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1 Weis Center for Research, Geisinger Medical Center, Danville, Pennsylvania 17822; 2 Department of Biochemistry and Molecular Biology, University of Calgary Health Sciences Center, Calgary, Alberta, Canada T2N 4N1; and 3 Department of Medicine, Medical University of South Carolina, and the Ralph H. Johnson Veterans Affairs Medical Center, Charleston, South Carolina 29403
The functional consequences of
overexpression of rat heart Na+/Ca2+ exchanger
(NCX1) were investigated in adult rat myocytes in primary culture. When
maintained under continued electrical field stimulation conditions,
cultured adult rat myocytes retained normal contractile function
compared with freshly isolated myocytes for at least 48 h.
Infection of myocytes by adenovirus expressing green fluorescent protein (GFP) resulted in >95% infection as ascertained by GFP fluorescence, but contraction amplitude at 6-, 24-, and 48-h
postinfection was not affected. When they were examined 48 h after
infection, myocytes infected by adenovirus expressing both GFP and NCX1
had similar cell sizes but exhibited significantly altered contraction amplitudes and intracellular Ca2+ concentration
([Ca2+]i) transients, and lower resting and
diastolic [Ca2+]i when compared with myocytes
infected by the adenovirus expressing GFP alone. The effects of NCX1
overexpression on sarcoplasmic reticulum (SR) Ca2+ content
depended on extracellular Ca2+ concentration
([Ca2+]o), with a decrease at low
[Ca2+]o and an increase at high
[Ca2+]o. The half-times for
[Ca2+]i transient decline were similar,
suggesting little to no changes in SR Ca2+-ATPase activity.
Western blots demonstrated a significant (P 
0.02)
threefold increase in NCX1 but no changes in SR Ca2+-ATPase
and calsequestrin abundance in myocytes 48 h after infection by
adenovirus expressing both GFP and NCX1 compared with those infected by
adenovirus expressing GFP alone. We conclude that overexpression of
NCX1 in adult rat myocytes incubated at high [Ca2+]o resulted in enhanced Ca2+
influx via reverse NCX1 function, as evidenced by greater SR Ca2+ content, larger twitch, and
[Ca2+]i transient amplitudes. Forward NCX1
function was also increased, as indicated by lower resting and
diastolic [Ca2+]i.
excitation-contraction coupling; fura 2; cardiac myocyte culture; video imaging
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