Adenoviral SERCA1a gene transfer to adult rat ventricular myocytes induces physiological changes in calcium handling.
OBJECTIVE
We examined the functional consequences of expressing adult rabbit fast skeletal sarcoplasmic reticulum (SR) Ca(2+)-ATPase (SERCA1a) in isolated adult rat ventricular myocytes.


METHODS
Myocytes were infected with a recombinant adenovirus harboring SERCA1a. Then 2 days after myocyte infection, protein expression was estimated using Western blot and SDS-PAGE analysis. We also measured the ATP-dependent oxalate-facilitated Ca(2+) uptake of myocyte homogenates and monitored Ca(2+) transient in myocytes loaded with the Ca(2+) dye, indo-1.


RESULTS
SERCA1a gene expression resulted in a 36% increase in the total SERCA protein level in infected myocytes compared to controls (P<0.01), while SERCA2 and phospholamban levels did not change. This increase was associated with a 42% rise in SR Ca(2+) uptake (P<0.01), while tau (the time constant of Ca(2+) transient decay), and the time to peak fell by 32% (P<0.01) and 38% (P<0.001), respectively. Increasing the frequency of stimulation from 0.2 to 2 Hz decreased tau in both cell types (P<0.01). However, the decrease was much smaller in infected (P<0.01) than in uninfected cells (P<0.001). Isoproterenol (1 microM) further decreased tau in infected myocytes by 23% (P<0.05). In these cells, the diastolic [Ca(2+)](i) decreased by 50% (P<0.05) while the systolic [Ca(2+)](i) increased by 19% (P<0.05). No difference was found in the speed of SR Ca(2+) reloading after caffeine washout between the two cell types.


CONCLUSIONS
Adenovirus-mediated SERCA1a gene transfer to adult rat ventricular myocytes enhances SR Ca(2+) handling to a degree similar to that observed following physiological stimulation.
Chossat
N
N
INSERM U 460, Faculté de Médecine Xavier Bichat, 16 rue Henri Huchard, 75018, Paris, France.
Griscelli
F
F
Jourdon
P
P
Logeart
D
D
Ragot
T
T
Heimburger
M
M
Perricaudet
M
M
Lompré
A
A
Hatem
S
S
Mercadier
J
J
D000256:Adenoviridae / Q000235:genetics*; D000704:Analysis of Variance; D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D002478:Cells, Cultured; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000008:administration & dosage*; D008297:Male; D018613:Microscopy, Confocal; D009206:Myocardium / Q000201:enzymology / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases
C506948:Atp2a2 protein, rat; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000252:Calcium-Transporting ATPases; D002118:Calcium