Russo Rebekah, Alber Bridget R, Mendelowitz David, Entcheva Emilia, Kay Matthew W
Department of Biomedical Engineering, The George Washington University.
Department of Pharmacology and Physiology, The George Washington University.
J Vis Exp. 2025 Mar 28(217). doi: 10.3791/67364.
A balance of cholinergic and catecholaminergic activation is necessary to maintain heart health. Interrogating the interaction between these pathways can be done using optogenetics through selective expression of channelrhodopsin-2 (ChR2) in cardiac autonomic neurons. Such cardiac applications of optogenetics allow for the study of the intrinsic release of neurotransmitters in a spatiotemporal manner. This method illustrates an ex vivo approach for specific optogenetic stimulation of cardiac neurons in perfused mouse hearts. Transgenic mice were bred to express ChR2 in either choline acetyltransferase (ChAT) or tyrosine hydroxylase (TH) neurons throughout the body. A micro-LED (465 nm) encased in a silicone elastomer was prepared for stimulating the neurons of the right atrium that innervate the sinoatrial node. The micro-LED was connected to a function generator set to pulse waves at 10 Hz with a 30 ms pulse width. Hearts with confirmed expression were excised and retrogradely perfused on a Langendorff system circulating Krebs-Henseleit solution. Electrocardiogram (ECG), temperature, and coronary flow rate were recorded using the LabChart software. Once the heart stabilized, the micro-LED was placed on the right atrium and tested for optimal heart rate response. An application of this approach combines the intrinsic release of cholinergic neurotransmitter (acetylcholine) during optogenetic activation of a ChAT-ChR2 mouse heart simultaneously with increasing exogenous catecholaminergic neurotransmitter (norepinephrine) added to the perfusate. The resulting changes in heart rate during the simultaneous cholinergic and catecholaminergic activation are presented. This method describes a valuable experimental approach for investigating the kinetics of sudden intrinsic autonomic neuron activation in perfused hearts and the interactions between cardiac cholinergic and catecholaminergic activity.
胆碱能和儿茶酚胺能激活之间的平衡对于维持心脏健康至关重要。通过在心脏自主神经元中选择性表达通道视紫红质-2（ChR2），利用光遗传学可以研究这些途径之间的相互作用。光遗传学在心脏方面的此类应用能够以时空方式研究神经递质的内在释放。该方法展示了一种用于在灌注的小鼠心脏中对心脏神经元进行特定光遗传学刺激的离体方法。培育转基因小鼠，使其在全身的胆碱乙酰转移酶（ChAT）或酪氨酸羟化酶（TH）神经元中表达ChR2。制备了一个封装在硅橡胶弹性体中的微型发光二极管（465nm），用于刺激支配窦房结的右心房神经元。微型发光二极管连接到一个函数发生器，设置为以10Hz的频率产生脉宽为30ms的脉冲波。切除确认有表达的心脏，并在循环Krebs-Henseleit溶液的Langendorff系统上进行逆行灌注。使用LabChart软件记录心电图（ECG）、温度和冠状动脉血流速度。一旦心脏稳定，将微型发光二极管放置在右心房上，并测试其对最佳心率反应的影响。这种方法的应用是在ChAT-ChR2小鼠心脏的光遗传学激活过程中，同时结合胆碱能神经递质（乙酰胆碱）的内在释放以及向灌注液中添加外源性儿茶酚胺能神经递质（去甲肾上腺素）的增加。呈现了同时进行胆碱能和儿茶酚胺能激活期间心率的变化。该方法描述了一种有价值的实验方法，用于研究灌注心脏中突然的内在自主神经元激活的动力学以及心脏胆碱能和儿茶酚胺能活性之间的相互作用。
