Both LeTx and ETx toxins, each of which includes two components consisting contribute to shock with anthrax. Common to each 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 (4, 5). 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 (i.e. the cellular energy producing part of each cell). Edema factor is 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 (1-6). 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 (Appendix 1). This model directly measures myocardial function in excised hearts that are allowed to continue to beat in an ex vivo system (7-13). The present study is being conducted in three parts. In the initial part presently underway, procedures are being developed to establish and determine the stability of the perfused rat heart model. These methods will be applied both to hearts from animals not previously undergoing intervention as well as in those with indwelling catheters undergoing in vivo hemodynamic measures including cardiac echocardiography (using sound waves to measure heart function) immediately prior to heart excision. In the second part, we will assess 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, we will assess 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 will have in vivo cardiopulmonary measures performed including blood pressure, heart rate, cardiac echo and arterial blood gas measures. Animals will be studied and their hearts removed for perfused heart preparation 8, 24, or 48 h after the initiation of a 24 h toxin infusion. For comparison throughout, we will also assess the effects of lipopolysaccharide (LPS, a toxin produced by commonly encountered types of bacteria) as a positive control or diluent ( negative control) challenge. During perfused heart studies, in addition to cardiac functional measures we will also collect perfusates 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, hearts will either be fixed with gluteraldehyde for EM studies including assessment of mitochondrial number and structure or they will be prepared for later mitochondrial, MAPKK, cAMP, gene microarray, quantitative PCR, and apoptosis analysis.
NIH ExPorter
NIH ExPorter