PARASYMPATHETIC DYSFUNCTION IN OBESITY: EFFECT OF CHOLESTEROL ON VAChT

描述(申请人提供)：肥胖在美国1和全球2是一个严重的公共健康问题，是高血压、心律失常、心肌缺血、心力衰竭1、3-6和糖尿病4、7的主要危险因素。在肥胖症中，心脏的副交感神经系统(PSNS)调节明显减弱8-10，这可能导致肥胖相关的心律失常和心肌缺血8、11-13。然而，到目前为止，肥胖症患者PSNS功能降低对心脏的作用机制仍然知之甚少。乙酰胆碱(Ach)是三叉神经节的主要神经递质。囊泡型乙酰胆碱转运体(Vacht)负责在PSNS胆碱能终末包装Ach。高血浆胆固醇(高胆固醇血症)会影响大脑14-16区的神经元，但对外周胆碱能系统的影响尚不清楚。我们最近的研究证实，单一泛素化参与了Vacht循环和Ach摄取活性。在高脂饮食(HFD)诱导的肥胖小鼠中，Vacht泛素化减少，同时PSNS对心脏的调节减弱。本研究旨在验证一个中心假说，即肥胖时PSNS对心脏的作用减弱与高胆固醇抑制泛素化介导的Vacht循环有关，从而导致突触小泡Vacht水平和功能降低，PSNS胆碱能终末Ach释放减少。目的1检测三七总皂苷对高脂诱导的肥胖小鼠心脏的影响是否与心脏副交感神经末梢Ach释放和Vacht表达减少有关。正常饮食(RD)或高脂饮食(HFD)喂养6周的小鼠将被用作肥胖模型。我们将观察迷走神经刺激(VS)引起的心动过缓和心房Ach释放，以及来自PSNS心脏末端的突触小泡的Vacht蛋白水平和Ach摄取活性。目的2阐明高胆固醇是否在心脏PSNS胆碱能功能障碍中起作用。小鼠饲喂RD、RD+他汀类、HFD、HFD+他汀类、RD+2%胆固醇、RD+2%胆固醇+他汀类药物。对VS和Vacht水平的反应以及PSNS心脏末端的Ach摄取将被检测并与胆固醇水平相关。胆固醇对Vacht表达和Ach摄取的直接影响也将在胆碱能细胞系SN56中进行测试。目标3将描述高胆固醇是否抑制单一泛素化介导的Vacht循环，从而在PSNS终末发挥作用。在Aim 2中，将在六组心房样本中分析血浆胆固醇水平与Vacht单一泛素化和Ach摄取活性的相关性；将使用SN56细胞测试胆固醇对Vacht单一泛素化、循环和活性的直接抑制作用。总体而言，这项研究的结果将证明，高胆固醇对泛素化介导的Vacht循环的负面影响有助于肥胖患者PSNS对心脏的胆碱能作用的减少。这一新概念可能会开辟一个新的研究领域，并可能为肥胖相关的自主神经和心血管疾病的预防和治疗提供新的策略。

DESCRIPTION (provided by applicant): Obesity is a serious public health issue in the United States 1 and worldwide 2 and is a major risk factor for hypertension, arrhythmia, myocardial ischemia, heart failure1, 3-6, and diabetes4, 7. In obesity, parasympathetic nervous system (PSNS) regulation of the heart is markedly attenuated 8-10, which may contribute to obesity-related arrhythmia and myocardial ischemia8, 11-13. To date, however, the mechanisms of the reduced PSNS function on the heart in obesity are still poorly understood. Acetylcholine (Ach) is a major neurotransmitter of PSNS. Vesicular acetylcholine transporters (VAChT) are responsible for Ach packaging In PSNS cholinergic terminals. High plasma cholesterol (hypercholesterolemia) affects neurons in the brain14-16 but its effect on the peripheral cholinergic system is unknown. Our recent study identified mono-ubiquitination is involved in VAChT recycling and Ach uptake activity. In high fat diet (HFD)-induced obese mice, VAChT ubiquitination is reduced, along with the attenuated PSNS regulation of the heart. This study is to test a central hypothesis that Attenuated PSNS action on the heart in obesity is related to the inhibitory effect of high cholesterol on the ubiquitination-mediated VAChT recycling, leading to the reduced VAChT level and function in synaptic vesicles and decreased Ach release in PSNS cholinergic terminals. Aim 1 will test whether the attenuated PSNS effect on the heart in HFD-induced obese mice involves reduced Ach release and VAChT expression at parasympathetic terminals in the heart. Mice fed a regular diet (RD) or HFD for 6 weeks will be used as an obesity model. Vagal stimulation (VS)-induced bradycardia and Ach release in atria, and VAChT protein levels and Ach uptake activity in synaptic vesicles from PSNS cardiac terminals will be examined. Aim 2 will elucidate whether high cholesterol plays a role in the PSNS cholinergic dysfunction in the heart. Mice fed RD, RD plus Statin, HFD, HFD plus Statin, RD plus 2% cholesterol and RD plus 2% cholesterol plus Statin will be used. Responses to VS and VAChT level and Ach uptake in the PSNS cardiac terminals will be examined and correlated with cholesterol levels. The direct effect of cholesterol on VAChT expression and Ach uptake will also be tested in a cholinergic cell line, SN56. Aim 3 will delineate whether high cholesterol inhibits the mono-ubiquitination-mediated VAChT recycling and thus function in the PSNS terminals. Correlation of plasma cholesterol levels with mono- ubiquitination and Ach uptake activity of VAChT will be analyzed in atria samples from the six groups in Aim 2; The direct inhibitory effect of cholesterol on VAChT mono-ubiquitination, recycling and activity will be tested using SN56 cells. Overall, results of this study will demonstrate that the negative effect of high cholesterol on the ubiquitination-mediated VAChT recycling contributes to the reduced PSNS cholinergic action on the heart in obesity. This novel concept may open a new area of research and may suggest novel strategies for prevention and treatments of obesity-related autonomic and cardiovascular disorders.