Simulated Ischemia Increases the Susceptibility of Rat Cardiomyocytes to Hypercontracture

Abstract

The hypothesis that rat cardiomyocytes become susceptible to hypercontracture after anoxia/reoxygenation was investigated. The cells were gradually overloaded with Ca 2+ after different periods of simulated ischemia (substrate-free anoxia, medium at pH 6.4) followed by 20 minutes of reoxygenation. The cytosolic Ca 2+ concentration (measured with fura 2) at which the cells developed maximal hypercontracture (Ca max ) was used as an index for their susceptibility to hypercontracture (SH). SH was increased in cardiomyocytes after prolonged periods of simulated ischemia; ie, these cells developed hypercontracture at significantly lower cytosolic Ca 2+ levels than did normoxic cells (Ca max , 0.80±0.05 μmol/L versus 1.27±0.05 μmol/L; P <.01). To find the possible cause of increased SH, the influence of Ca 2+ overload, acidosis, and protein dephosphorylation were studied. Prevention of cytosolic Ca 2+ overload in anoxic cardiomyocytes or imitation of ischemic acidosis in normoxic cells did not influence Ca max . In contrast, use of 10 μmol/L cantharidin (inhibitor of protein phosphatases 1 and 2A) during anoxic superfusion prevented the reduction of Ca max . Furthermore, treatment of normoxic cardiomyocytes with 20 mmol/L of the chemical phosphatase 2,3-butanedione monoxime reduced Ca max . Therefore, prolonged simulated ischemia increases susceptibility of cardiomyocytes to hypercontracture. This seems to be due to protein dephosphorylation.