Embryonic Stem Cell Derived Biological Pacemaker for the Heart

描述（由申请人提供）：植入式人工起搏器是治疗窦房结功能障碍和传导异常的最新技术。然而，它们有限的神经体液反应性和手术侵入性修复引起的并发症促使生物起搏器的发展，这些起搏器对神经体液刺激作出反应，并无限期地融入心肌。在成人心肌的一个区域中诱导自发活动只是不能充分纠正这个问题，因为新形成的起搏器仍然没有对i)周围心肌细胞的超极化影响和ii)心律失常兴奋的反向传播的保护。因此，我们建议使用具有明确连接蛋白表达的胚胎干细胞衍生心肌细胞（ESdCs）聚集体，这些连接蛋白表达反映了窦房结中建立的细胞间偶联的功能特性。该研究的具体目的是：1)确定胚胎干细胞衍生心肌细胞中起搏器活性的机制2)确定间隙连接通道的功能特性，这些通道允许ESdC聚群在多细胞心肌细胞制备中获得并维持起搏器优势！3)确定促进esdc异细胞对与成人心肌细胞之间频率和波形夹带的机制，而不诱导成人组织中的心律失常活动！

DESCRIPTION (provided by applicant): Implantable artificial pacemakers are the state-of the art treatment for sinoatrial node dysfunction and conduction abnormalities. However, their limited neurohumoral responsiveness and complications arising from surgically invasive repair have prompted the development of biological pacemakers that respond to neurohumoral stimuli and indefinitely integrate into the myocardium. The induction of spontaneous activity in an area of the adult cardiac muscle only insufficiently corrects the problem since the newly formed pacemaker remains without protection against i) the hyperpolarizing influences of surrounding cardiomyocytes, and ii) the back propagation of arrhythmic excitation. Therefore we propose the use of aggregates of embryonic stem cell derived cardiomyocytes (ESdCs) with defined connexin expression that reflect the functional properties of the intercellular coupling established in the sinoatrial node. The specific aims of the proposed study are: 1) to determine the mechanism of pacemaker activity in embryonic stem cell derived cardiomyocytes 2) to determine the functional properties of gap junction channels that allow ESdC aggregates to gain and maintain pacemaker dominance in multicellular cardiomyocyte preparations! 3) to determine mechanisms that promote frequency and waveform entrainment between heterocellular pairs of ESdCs and adult cardiac myocytes without induction of arrhythmic activity in the adult tissue! To achieve these aims different experimental techniques will be used. High resolution Ca imaging by laser scanning confocal microscopy in single ESdCs and in cell pairs of ESdCs and adult cardiomyocytes; novel FRET based fluorescent IPS sensors to study the role of IPS in pacemaker activity or during the induction of arrhythmic spontaneous activity in the adult cardiomyocytes; Single cell current clamp measurements will be employed to evaluate the mechanism of spontaneous activity in ESdCs and double whole cell voltage clamp measurements will be used to characterize the intercellular coupling resistance and voltage dependent gating between the ESdCs used as biological pacemakers and the host cardiac tissue (either HL-1 cells or adult cardiomyocytes). The electrophysiological properties of the multicellular in-vitro pacemaker model however will be determined by multielectrode measurements of heterocellular cultures of HL-1 monolayers and ESdC pacemaker aggregates. We will furthermore use stably transfected ES cell lines that are either deficient for the expression of Cx43 or Cx45 or overexpress Cx45.