Project: Research project

Grant Details


The heart is susceptible to arrhythmia initiation when cell-cell coupling
is reduced to levels near or below the "margin of safety" for successful
propagation. A high incidence of cardiac arrhythmias is associated with
acute myocardial ischemia and reperfusion. The long term goal of the
proposed studies is to define the contribution of gap junctions to the
increased susceptibility of the heart to arrhythmia initiation during and
following ischemia. Towards this end, molecular and electrophysiological
techniques will be used to study quantitatively, the incidence and
functionality of gap junctions during perturbations that simulate changes
occurring during ischemia, e.g. alterations of second messenger levels
and accumulation of non-esterified fatty acids (NEFA). Results obtained
in cultured heart cells will be compared to those obtained in A7r5 cells,
a cell line that expresses the same gap junction protein as heart, i.e.
Cx43, as well as Cx40. A7r5 cells whose expression of Cx43 or Cx40 has
been blocked using antisense to the corresponding mRNA will also be
examined. Comparison of these cells types will allow us to determine 1)
whether unitary conductance and gating of gap junction channels is
regulated in a tissue or connexin specific fashion, and 2) whether the
strategies cells use to control the extent to which they are coupled are
tissue or connexin dependent. The hypotheses to be tested build on
several observations. 1) Heart, which expresses Cx43, exhibits an
increase in junctional conductance when either protein kinase A or C is
stimulated, whereas A7r5 cells, which express Cx43 and Cx40, exhibit a
decrease. 2) Heart, which expresses Cx43 as 95pS channels, is uncoupled
by oleic acid (OA), whereas A7r5 cells which express Cx40 as 65 pS
channels and Cx43 as predominantly 134 pS cells, are only partially
uncoupled by OA. 3) The Cx43-134 pS channel in the A7r5 cells appears to
be resistant to closure by OA suggesting that its sensitivity to OA
differs from that of Cx43-95pS and from Cx40 channels. 4) Elimination of
functional Cx43 channels induces A7r5 cells to up-regulate Cx40
expression. These observations lead us to hypothesize that protein
kinases A and C influence channel number, channel unitary conductance and
channel open time probability, and that OA influences channel number and
channel open time probability. The effects of these agents on channel
number occur with a significantly longer time course (hours) than effects
on open time probability or conductance (seconds to minutes). In the
proposed studies we will determine if this general hypothesis is correct
using electrophysiological and molecular approaches. In addition, we
will begin to explore the specific domains of the Cx40 and Cx43 proteins
that confer onto them sensitivity to OA. By comparing heart cells to cell
types that express the same gap junction protein, we hope over the long
term, to gain an understanding of tissue vs. connexin specific regulation
of gap junction function and to define the probable role of gap junctions
in cardiac dysfunction.
Effective start/end date7/1/833/31/98


  • Medicine(all)


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