Maitotoxin-induced myocardial cell injury: Calcium accumulation followed by ATP depletion precedes cell death

Abstract

Maitotoxin, the most potent marine toxin, is known to increase the uptake and the accumulation of Ca 2+ into cells, and was used in the present study to investigate the mechanisms of myocardial cell damage induced by Ca 2+ overload. In cultured cardiomyocytes, isolated from 2-day-old rats, maitotoxin affected cell viability, as indicated by the leakage of the cytosolic enzyme lactate dehydrogenase (LDH) and of radiolabeled adenine nucleotides into the extracellular medium. Maitotoxin-induced leakage of LDH steadily increased between 30 min and 24 hr, and was preceded by a marked depletion of intracellular ATP. Addition of maitotoxin resulted in a rapid influx of extracellular Ca 2+, as detected by preincubating the cells in the presence of 45Ca; this effect evolved in a few minutes, thus preceding the signs of cell death. Cytosolic levels of free Ca 2+ ([Ca 2+] i ) were monitored by loading freshly isolated, suspended cardiomyocytes with the intracellular fluorescent probe fura-2; in these cells, maitotoxin induced a dose-dependent increase in [Ca 2+] i , with a lag phase of less than a minute. All these effects of maitotoxin were inhibited by reducing Ca 2+ concentration in the culture medium or by incubating the cells with the calcium-channel blocking drug verapamil. It is thus demonstrated that maitotoxin-induced cardiotoxicity is secondary to an inordinate influx of Ca 2+ into the cells. It is also suggested that, in those conditions that lead to an inordinate accumulation of Ca 2+ into myocardial cells, the unmatched demands of energy and the depletion of ATP play a primary role in the irreversible stage of cell damage.