Creation of Optical Biosensor Mice for Longitudinal Studies of Vascular Function

 描述(申请人提供)：高血压涉及血管阻力升高，仅存在于活体动物中，其中血管张力的生理调节(即神经元活动、血液流动和内分泌因素)是完整的。因此，在体内探索动脉平滑肌细胞收缩的分子调控机制，对高血压的研究将大有裨益。我们的总体目标是首次在清醒动物中阐明实验性盐依赖型高血压期间血管阻力增加的机制。使用清醒的动物(而不是麻醉的动物)是理解交感神经活动(SNA)假定作用的关键，SNA越来越被认为是盐依赖型高血压的关键机制。这一总体目标是通过对清醒的光学生物传感器小鼠小动脉中的分子信号进行非侵入性的荧光成像来实现的。鉴于肌球蛋白调节轻链的磷酸化是平滑肌收缩的关键决定因素，特定的目标1和2是开发光学生物传感器小鼠，表达新型的、遗传编码的活性生物传感器，用于平滑肌肌球蛋白磷酸化的关键分子调控分子：a)肌球蛋白轻链激酶(MLCK)，b)肌球蛋白轻链磷酸酶(MLCP)，以及c)MLCP的关键上游调控因子，小GTP酶，RhoA。在目标3中，我们将利用这些光学生物传感器在体内检测：1)某些可能与高血压有关的血管G蛋白偶联受体(α)对MLCK、MLCP和RhoA的调节，包括肾上腺素受体(AT1-AR)、血管紧张素II受体(AT1-R)、内皮素-1受体(ETa)和鞘氨醇-1-磷酸受体(S1P1)。SNA对MLCK、MLCP和RhoA的调节将通过在清醒小鼠中使用完全自主神经节阻滞剂(六甲溴铵)来确定。老鼠将被植入遥测动脉血压传感器，以便在成像期间(以及所有其他时间)连续测量动脉血压。在特定目标4中，将测量在血管紧张素14天内清醒个体小鼠耳小动脉中MLCK、MLCP和RhoA的激活水平的时间进程(即纵向研究)。 II/盐性高血压。小鼠长期注射血管紧张素II，并以高盐(氯化钠)饮食喂养。在这种盐依赖高血压模型中，血管阻力的增加被认为涉及到来自盐敏感的中枢心血管控制区的高SNA(交感兴奋)，强制使用清醒的小鼠。这些具体目标将在这个多PI项目中在两名首席研究人员的指导下进行，他们具有必要的专业知识来生成建议的传感器(RIZO)并执行生理实验(WIER)。综上所述，我们希望首次在活体动物中实现肌球蛋白磷酸化调节过程在盐敏感性高血压形成过程中的动态成像。这些研究将揭示高血压血管阻力增加的分子基础，因为它实际上发生在活动物身上。

 DESCRIPTION (provided by applicant): Hypertension involves elevated vascular resistance and only exists in a living animal, where the physiologic regulators of vascular tone (i.e. neuronal activity, blood flow, and endocrine factors) are intact. Therefore, hypertension research will greatly benefit from the in vivo exploration of the molecular regulators of arterial smooth muscle cell contraction. Our overall goal is to elucidate mechanisms of increased vascular resistance, for the first time, in conscious animals, during experimental salt-dependent hypertension. The use of conscious animals (as opposed to anesthetized) is key to understanding the putative role of sympathetic nerve activity (SNA), increasingly recognized as a key mechanism of salt-dependent hypertension. This overall goal is to be achieved through the use of non-invasive, fluorescence imaging of molecular signaling in arterioles of conscious optical biosensor mice. Given that phosphorylation of myosin regulatory light chains is the critical determinant of smooth muscle contraction, Specific Aims 1 and 2 are to develop optical biosensor mice that express novel, genetically-encoded activity biosensors for the key molecular regulators of smooth muscle myosin phosphorylation; a) myosin light chain kinase, (MLCK), b) myosin light chain phosphatase, MLCP), and c) a key upstream regulator of MLCP, the small GTPase, RhoA. In Aim 3, we will use these optical biosensor mice to determine, in vivo, 1) the regulation of MLCK, MLCP and RhoA by certain vascular G-protein coupled receptors (GPCR) putatively involved in hypertension, including adrenoceptors (α1-AR), Angiotensin II receptors (AT1-R), endothelin-1 receptors, (ETA), and sphingosine-1-phosphate receptors (S1P1). The regulation of MLCK, MLCP and RhoA by SNA will be determined through the use of complete autonomic ganglionic blockade (hexamethonium) in conscious mice. Mice will be implanted with telemetric arterial blood pressure transducers to allow continuous measurement of arterial blood pressure during imaging (and all other times). In Specific Aim 4, the time course of the activation levels of MLCK, MLCP and RhoA will be measured in ear arterioles of conscious individual mice (i.e. a `longitudinal' study) during 14 days of Angiotensin II/salt hypertension. Mice are infused chronically with Angiotensin II and fed a high-salt (NaCl) diet. Increased vascular resistance in this model of salt-dependent hypertension is believed to involve heightened SNA (`sympathoexcitation) emanating from salt-sensitive CNS cardiovascular control regions, mandating use of conscious mice. These Specific Aims will be performed in this multi-PI project under the direction of two Principal Investigators with the necessary expertise to generate the proposed sensors (Rizzo) and perform the physiologic experiments (Wier). In summary, we expect to achieve dynamic imaging of myosin phosphorylation regulatory processes during the development of salt-sensitive hypertension in a living animal for the first time. These studies will reveal new insights on the molecular basis of increased vascular resistance in hypertension, as it actually occurs in living animals.