Mechanisms mediating age-dependent inhibition of cerebrovascular MLCK activity and contractility by chronic hypoxia

心血管不稳定是NICU婴儿的常见特征，通常会导致脑部受损 自我调节、缺氧缺血性脑损伤和脑室出血。我们最近的研究表明 出生后心血管不稳定涉及肌球蛋白轻链激酶(MLCK)功能低下， 负责启动和调节血管收缩的限制性酶。因为信使核糖核酸 MLCK的转录随年龄和低氧变化不大，我们的结果暗示了mRNA翻译的变化， MLCK降解和MLCK活性是控制新生儿MLCK功能的主要机制。第一, 我们将研究microRNAs对MLCK翻译的影响。低氧诱导了大量的微小RNA 并通过与转录本的结合直接影响收缩蛋白的表达，通过影响转录间接影响收缩蛋白的表达 平滑肌分化。为了探索这些机制，我们开发了外科方法， 使早产羔羊能够在体内、在子宫内进行腺病毒转染术。这种方法提供了 探索微小RNA在胎儿对低氧应激反应中的分子作用的前所未有的机会， 尤其是与MLCK功能调节有关的基因。第二，我们将研究泛素化和泛素化的作用 胎儿血管对低氧反应中的蛋白质降解。尽管人们认识到 泛素化，尚未在胎羊、它们的大脑动脉或它们对缺氧的反应中进行研究。我们的 研究结果表明，一些泛素连接酶的表达与年龄有关，而对另一些泛素连接酶的表达则是有效的 因慢性缺氧而升高。这些结果提出了一种新的观点，即蛋白质降解的变化 密切参与胎儿血管对慢性低氧的适应。第三，我们将研究低氧对 MLCK活性，原位测定。使用新方法测量细胞内钙和肌球蛋白的高速瞬变 轻链在整个动脉中的磷酸化，我们发现MLCK的速度在慢性 胎儿动脉缺氧，但成人动脉不缺氧。我们的共聚焦方法进一步表明MLCK与 其底物在胎儿动脉中比成人动脉中更强，并在慢性低氧中显著改变，提示 MLCK区划在胎儿脑血管收缩调节中的新作用总体而言，进一步 对我们最近工作确定的机制的研究有望揭示出多种重要的新特征 MLCK功能的分子、细胞和组织水平的调节，并提供了对如何 这些机制可能被用来改善出生后心血管不稳定的临床管理。

Cardiovascular instability is a common feature of NICU infants that often leads to compromised cerebral autoregulation, hypoxic-ischemic brain injury, and intraventricular hemorrhage. Our recent work suggests that postnatal cardiovascular instability involves depressed function of Myosin Light Chain Kinase (MLCK), the rate- limiting enzyme responsible for initiation and regulation of vascular contraction. Because rates of mRNA transcription for MLCK vary little with age and hypoxia, our results implicate changes in mRNA translation, MLCK degradation, and MLCK activity as the main mechanisms that govern neonatal MLCK function. First, we will examine effects of micro-RNAs on MLCK translation. Numerous micro-RNAs are induced by hypoxia and influence contractile protein expression directly through binding to transcripts, and indirectly by influencing smooth muscle differentiation. To explore these mechanisms we have developed surgical methods that enable the in vivo adenoviral transfection of pre-term fetal lambs, in utero. This approach offers unprecedented opportunities to explore the molecular roles of micro-RNAs in fetal responses to hypoxic stress, particularly as related to regulation of MLCK function. Second, we will examine the roles of ubiquitination and protein degradation in fetal vascular responses to hypoxia. Despite the recognized importance of ubiquitination, it has not been studied in fetal lambs, their cerebral arteries or their responses to hypoxia. Our findings demonstrate that expression of some ubiquitin ligases is age-dependent and for others is potently upregulated by chronic hypoxia. These results advance the novel idea that changes in protein degradation are intimately involved in fetal vascular adaptation to chronic hypoxia. Third, we will examine effects of hypoxia on MLCK activity, in situ. Using novel methods to measure high-speed transients in cytosolic calcium and myosin light chain phosphorylation in whole arteries, we have found that MLCK velocity is enhanced by chronic hypoxia in fetal but not adult arteries. Our confocal methods further suggest that colocalization of MLCK with its substrate is stronger in fetal than adult arteries, and is significantly altered by chronic hypoxia, suggesting a new role for MLCK compartmentalization in regulation of fetal cerebrovascular contractility. Overall, further study of the mechanisms identified by our recent work promises to reveal multiple important new features of the molecular, cellular, and tissue level regulation of MLCK function, and offers new understanding of how these mechanisms might be leveraged to improve clinical management of postnatal cardiovascular instability.