including assessment of mitochondrial number and structure or they are prepared for mitochondrial, MAPKK, cAMP, gene microarray, quantitative PCR, and apoptosis analysis. Experiments have been completed for this part and a manuscript is in preparation for submission.