Potential Pacemaker Function And ?-adrenergic Signals Of Pnmt~+ Cardiomyocytes

Objective:1.This study intends to study the membrane potential(Vm)and intracellular Ca²⁺transients(Ca T)in sinoatrial preparation with Pnmt⁺cardiomyocytes using optogenetics pacing to elucidate the potential pacemaker function of Pnmt⁺cardiomyocytes.2.The purpose of this study was to determine the electrophysiological characteristics and the?adrenergic signal characteristics of Pnmt⁺cardiomyocytes.Methods:1.Potential pacemaker function of Pnmt⁺cardiomyocytes:In this study,the sinoatrial preparation containing two atria and the SAN region of adult optogenetic transgenic mice Pnmt^{Cre/Ch R2} were micro-dissected under a stereomicroscope.The SAN region was identified from anatomical landmarks,including the superior vena cava,the crista terminalis,and the interatrial septum.And the SAP tissues were loaded with the Ca²⁺sensitive dye Rhod-2 AM and the voltage sensitive dye Di-4-ANBDQPQ.Dual dye imaging was performed after loading using optical mapping system and calcium transient and action potential of SAP were recorded synchronously after optogenetic pacing with blue light to investigate the potential pacemaker function of Pnmt⁺cardiomyocytes.2.?-adrenergic signals of Pnmt⁺cardiomyocytes:In this study,method was used to cardiomyocytes of adult transgenic Pnmt^{Cre/Ch R2}mice(12-16 weeks old)were isolated using acuteenzyme separation method in a Langendorff perfusion system.Immunofluorescence was used to detect the expression of?1-ARs and?2-ARs in Pnmt⁺cardiomyocytes.The visual edge detection system was used to record the contraction and calcium signals of Pnmt⁺cardiomyocytes under blue light/electric stimulation at different frequencies and the differences between them and conventional cardiomyocytes.Then dobutamine hydrochloride(?1-AR selective agonist),and toelotrol hydrochloride(?2-AR selective agonist)treated the cells respectively to observe the function of?1-or?2-ARs for contraction and calcium signals in Pnmt⁺cardiomyocytes.Addtionally,the electrophysiological characteristics of Pnmt⁺cardiomyocytes were recorded using patch clamp technique and compared with those of conventional cardiomyocytes.Results:1.The spontaneous sinus rhythmic Ca²⁺transients(by Rhod-2 AM)were recorded in SAP with the optical mapping system.And the early activation point of sinoatrial tissue is located within the intercaval region near to where the SAN is located.However,the calcium activation pattern and atrial conduction changed after stimulating with blue light.The leading pacemaker site was shifted to the location of left atrium,suggesting that Pd CMs were activated earlier than the SAN pacemaker cells and optogenetic pacing of a SAP can shift the activation point to the left,creating a new leading pacemaker site or“neonode.”2.Immunofluorescence staining results showed that Pnmt⁺cardiomyocytes express the?1-,and?2-ARs.3.Further studies showed that dobutamine hydrochloride,a selective?1-AR agonist,increased the contraction and calcium signals of Pnmt⁺cardiomyocytes and Pnmt⁺cardiomyocytes contracted and the calcium signals stronger than that of conventional cardiomyocytes.The selective?2-AR agonist toloterol hydrochloride increased the calcium signals of Pnmt⁺cardiomyocytes and ordinary cardiomyocytes,the contraction and calcium signals of Pnmt⁺cardiomyocytes and Pnmt⁺cardiomyocytes are similar.Interestingly,the increase of contraction and calcium signals in Pnmt⁺cardiomyocytes was higher than that of in conventional cardiomyocytes.These results suggest that the functions of?1-ARs and?2-ARs in Pnmt⁺cardiomyocytes are not completely imilar to conventional cardiomyocytes.And?2-ARs of Pnmt⁺cardiomyocytes may play a key role in calcium signal regulation under stress.4.The AP shap of Pnmt⁺cardiomyocyte almostly like that of conventional cardiomyocyte and there was no significant difference in APD50 and APD90between Pnmt⁺cardiomyocytes and conventional cardiomyocytes.Conclusion:1.Pnmt⁺cardiomyocytes in the atrium have potential pacemaker function.2.Pnmt⁺cardiomyocytes express both the?1-,and?2-ARs,but their physiological activities in Pnmt⁺cardiomyocytes are not consistent with those of ordinary cardiomyocytes,suggesting that Pnmt⁺cardiomyocytes play an important role under stress.3.The AP of Pnmt⁺cardiomyocyte almostly like that of conventional cardiomyocyte...