Overexpression of Na+/Ca2+ exchanger alters contractility and SR Ca2+ content in adult rat myocytes.
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 < or = 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.
Zhang
X Q
XQ
Weis Center for Research, Geisinger Medical Center, Danville, Pennsylvania 17822-2619, USA.
Song
J
J
Rothblum
L I
LI
Lun
M
M
Wang
X
X
Ding
F
F
Dunn
J
J
Lytton
J
J
McDermott
P J
PJ
Cheung
J Y
JY
D000256:Adenoviridae / Q000235:genetics; D000367:Age Factors; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D004558:Electric Stimulation; D005456:Fluorescent Dyes; D016257:Fura-2; D015870:Gene Expression / Q000502:physiology; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D007202:Indicators and Reagents / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics; D008297:Male; D018715:Microscopy, Video; D018485:Muscle Fibers, Skeletal / Q000166:cytology / Q000502:physiology*; D009200:Myocardial Contraction / Q000502:physiology; D009206:Myocardium / Q000166:cytology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*; D019831:Sodium-Calcium Exchanger / Q000235:genetics* / Q000378:metabolism
D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D019831:Sodium-Calcium Exchanger; D049452:Green Fluorescent Proteins; D002118:Calcium; D016257:Fura-2