应激诱导“Pnmt细胞分化的心肌细胞”分泌肾上腺素对小鼠心肌细胞兴奋-收缩偶联钙转运的调控作用

Adrenaline (Adr) is secreted by the adrenergic cells located the adrenal medulla and specific regions of the brain, in fact, the heart is able to release Adr as well. It has been previously showed that there are intrinsic cardiac adrenergic (ICA) cells with capability of secreting Adr in the heart. Unexpectedly, our recent studies also showed that Pnmt cell-derived cardiomyocytes (PdCMs), a newly identified cardiompycyte population, presented in the heart. These PdCMs are similar to conventional cardiomyocytes in term of their morphology, excitability and contraction. Remarkably, some of PdCMs express Pnmt, the enzyme converting noradrenaline to Adr as the mark of adrenergic cells. . .Based on our recent findings, this project proposes a hypothesis "stress-induced adrenaline secretion from PdCMs activates β-AR/Ca2+ to regulate excitation-contraction Coupling（ECC）of cardiomyocytes”. Using PnmtCre/ChR2 mice model with cell-type specific express channelrhodopsin 2 (ChR2) under promoter gene Pnmt, series of studies will be conducted. Firstly, we will determine the endocrinal capability of PdCMs to secrete Adr, which will be done by separating them from normal myocardial cells with flow cytometry and detecting Adr by liquid chromatography-mass spectrometry chromatography; secondly, the calcium transients, the action potentials and contractility of PDCMs will be investigated at both tissue and cellular levels with optogenetic light stimulation using optical mapping imaging technique, edge detection system and patch clamping techniques respectively. Thirdly, The L-type Ca2+ channel current, Ca2+ transients of both PdCMs and conventional myocytes will be studied at both baseline and adrenergic stress conditions with or without β-AR agonist or blocker.. .Therefore, the proposed project will for the first time determine the endocrine function of PdCMs and define their electrophylogical, Ca2+ handling and E-C coupling characteristics and their distinctions to conventional cardiac myocyte. This project will provide a experimental evidence for establishing a new theoretical basis of the adrenergic endocrine function of the specific cardiac myocytes population in the heart and the implication of such function under physiological and pathophysiological conditions.

应激状态下心交感神经兴奋引起肾上腺髓质分泌肾上腺素（Adr），研究发现心脏肾上腺素能细胞ICA也分泌Adr。我们发现“苯乙醇胺N-甲基转移酶（Pnmt）细胞分化的心肌细胞”即PdCMs表达Pnmt，有心肌细胞样的形态结构和兴奋收缩功能。但PdCMs兴奋-收缩偶联（ECC）的钙转运机制尚未明确。基于PdCMs表达Pnmt—合成Adr关键酶特征，我们提出“应激诱导PdCMs分泌Adr参与ECC钙转运”。本研究以PnmtCre/ChR2小鼠为模型，流式分选PdCMs，采用液质联用确定PdCMs分泌Adr。光电标测、钙信号与收缩同步记录、膜片钳等技术观察应激状态下PdCMs的钙信号、收缩、AP、ICa,L、钙库钙容量，阐明应激状态下PdCMs调节钙转运的特征及与心肌细胞的差异，确定β-AR对PdCMs 钙转运的作用。项目将为应激心脏的内分泌功能及PdCMs对应激诱发的钙转运特征提供新的理论依据。

心脏受交感神经和迷走神经的双重支配，当交感神经兴奋时，刺激心交感神经末梢释放去甲肾上腺素（NE）和肾上腺髓质分泌肾上腺素(Adr)，引发心脏一系列的生理性变化。传统观点认为Adr来源于肾上腺髓质和大脑特定区域中的肾上腺素能细胞。但我们前期研究发现心脏中存在一类表达苯乙醇胺N-甲基转移酶编码基因(Pnmt)的细胞，它们有心肌细胞样的形态结构和兴奋收缩功能，被称为Pnmt细胞分化的心肌细胞（PdCMs)。基于PdCMs表达NE转化成Adr关键酶编码基因Pnmt的特征，我们推测PdCMs分泌Adr并参与心功能调节。本项目以PnmtCre/ChR2和心脏条件性敲除PnmtCKO小鼠为对象，采用碳纤电极、液质联用、ELISA等技术证明PdCMs含有NE和Adr。单细胞测序结果进一步证实PdCMs中的NE是通过OCT3而非NET途径转运至胞内，进而通过Pnmt转化成Adr。钙信号与收缩同步记录、膜片钳等实验结果发现，正常情况下PdCMs细胞的收缩、钙信号和AP与普通心肌细胞（CMs）比较无明显差异。PdCMs和CMs对1 nM ISO刺激时收缩和钙信号均增强，有意思的是PdCMs的收缩和钙信号对β1-ARs激动剂盐酸多巴酚丁胺的敏感性更强，而对β2-ARs激动剂盐酸妥洛特罗的反应则相反。但免疫荧光实验并没有观察到PdCMs和CMs在β1/β2-ARs表达上的明显差别。众所周知，儿茶酚胺对提高心输出量，促进心肌细胞生长有重要作用。心脏局部自身产生的Adr可能对心脏的结构和功能发挥重要作用。我们进一步研究发现主动脉缩窄（TAC）后，左心肌细胞（PdCMs比较丰富）的NE比右心肌细胞（PdCMs较少）增加更明显，但Adr在左、右心肌细胞中的变化不明显，这进一步证实PdCMs摄取了交感神经因TAC应激反应释放的大量NE，并在胞内将其转化成Adr，后者可能进一步代谢成别的物质。当采用白喉毒素损毁PdCMs后小鼠的ECG结果显示房室传导减慢，并出现心律不齐，提示PdCMs可能对心脏的传导和调节心功能的贡献。条件性敲除心脏Pnmt后，心脏的APD90和传导速度（CV）缩短，而CaTD90延长；但对ISO刺激APD90和CaTD90均无反应，而CV变慢，且比对照组慢得多。进一步证明PdCMs在心脏的传导和调节心功能调节中起重要作用。