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

LeTx和ETx毒素都会导致炭疽休克。每种毒素的共同点是一种称为保护性抗原（PA）的组分，它是细胞摄取毒性组分所必需的; LeTx的致死因子（LF）和ETx的水肿因子（EF）。这两种因子都能够破坏基本但非常不同的细胞内信号传导途径中的关键步骤。致死因子是一种蛋白酶，其抑制应激激酶途径，并已显示出抑制宿主防御和宿主生存所必需的其他反应。新出现的数据还表明，LF可能对线粒体功能有直接的抑制作用。水肿因子具有腺苷酸环化酶活性，并将细胞内cAMP水平提高到非常高的水平。虽然水肿因子能够抑制宿主防御，但它也可能通过其腺苷酸环化酶活性对心肌和血管功能产生直接影响。 我们现在在毒素攻击的大鼠和犬模型中进行的研究结果提出了关于LeTx和ETx作用的几个问题。首先，LeTx是否会降低心率或心脏泵送的强度。第二，LeTx是否会引发导致后来心力下降的反应，尽管停止了毒素给药？第三，ETx会直接增加心率吗？第四，ETx是否会降低犬模型对检测不敏感的心脏强度？本研究采用Langendorff再循环灌注大鼠心脏模型，进一步研究LeTx和ETx对心肌功能的影响。该模型直接测量允许在离体系统中继续跳动的切除心脏中的心肌功能。 本研究分三个部分进行。在初始部分中，开发了用于建立和确定灌流的大鼠心脏模型的稳定性的程序。第二部分评估了LeTx和ETx单独或共同加入灌注回路对正常动物心脏心率和收缩力的直接影响。在第三部分中，我们正在评估之前用LeTx或ETx单独或一起挑战的动物的心率和心脏收缩性。在最后一部分，在取出心脏进行灌注之前，对动物进行体内心肺测量，包括血压、心率、心脏回波和动脉血气测量。在灌注心脏研究期间，除了心脏功能测量之外，还以定时间隔收集灌注液以测量心脏损伤的标志物（肌酸酐磷酸激酶、心肌肌钙蛋白I、心肌肌球蛋白轻链-1）、一氧化氮和细胞因子。在灌注完成时，在一些研究中，心脏用戊二醛固定用于EM研究，包括线粒体数量和结构的评估，或者它们被制备用于线粒体、MAPKK、cAMP、基因微阵列、定量PCR和凋亡分析。 这一部分的实验已经完成，目前正在编写一份草稿以供提交。

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.