Molecular basis of age-dependent changes in airway smooth muscle functions

描述(由申请人提供)：临床研究表明，随着年龄的增长，呼吸功能显著丧失，导致生活质量下降，其他疾病的倾向增加，治疗阻塞性呼吸道疾病的气雾剂药物输送无效。然而，这种年龄相关性呼吸丧失的细胞和分子基础尚不清楚。气道平滑肌(ASM)收缩状态通过影响呼吸道内径对呼吸起着重要的调节作用，因此ASM是治疗阻塞性呼吸道疾病的药物靶点。在这里，我们建议使用从大鼠和人类受试者身上获得的呼吸道和ASM来建立导致老年人呼吸功能丧失的ASM中发生的分子变化。初步研究表明，与“年轻”和“早期”大鼠相比，老年(“老年”)大鼠ASM的收缩和松弛反应减弱。更多的数据表明，衰老促进了ASM的表型转换，在ASM中，平滑肌收缩表型转变为增殖/合成表型。肌球蛋白重链和平滑肌α-肌动蛋白(收缩表型标记蛋白)在老年大鼠ASM中的表达低于青年大鼠。此外，ASM细胞的整体转录组分析显示，Myostatin的表达减少，Myostatin是已知的抑制心肌细胞增殖的转化生长因子-b家族的成员。最后，来自ASM细胞的初步数据表明，随着年龄的增长，激活ASM上G蛋白偶联受体(GPCRs)的收缩和松弛药物的细胞内信号减弱。基于这些研究，我们假设ASM随着年龄的增长经历了表型调整，导致收缩和松弛反应性降低。我们进一步假设，ASM收缩的3个关键调控特征的改变是这种表型转换的基础：1)GPCR信号/药物-机械耦合；2)机电耦合；3)动态细胞骨架组装。最后，我们假设ASM中Myostatin的表达减少是推动表型随年龄变化的主要上游机制。在具体目标1中，我们建议使用肌图仪、光磁扭曲细胞术和牵引显微镜等新工具，利用大鼠和人的呼吸道以及来自三个不同年龄组的ASM细胞来建立ASM表型的变化。在具体目标2中，我们建议通过识别与年龄相关的GPCR信号和药物-机械耦合的变化，膜去极化/超极化及其效应器的调节，以及ASM细胞骨架组装的调节，来建立AGE导致ASM收缩/松弛能力丧失的机制基础。目的3将探讨肌肉抑制素作为ASM表型随年龄变化的主要上游驱动因素的作用机制，以及目标2中详细描述的收缩功能的潜在机制。总之，这些研究试图确定ASM功能中与年龄相关的分子变化，这些变化有助于老年人口呼吸功能的年龄相关下降。我们的发现可能有助于开发改善呼吸功能的工具，并修改老年人阻塞性肺病的诊断和治疗方案。

DESCRIPTION (provided by applicant): Clinical studies have demonstrated a significant loss of respiratory function with age that results in reduced quality of life, increased propensity for other diseases, and ineffective aerosol drug delivery for the treatment of obstructive respiratory diseases. However, the cellular and molecular basis for this age-dependent loss of respiration is unknown. Airway smooth muscle (ASM) contractile state plays a significant role in regulating respiration by influencing the airway diameter and hence ASM is the target of drugs used in the treatment of obstructive airway diseases. Herein we propose to establish molecular changes that occur in the ASM that account for loss of respiratory function in the elderly using airways and ASM obtained from rats and human subjects. Preliminary studies demonstrate diminished contractile and relaxation responses of ASM in aged ("Old") rats compared to "Young" and "Early Aged" rats. Additional data suggest aging promotes phenotype "switching" in ASM in which the smooth muscle contractile phenotype is changed to a proliferative/synthetic phenotype. Expression of myosin heavy chain and smooth muscle a-actin (contractile phenotype marker proteins) is lower in ASM obtained from Old rats compared to Young rats. Furthermore, global transcriptome analyses of ASM cells reveal decreased expression of myostatin, a member of the TGF-b family known to inhibit the proliferation of myocytes. Lastly, preliminary data from ASM cells suggest diminished intracellular signaling with age to both contractile and relaxant agents that activate G protein-coupled receptors (GPCRs) on ASM. Based on these studies we hypothesize that ASM undergoes phenotype modulation with age that results in decreased contractile and relaxant responsiveness. We further hypothesize that alterations in 3 key regulatory features of ASM contractile underlie this phenotype switch: 1) GPCR signaling/pharmaco-mechanical coupling; 2) electromechanical coupling; and 3) dynamic cytoskeleton assembly. Lastly, we hypothesize that reduced expression of ASM myostatin in ASM is the principal upstream mechanism driving phenotype modulation with age. In Specific Aim 1, we propose to establish changes in ASM phenotype using rat and human airways and ASM cells from three distinct age groups, using novel tools such as myograph, optical magnetic twisting cytometry and traction microscopy. In Specific Aim 2, we propose to establish the mechanistic basis by which age causes a loss of the contractile/relaxant capacity of ASM by identifying age-related changes in GPCR signaling and pharmaco-mechanical coupling, regulation of membrane de/hyper-polarization and its effectors, and in the regulation of ASM cell cytoskeleton assembly. Aim 3 will explore the mechanistic role of myostatin as the principal upstream driver of the age-related change in ASM phenotype and those mechanisms underlying contractile function detailed in Aim 2. Collectively, these studies seek to identify age-dependent molecular changes in ASM function that contribute to the age-associated decline in respiratory function in the elderly population. Our findings may help develop tools to improve respiratory functions, and modify diagnostic and treatment regimens for obstructive pulmonary diseases in the elderly.