The long range objective of this research project is to determine the electrophysiological mechanisms, functional properties and morphological characteristics of atrial subsidiary pacemakers, and to compare these findings to those found in primary (SA node) pacemakers of the same species. The methods used in this study will involve recording voltage and ionic currents from single pacemaker cells isolated from the eustachian ridge and SA node of the cat right atrium. Cells will be isolated by retrograde (Langendorf) perfusion of the whole heart with enzyme (collagenase) solutions. Signals will be recorded using a single suction glass pipette in the whole cell configuration. Single cells will be voltage clamped using a discontinuous voltage clamp method and Pclamp software programs. The morphology of single cells will be determined using electron microscopic techniques.
The specific aims of this study will include a series of experiments to determine the ionic current mechanisms responsible for subsidiary pacemaker automaticity. This will involve a voltage clamp analysis of the i-f pacemaker current, the iK potassium current, the T- and L-type slow inward Ca 2+ currents and leak currents. A second series of experiment will determine the ionic current mechanisms responsible for the response of subsidiary pacemakers to adrenergic, cholinergic, purinergic and peptidergic receptor stimulation. This will include a voltage clamp analysis of the ionic current changes that occur in response to isoproterenol, methoxamine, acetylcholine, adenosine and calcitonin gene related peptide (CGRP). A third series of experiments will determine the ionic current mechanisms responsible for the effects of alterations in potassium, calcium and magnesium ion concentrations. Where appropriate, both intracellular and extracellular changes in ionic composition will be analyzed.
A fourth aim of this research is to determine the morphological (ultrastructural) characteristics of atrial subsidiary pacemaker cells. Finally, the findings in atrial subsidiary pacemakers (as described above) will bc compared with those found in primary (SA node) pacemakers. In this way, these experiments will directly compare the electrophysiology, function and morphology of subsidiary versus primary pacemaker cells isolated from hearts of the same species. At present, there is little known about the cellular mechanisms that govern or regulate atrial subsidiary pacemaker activity or how these factors change under abnormal conditions. This information is essential for an understanding of normal and abnormal atrial f unction. These experiments will provide new information toward a better understanding of the fundamental mechanisms that control and regulate atrial pacemaker activity.
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