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州联邦大学与Silvia Guatimosim博士合作完成，作为NIH资助号P01 HL67849 （A.R. Marks）项目3的延伸，4/1/2006-3/31/2011。W. Jonathan Lederer博士是项目负责人。拟议中的FIRCA项目旨在为由Silvia Guatimosim博士领导的巴西杰出研究小组提供新型实时成像和细胞生物学实验。这项新工作将利用PI在马里兰大学生物医学工程与技术中心最先进的设施，以新方法培训Guatimosim博士和她的同事。总的来说，共同研究人员将研究心脏胆碱能张力如何影响心肌细胞和线粒体功能中的Ca2+信号。成像和生物物理方法将与一种新的小鼠系（VaChT KDHOM小鼠）一起使用，该小鼠系减少了囊泡乙酰胆碱转运蛋白的表达。这种新颖的小鼠系是由先前的FIRCA资助M. Prado博士开发的，将允许共同研究者表征胆碱能功能减退对心脏细胞行为的影响。Guatimosim博士的初步结果表明，VAChT kdhm小鼠心力衰竭包括心肌力下降，心室钙处理和分子重塑改变（Lara等人，molecular & Cellular Biology， 2010年出版），包括线粒体生物学和酶水平的改变以及活性氧（ROS）的产生增加（见初步结果）。使用胆碱酯酶抑制剂（吡哆斯的明）治疗后，大部分功能障碍得以逆转。由于线粒体被认为是细胞的动力源，这个FIRCA项目的中心假设是线粒体动力学的改变导致了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Ca2+水平和膜电位（[Ca2+]mito）的实时成像来测量来自VAChT突变体的贴片夹紧心室肌细胞的线粒体动力学。实时成像获得的功能数据将提供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。这项FIRCA提案将为在米纳斯吉拉斯州联邦大学建立新的研究能力提供手段，同时对线粒体进行膜片钳和实时成像，并将这些最先进的技术纳入Guatimosim博士的研究中。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.