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.
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