Stress-induced loss of BIV-spectrin regulates cardiac fibroblast function and long-range communication

项目摘要 心肌梗死(MI)是世界范围内死亡和残疾的主要原因，影响着大约80万美国人 每年一次。受损组织的最佳愈合需要在空间和时间上保持微妙的平衡 炎症和修复机制造成纤维性瘢痕。心脏成纤维细胞(CFs)是主要的 是纤维化重塑的诱因。在缺血性损伤后，CFS转变为激活的表型，即 以增加增殖、向梗死区迁移和分泌纤维蛋白和 旁分泌信号。同时，CF对损伤反应的失调可以促进病理性纤维化， 心律失常和心脏功能不全的风险增加。虽然已经有许多研究探索了不同的 导致CF激活的信号级联和应激源，这些应激源如何调节CF表型和 旁分泌信号的产生，无论是在空间上还是在时间上，仍然难以捉摸。 最近的研究发现，应激诱导的细胞骨架蛋白βIV-SPECTIN的丢失是CF的重要步骤 活化与纤维化。此外，还发现βIV-血影蛋白的丢失依赖于钙/钙调蛋白依赖的蛋白 激酶II(CaMKII)。βIV-Spectrin/CaMKII在调节CF基因表达中的广泛作用已被发现 通过与信号转导分子的相互作用和信号分子转录3(STAT3)3，4的激活 以及促进促纤维化机制的转录因子。具体来说，CaMKII被激活并促进 βIV-SPECTIN的丢失和STAT3的重新分布到细胞核，导致基因表达的变化。一起， 这导致了一种假设，即βIV-SPECTIN/STAT3复合体作为信号转导节点，是 调节心肌梗死后心脏成纤维细胞的激活、募集和瘢痕形成。为了评估这一假设，Aim 1将确定βIV-SPECTIN/STAT3复合体在CF激活和远程通信中的作用。CFS 将受到生物力学拉伸和神经激素刺激，与心肌梗死病理生理学相关，以 评价其对CF激活和外切体分泌的影响。要了解远程CFS如何迁移到 梗死区，来自血影蛋白缺乏的CFS的远程通信信号将被表征和培养 使用新鲜的CFs，看看它们是否会导致激活。此外，该项目将提供对 血影蛋白在慢性阻塞性肺疾病外体分泌调控中的时空调节作用 压力。最后，Aim 2将使血影蛋白保留和血影蛋白缺陷的小鼠接受心肌梗死，并评估其效果 关于疤痕的形成和成熟。这些研究将为深入了解特定的压力组合如何调整 心肌梗死后纤维化重塑的过程，以及这些调节蛋白如何影响心肌梗死后的整体预后 心肌梗塞患者。

Project Summary Myocardial infarction (MI) is a major cause of death and disability worldwide, affecting ~800,000 Americans annually. Optimal healing of the damaged tissue requires the delicate balance, both spatially and temporally, of inflammatory and reparative mechanisms to create the fibrotic scar. Cardiac fibroblasts (CFs) are the main contributor to fibrotic remodeling. Following ischemic injury, CFs transition into an activated phenotype that is characterized by increased proliferation, migration to the infarct region, and secretion of fibrotic proteins and paracrine signals. At the same time, dysregulation of the CF response to injury can promote pathological fibrosis, increased risk for arrhythmia, and cardiac dysfunction. While there has been many studies exploring the diverse signaling cascades and stressors that cause CF activation, how these stressors regulate the CF phenotype and paracrine signal generation, both spatially and temporally, remain elusive. Recent work identified stress-induced loss of the cytoskeletal protein, βIV-spectrin, to be an important step in CF activation and fibrosis3. Further, loss of βIV-spectrin was found to depend on Ca2+/ calmodulin-dependent protein kinase II (CaMKII). A broader role has been identified for βIV-spectrin/CaMKII in regulating CF gene expression through an interaction with signal transducer and activation of transcription 3 (STAT3)3,4, a signaling molecule and transcription factor that promotes profibrotic mechanisms. Specifically, CaMKII is activated and promotes loss of βIV-spectrin and redistribution of STAT3 to the nucleus that lead to changes in gene expression. Together, this leads to the hypothesis that the βIV-spectrin/STAT3 complex acts as a signaling node that is necessary for regulating cardiac fibroblast activation, recruitment, and scar formation post MI. To evaluate this hypothesis, Aim 1 will identify the role of the βIV-spectrin/STAT3 complex in CF activation and long-range communication. CFs will be subjected to both biomechanical stretch and neurohormonal stimuli, correlating to MI pathophysiology, to evaluate the effects on CF activation and exosome secretion. To understand how remote CFs migrate to the infarct area, long-range communication signals from spectrin-deficient CFs will be characterized and cultured with fresh CFs to see if they lead to activation. Additionally, this project will offer mechanistic insight into the spatiotemporal regulatory role of spectrin-based proteins in modulating exosome secretion following chronic stress. Lastly, Aim 2 will subject spectrin-preserved and spectrin-deficient mice to MI and evaluate the effects on scar formation and maturation. These studies will offer insight into how specific stress combinations tune the process of fibrotic remodeling following MI, and how these regulatory proteins can affect the overall outcome of MI patients.