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

LeTx和ETX毒素都会导致炭疽病休克。每种毒素都有一种称为保护性抗原(PA)的成分，这是细胞摄取有毒成分所必需的；LeTx的致死因子(LF)和ETX的水肿因子(EF)。这两个因素都能够扰乱基本但截然不同的细胞内信号通路的关键步骤。致死因子是一种蛋白水解酶，它抑制应激激酶通路，并已被证明抑制宿主防御和其他宿主生存所必需的反应。新出现的数据还表明，LF可能对线粒体功能有直接抑制作用。水肿因子具有腺苷环化酶活性，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环化酶活性对心肌和血管功能产生直接影响。 我们目前在毒素攻击的大鼠和犬模型上进行的研究结果提出了几个关于LeTx和ETX影响的问题。首先，LeTx是否会降低心率或心脏泵血的力量。第二，尽管停止给毒素，LeTx是否会引发导致后来心脏力量减弱的反应？第三，ETX是否直接提高心率？第四，ETX是否会导致犬类模型检测不到的心脏强度下降？本研究旨在进一步研究LeTx和ETX对大鼠心脏再循环Langendorff灌流模型的影响。这个模型直接测量了在体外系统中允许继续跳动的切除心脏的心肌功能。 本研究分三部分进行。在最初的部分中，开发了建立和确定大鼠心脏灌流模型的稳定性的程序。第二部分评估了LeTx和ETX单独或联合加入灌流回路对正常动物心脏的心率和收缩能力的直接影响。在目前正在进行的第三部分中，我们正在评估之前单独或联合使用LeTx或ETX挑战的动物的心率和心脏收缩能力。在最后这一部分，在取出心脏进行灌流之前，动物正在进行活体心肺测量，包括血压、心率、心脏回声和动脉血气测量。在灌流心脏研究中，除了心功能测量外，还定期收集灌流液，以测量心脏损伤的标志物(肌酸磷酸酶、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。在一些研究中，在灌流结束时，用丙醛固定心脏进行EM研究，包括评估线粒体的数量和结构，或者准备进行线粒体、MAPKK、cAMP、基因芯片、定量PCR和细胞凋亡分析。这一部分的实验已经完成，并出版了手稿。

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 has been published.