© 1995 by European Society of Cardiology
Copyright © 1995, European Society of Cardiology
Effect of osmotic stress on sarcolemmal integrity of isolated cardiomyocytes following transient metabolic inhibition
Servicio de Cardiología, Hospital General Universitario Vall d'Hebron, Paseo Vall d'Hebron 119-129, Barcelona 08035, Spain
* Corresponding author.
Objective: Exposure to hypotonic medium induces sarcolemmal rupture in metabolically inhibited cardiomyocytes. This study investigated the effect of osmotic stress applied during reoxygenation and the possible cooperation between cell swelling and hypercontracture to produce sarcolemmal disruption. Methods: Freshly isolated adult rat myocytes were submitted to 60 min of metabolic inhibition (NaCN 2 mM). Reoxygenation was simulated by changing to one of 3 inhibitor free buffers: (1) normo-osmotic (312 mOsm); (2) hypo-osmotic (80 mOsm); (3) low Na+ normo-osmotic (312 mOsm). The contribution of hypercontracture-induced reoxygenation on sarcolemmal rupture was investigated in myocytes submitted to hypo-osmotic reoxygenation in presence of 2,3-butanedione monoxime 30 mM, a blocker of contractility. Recovery from mechanical fragility was studied by exposing cells to hypotonic buffer 20 or 40 min after restoration of metabolic activity, in either presence or absence of 2,3-butanedione monoxime. Two control groups without metabolic inhibition were used. One was exposed to osmotic stress after 60 min incubation in control conditions, the other was induced to hypercontract by exposure to hypo-osmotic, high-calcium buffer. Cell viability was assessed by the Trypan blue test. Results: Before any intervention 81.9(1.2)% of cells were rod-shaped. After 60 min of metabolic inhibition most cells developed rigor contracture and only 16.4(1.8)% remained rod-shaped. Restoration of metabolic activity induced hypercontracture of most cells with rigor independently of buffer osmolality. Cell viability, however, significantly differed among groups: only 25.9(4.4)% of cells reoxygenated with hypo-osmotic buffer were viable vs. 74.1(7.6)% in the normo-osmotic reoxygenation group, and 82.9(2.9)% in the control group. Addition of 2,3-butanedione monoxime 30 mM during hypo-osmotic reoxygenation prevented hypercontracture and preserved cell viability. Delaying osmotic stress 20 or 40 min after the onset of reoxygenation did not improve viability [19.3(3.9) and 34.9(1.3)%, respectively]. Contractile blockade with 2,3-butanedione monoxime during the first 20 or 40 min of reoxygenation was associated with a reduction in the number of hypercontracted cells after the removal of the inhibitor but did not increase the proportion of hypercontracted viable cells (25% and 27%, respectively). Conclusions: (1) Osmotic stress following transient metabolic inhibition produces sarcolemmal disruption, and this effect is not related to the low Na+ concentration present in the hypo-osmotic buffer; (2) reoxygenation-induced hypercontracture cooperates with cell swelling to produce sarcolemmal disruption; and (3) osmotic fragility persists for at least 40 min after restoration of metabolic activity.
KEYWORDS Osmotic swelling; Reoxygenation; Reperfusion; Cell viability; Myocardial ischemia; Myocardial infarction
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