Decreased Cholinergic Tone and Mitochondrial Dysfunction in Heart

描述（由申请人提供）：本研究将主要在巴西米纳斯吉拉斯联邦大学与Silvia Guatimosim博士合作完成，作为NIH资助号P01 HL 67849（A.R. Marks），4/1/2006-3/31/2011. W博士Jonathan Lederer是项目负责人。拟议的FIRCA项目旨在为巴西Silvia Guatimosim博士领导的一个杰出研究小组提供新颖的实时成像和细胞生物学实验。这项新的工作将通过使用PI在马里兰州大学生物医学工程和技术中心的最先进的设施来培训Guatimosim博士和她的同事们使用新的方法来实现。广泛地说，共同研究者将研究心脏中的胆碱能张力如何影响心肌细胞和线粒体功能中的Ca 2+信号传导。成像和生物物理学方法将沿着使用一种新的小鼠品系（VaChT KDHOM小鼠），其囊泡乙酰胆碱转运蛋白的表达降低。这一新型小鼠系是在FIRCA之前的资助下开发的。普拉多，并将允许共同研究者的特点，胆碱能功能减退的后果对心脏细胞的行为。Guatimosim博士的挑衅性初步结果表明，VAChT KDHOM小鼠具有心力衰竭，包括心肌力降低、心室钙处理改变和分子重塑（Lara等人，Molecular & Cellular Biology，2010年出版），包括改变线粒体生物学和酶水平以及增加活性氧（ROS）的产生（见初步结果）。这种功能障碍大部分通过胆碱酯酶抑制剂（吡啶斯的明）治疗而逆转。由于线粒体被认为是细胞的动力来源，因此FIRCA项目的中心假设是线粒体动力学的改变导致VAChT突变小鼠的心脏功能障碍。为了验证这一假设，我们将通过结合线粒体Ca 2+水平和膜电位（[Ca 2 +]mito）的实时成像来测量来自VAChT突变体的膜片钳心室肌细胞中的线粒体动力学。通过实时成像获得的功能数据将提供对线粒体在胆碱能张力降低引起的心脏病中的作用的综合理解。该FIRCA提案将提供在米纳斯吉拉斯联邦大学建立新的研究能力的手段，用于线粒体的同步膜片钳和实时成像，并将这些最先进的技术纳入Guatimosim博士的研究。Lederer博士将在巴尔的摩为Guatimosim博士及其同事提供培训，并在巴西提供现场指导。

DESCRIPTION (provided by applicant): This research will be done primarily in Brazil at Universidade Federal de Minas Gerais in collaboration with Dr. Silvia Guatimosim, as an extension of Project 3 of NIH Grant number P01 HL67849 (A.R. Marks), 4/1/2006-3/31/2011. Dr. W. Jonathan Lederer is the project leader. The proposed FIRCA project seeks to enable novel real-time imaging and cell biology experiments for an outstanding research group in Brazil headed by Dr. Silvia Guatimosim. The new work will be made possible by using the PI's state-of-the-art facilities at the University of Maryland Center for Biomedical Engineering and Technology to train Dr. Guatimosim and her colleagues in new methods. Broadly the co-investigators will investigate how the cholinergic tone in heart affects Ca2+ signaling in the cardiac myocytes and mitochondrial function. Imaging and biophysical methods will be used along with a novel mouse line (VaChT KDHOM mice) that has reduced expression of the vesicular acetylcholine transporter. This novel mouse line was developed with previous FIRCA funding to Dr. M. Prado and will permit the co-investigators to characterize the consequences of cholinergic hypofunction on heart cell behavior. Provocative preliminary results by Dr. Guatimosim show that the VAChT KDHOM mouse has heart failure including decreased myocardial force, altered ventricular calcium handling and molecular remodeling (Lara et al., Molecular & Cellular Biology, 2010 in press), including altered mitochondrial biology and enzyme levels and increased production of reactive oxygen species (ROS) (see Preliminary Results). Much of this dysfunction was reversed by treatment with a cholinesterase inhibitor (pyridostigmine). Since mitochondria are considered the powerhouse of the cell, the central hypothesis of this FIRCA project is that alterations in mitochondrial dynamics contribute to cardiac malfunction in VAChT mutant mice. To test this hypothesis, we will measure mitochondrial dynamics in patch-clamped ventricular myocytes from VAChT mutants by using a combination of real-time imaging of mitochondrial Ca2+ levels and membrane potential ([Ca2+]mito )The functional data obtained by real-time imaging will provide an integrated understanding of mitochondria' s role on heart disease caused by reduced cholinergic tone. This FIRCA proposal will provide the means to build new research capabilities at the Universidade Federal de Minas Gerais site for the simultaneous patch-clamp and real-time imaging of mitochondria, and to incorporate these state-of-the-art techniques into Dr. Guatimosim's research. Dr. Lederer will provide training in Baltimore for Dr. Guatimosim and her co-workers and on-site instruction in Brazil.