The long term objective of our planned studies remains as before, that is, to increase our understanding of the relationship between Ca2+ waves, altered Ca, cycling in the mechanisms of arrhythmias. The overall hypothesis is that increased probability of the fundamental Ca2+ release-event (the Ca2+ spark) leads to local summation of events(macro sparks) and, then by CICR to coupling of Ca2+ release from different SR-sites causing Ca2+ waves of increasing, amplitude, spatial extent and propagation velocity. Ca2+ waves, in proportion to their amplitude and extent, cause delayed afterdepolarizations (DADs) and therefore are arrhythmogenic. We propose that increased probability of Ca2+ release both due to transient elevations of SR-Ca2+ content and nonuniformity of the myocardium is involved in several diseases and contributes to the arrhythmias. We will use two approaches. One approach will be to use Purkinje cells dispersed from the arrhythmogenic infarcted heart (IZPCs) with electrical, epifluorescent and confocal imaging techniques. In our second approach, we use a multicellular trabecular preparation that is arrhythmogenic (due to nonuniformity of EC coupling and to CHF). The initiation and propagation of Ca2+ waves will be examined using epi- and confocal microscopy, electronmicoroscopy.
Aim#1 We hypothesize that nonuniform Ca2+ transients and abnormal micro Ca2+ wave events in IZPCs are due to differences a) in Ca2+ spark/event characteristics vs. those of normal cells, b) in SR content by subcellular region, c) in different spatial/regional types of Ca2+ release channels (e g. lP3R, RYR2, RYR3) /SERCA and NCX isoforms, d) in Ca2+ dependence of Ca2+ release in the subsarcolemmal vs. core compartments and sensitivity to IP3R agents, e) in response of Ca2+ release in these two cell compartments to voltage.
In Aim#2, we will determine whether Ca2+ dissociation from the filaments determines the initial Ca transient (a surge) that leads to Ca2+ waves and arrhythmias in the nonuniform muscle; whether Ca2+ wave propagation requires SR Ca2+ release and diffusion to adjacent SR release sites using epi- and confocal microscopy. Finally, we will determine whether the threshold for initiation and propagation of Ca2+ waves in trabeculae from hearts of animals with CHF is substantially lower than that in muscles from animals without CHF. In sum, these studies will provide a subcellular basis for the relationship between diseased-induced nonuniformities of Ca2+ cycling within a myocyte, Ca2+ waves and arrhythmias and thus will help in identifying new molecular targets for Ca2+ dependent nonreentrant arrhythmias.
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