Tsg101 and endosomes in cardiac surgery-induced injury

冠状动脉旁路移植术(CABG)和心脏移植的心脏手术通常涉及心脏 缺血/再灌注(I/R)，导致心肌能量来源从脂肪酸β- 氧化为厌氧糖酵解。作为一种适应，GLUT-4，葡萄糖转运体的一个主要亚型，在 心脏，被招募到心肌细胞表面(也称为肌膜)吸收葡萄糖并刺激 心脏三磷酸腺苷的产生。然而，这种代偿性GLUT4易位不足以满足心脏疾病 在I/R心脏中，葡萄糖需要产生ATP，从而导致能量危机。值得注意的是，最近 多项涉及向患者输注葡萄糖-胰岛素-钾(GIK)溶液的大型临床试验 正在接受心脏手术的患者没有显示出任何积极的结果(甚至更糟)。因此，探索如何 为了增加Glut-4的易位和葡萄糖的利用，不依赖胰岛素，迫切需要 计数器I/R引发的心脏能量损失。我们最近有了新的发现，肿瘤的易感性 基因101(Tsg101)，ESCRT的中心成分(运输所需的内体分选复合体) 能够调节动物心脏内膜受体的内体循环。我们最新的 数据进一步表明：1)Tsg101直接与成年小鼠心脏中的GLUT-4结合；2)强迫表达 培养的心肌细胞中的Tsg101导致更高水平的肌膜GLUT-4，并在以下情况下改善细胞存活 3)Tsg101上调Rab11a和FIP3的表达 (Rab11-家族相互作用蛋白3)，这是参与内体循环的两个关键因素。最重要的是，我们的 试点数据还显示，一组自然产生的纳米囊泡，即外周小体，由骨骼- 骨髓干细胞，可以有效地将Tsg101导入心肌细胞。根据这些初步调查结果，我们 假设Tsg101可通过促进Rab11a/Tsg101减少或预防心脏I/R所致的能量危机/损伤 FIP3介导的GLUT-4内体循环。含Tsg101外切体对小鼠心脏的治疗作用 缺血前或再灌注早期可升高心肌Tsg101，从而限制I/R触发 心脏受损。这里提出的工作将解决三个具体目标：1)确定Tsg101在 葡萄糖依赖的能量产生和对I/R损伤的心脏保护，使用心脏特异性的 Tsg101过表达和可诱导的基因敲除小鼠模型；2)鉴定Tsg101是否诱导了 心脏保护依赖于Rab11a/FIP3介导的GLUT-4的内体循环； 探讨Tsg101预防/减轻I/R所致心脏能量应激的治疗潜力 和损伤，使用Tsg101负载的外切体。拟议的研究预计将确定Tsg101是一种 GLUT-4易位的新调节因子，作为对抗I/R诱导的能量应激的主要心脏保护器。如果 经过验证，这项提案的发现应该会为增加能源发电量提供新的安全策略 并希望将外科手术引起的心脏I/R损伤降至最低。

Heart surgery for both coronary artery bypass graft (CABG) and transplantation often involves cardiac ischemia/reperfusion (I/R), which leads to a switch of the myocardial energy source from fatty acid β- oxidation to anaerobic glycolysis. As an adaptation, Glut-4, a major isoform of the glucose transporters in the heart, is recruited to the cardiomyocyte surface (also called sarcolemma) to take up glucose and stimulate cardiac ATP production. Nonetheless, such compensatory Glut4 translocation is not sufficient to meet cardiac glucose demands for ATP generation in I/R hearts and thereby, results in an energy crisis. Notably, recent multiple large clinical trials involving infusion of glucose-insulin-potassium (GIK) solution into patients undergoing cardiac surgery have not shown any positive results (or even worse). Therefore, exploring how to augment Glut-4 translocation and glucose utilization, independent of insulin, is desperately needed to counter I/R-triggered cardiac energy loss. We recently made the novel findings that the tumor susceptibility gene 101 (Tsg101), a central component of the ESCRT (endosomal sorting complexes required for transport) machinery, is able to regulate the endosomal recycling of membrane receptors in animal hearts. Our newest data further showed that: 1) Tsg101 binds directly to Glut-4 in adult mouse hearts; 2) forced expression of Tsg101 in cultured myocytes resulted in higher levels of sarcolemma Glut-4 and improved cell survival when challenged with hypoxia/reoxygenation; and 3) Tsg101 up-regulates the expression of Rab11a and FIP3 (Rab11-family interacting protein 3), two key factors involved in endosomal recycling. Most importantly, our pilot data also showed that a group of naturally-occurring nano-vesicles, exosomes, released by bone- marrow stem cells, can effectively deliver Tsg101 into cardiac myocytes. Based on these initial findings, we hypothesize that Tsg101 can reduce or prevent cardiac I/R-induced energy crisis/injury by promoting Rab11a/ FIP3-mediated endosomal recycling of Glut-4. Treatment of mouse hearts with Tsg101-containing exosomes before ischemia or during early reperfusion can elevate myocardial Tsg101, thereby limiting I/R-triggered cardiac damage. The work proposed here will address three specific aims: 1) Define the role of Tsg101 in glucose-dependent energy generation and cardio-protection from I/R injury, using both heart-specific Tsg101-overexpressing and inducible knockdown mouse models; 2) Identify whether Tsg101-induced cardio-protection is dependent on Rab11a/FIP3-mediated endosomal recycling of Glut-4; and 3) Investigate the therapeutic potential of Tsg101 to prevent/reduce I/R-induced cardiac energy stress and injury, using Tsg101-loaded exosomes. The proposed studies are expected to identify Tsg101 as a novel regulator of Glut-4 translocation and as a major cardio-protector against I/R-induced energy stress. If verified, the findings from this proposal should provide new and safe strategies to increase energy generation in I/R hearts and hopefully, minimize surgically induced cardiac I/R injury.