Mechanisms of cardiomyocyte dysfunction in pediatric septic shock

项目总结/摘要 标题：小儿感染性休克中心肌细胞功能障碍的机制 尽管在儿科败血症方面进行了数十年的研究，但患有败血症的儿童的死亡率仍约为25%。 败血性休克脓毒症相关性心肌功能障碍（SAMD）在儿童中很常见， 死亡率不仅仅是疾病严重程度的反映。由于没有疾病修饰疗法存在， SAMD，有一个关键的需要，以了解脓毒症心肌细胞功能障碍的生物学基础。 此外，考虑到未能实现人体模型的功能，需要使用源自患者的材料的新型人体模型。 将败血症小鼠模型中的分子发现转化为人类器官损伤的改善。我们 目的是建立人诱导多能干细胞（hiPSC）衍生的心肌细胞（hiPSC-CM）， 作为儿科败血症心肌细胞功能障碍的模型，并检查宿主遗传因素的作用。 背景和血清因素在SAMD发病机制中的作用。我们建立了我们的大， 脓毒症儿童血清生物库研究心肌细胞对脓毒症血清库的反应， 患有和未患有SAMD的儿童。我们已经发现，hiPSC-CM的收缩性受到抑制， 用SAMD患儿血清库检测，而用非SAMD患儿的对照脓毒症血清检测不出。这 去除血清后，细胞死亡效应是可逆的，表明这并不反映细胞死亡。 此外，我们还发现，在脓毒症患儿中，白细胞介素-8（IL-8）与SAMD显著相关。 休克，并发现重组人IL-8抑制hiPSC-CM收缩性。我们的提案将解决 作为具体目标的三个重要问题：首先，我们将确定hiPSC-CM和心肌细胞 来自相同儿童的基因具有共同的功能和转录反应模式， 暴露在储存的脓毒血清中我们将提供hiPSC-CM与离体 通过从接受心脏手术的儿童的废弃手术组织中分离心肌细胞， 通过手术和从这些相同的患者产生hiPSC-CM。其次，我们将确定 哪些宿主遗传背景导致SAMD中的心肌细胞功能障碍。我们将开发hiPSC- 来自患有和不患有SAMD的儿科患者的CM，以确定对脓毒症血清的反应，从而确定模式 与SAMD易感性相关的功能和转录反应。第三，我们将剖析 IL-8信号在脓毒症心肌细胞功能障碍中的作用我们将采用IL-8调节 hiPSC-CM中的血清和IL-8受体（CXCR 1和CXCR 2）敲除，为IL-8 阻断作为SAMD的潜在治疗靶点。这个职业发展计划将建立在我的基础上， 在心肌功能障碍的转化研究背景，以获得新的专业知识，在心肌细胞 功能分析、hiPSC生成和分化以及下一代RNA测序技术， 促进我作为一名专注于阐明和瞄准的物理学家和科学家的独立过渡 小儿SAMD的机制。

PROJECT SUMMARY / ABSTRACT Title: Mechanisms of cardiomyocyte dysfunction in pediatric septic shock Despite decades of research in pediatric sepsis, mortality remains at approximately 25% for children with septic shock. Sepsis-associated myocardial dysfunction (SAMD) is common in children and has an association with mortality that is not simply a reflection of the severity of illness. As no disease-modifying therapies exist for SAMD, there is a critical need to understand the biologic basis of cardiomyocyte dysfunction in sepsis. Furthermore, there is a need for novel human modeling with patient-derived materials given the failure to translate molecular discoveries in murine models of sepsis to improvements in human organ injury. Our objectives are to establish human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (hiPSC-CMs) as a model for cardiomyocyte dysfunction in pediatric sepsis and to examine the roles of host genetic background and serum factors in the pathogenesis of SAMD. We have built upon our large, established biobank of serum from children with sepsis to study the cardiomyocyte response to septic serum banked from children who did and who did not have SAMD. We have found that the contractility of hiPSC-CMs is depressed by serum banked from children with SAMD but not by control septic serum from children without SAMD. This depressant effect was reversible after removal of the serum, suggesting this was not reflective of cell death. Furthermore, we identified a significant association of interleukin-8 (IL-8) with SAMD in children with septic shock and found that recombinant human IL-8 depresses hiPSC-CM contractility. Our proposal will address three important questions as specific aims: First, we will determine whether hiPSC-CMs and cardiomyocytes derived from the same children share common functional and transcriptional patterns of responses when exposed to banked septic serum. We will provide a comprehensive comparison of hiPSC-CMs to ex vivo cardiomyocytes by isolating cardiomyocytes from discarded surgical tissue from children undergoing cardiac surgery and by generating hiPSC-CMs from these same patients. Second, we will determine the degree to which host genetic background contributes to cardiomyocyte dysfunction in SAMD. We will develop hiPSC- CMs from pediatric patients with and without SAMD to determine responses to septic serum to identify patterns of functional and transcriptional responses associated with susceptibility to SAMD. Third, we will dissect the role of IL-8 signaling in cardiomyocyte dysfunction in sepsis. We will employ a combination of IL-8 modulation in serum and IL-8 receptor (CXCR1 and CXCR2) knockouts in hiPSC-CMs, providing evidence for IL-8 blockade as a potential therapeutic target in SAMD. This career development proposal will build on my background in translational research in myocardial dysfunction to gain new expertise in cardiomyocyte functional analysis, hiPSC generation and differentiation, and next-generation RNA sequencing technology to facilitate my transition to independence as a physician-scientist focused on elucidating and targeting mechanisms of pediatric SAMD.