[Effects of reoxygenation-induced osmotic edema on cell viability. Study using isolated myocytes].
OBJECTIVE
To investigate the hypothesis that reperfusion edema may kill myocytes.


METHODS
Adult Sprague-Dawley rat hearts were perfused with a calcium free dissociation buffer containing collagenase 0.03% in a Langedorff system. Intact cells were selected and myocytes were cultured in adherent pretreated dishes. After 3 hours, 80% of cells were rod-shaped. Anoxia was simulated by means of metabolic inhibition by adding NaCN 2 mM to the control media, and reoxygenation by substituting this media with one of the following media non containing NaCN: 1) normo-osmotic (312 mOsm); 2) hypoosmotic (80 mOsm); 3) normo-osmotic with low Na+ (312 mOsm). A group of cells was kept with control media without metabolic inhibition and then submitted to simulated reoxygenation with hypoosmotic media (control group). The number of rod, square and round-shaped cells was monitored, and cell viability was assessed after 5 min of reoxygenation by the Trypan blue test.


RESULTS
After 60 min of metabolic inhibition there were no differences in the % of cells without hypercontracture among groups reoxygenated with normo-osmotic, hypoosmotic, low Na+ normo-osmotic and control media (84 +/- 16, 74 +/- 10, 76 +/- 14 and 90 +/- 6% respectively (p = NS). After 5 min of reoxygenation, these values decreased (p < 0.001) to 19 +/- 6, 11 +/- 9 and 13 +/- 3% (p = NS), respectively, in groups with normo-osmotic, hypoosmotic, and low Na+ normo-osmotic reoxygenation, but were not modified in the control group (78 +/- 4). The % of viable cells (Trypan negative) preserved after 5 min of reoxygenation was 67 +/- 29% in the group with normo-osmotic reoxygenation, 31 +/- 23% in the group with hypoosmotic reoxygenation, and 85 +/- 12% in the group with low Na+ normo-osmotic reoxygenation (p < 0.001). Exposing cells without metabolic inhibition to hypoosmotic media resulted in no significative reduction of cell viability.


CONCLUSIONS
Hypoosmotic reoxygenation following prolonged metabolic inhibition may kill viable myocytes. This effect is not due to the low Na+ concentration in the hypoosmotic medium.
Ruiz-Meana
M
M
Servicio de Cardiología, Hospital General Universitario Vall d'Hebron, Barcelona.
García-Dorado
D
D
González
M A
MA
Barrabés
J A
JA
Oliveras
J
J
Soler-Soler
J
J
D000818:Animals; D015687:Cell Hypoxia; D002470:Cell Survival; D002478:Cells, Cultured; D003470:Culture Media; D004489:Edema, Cardiac / Q000378:metabolism / Q000473:pathology*; D008297:Male; D015428:Myocardial Reperfusion Injury / Q000378:metabolism / Q000473:pathology; D009206:Myocardium / Q000166:cytology* / Q000378:metabolism; D009995:Osmosis; D010100:Oxygen / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D013997:Time Factors
D003470:Culture Media; D010100:Oxygen
Efecto del edema osmótico durante la reoxigenación sobre la viabilidad celular. Estudio en el miocito aislado.