In ischemia the cytosol of cardiomyocytes acidifies; this is reversed upon reperfusion. One of the major pH(i)-regulating transport systems involved is the Na+/H+ exchanger. Inhibitors of the Na+/H+ exchanger have been found to more effectively protect ischemic-reperfused myocardium when administered before and during ischemia than during reperfusion alone. It has been hypothesized that the protection provided by pre-ischemic administration is due to a reduction in Na+ and secondary Ca2+ influx. Under reperfusion conditions Na+/H/ exchange inhibition also seems protective since it prolongs intracellular acidosis which can prevent hypercontracture. In detail, however, the mechanisms by which Na+/H+ exchange inhibition provides protection in ischemic-reperfused myocardium are still not fully identified.
Halothane protects cardiomyocytes against reoxygenation-induced hypercontracture by preventing oscillations of intracellular Ca2+ during the early phase of reoxygenation.
The hypothesis that rat cardiomyocytes become susceptible to hypercontracture after anoxia/reoxygenation was investigated. The cells were gradually overloaded with Ca2+ after different periods of simulated ischemia (substrate-free anoxia, medium at pH 6.4) followed by 20 minutes of reoxygenation. The cytosolic Ca2+ concentration (measured with fura 2) at which the cells developed maximal hypercontracture (Camax) 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 Ca2+ levels than did normoxic cells (Camax, 0.80 +/- 0.05 mumol/L versus 1.27 +/- 0.05 mumol/L; P< .01). To find the possible cause of increased SH, the influence of Ca2+ overload, acidosis, and protein dephosphorylation were studied. Prevention of cytosolic Ca2+ overload in anoxic cardiomyocytes or imitation of ischemic acidosis in normoxic cells did not influence Camax. In contrast, use of 10 mumol/L cantharidin (inhibitor of protein phosphatases 1 and 2A) during anoxic superfusion prevented the reduction of Camax. Furthermore, treatment of normoxic cardiomyocytes with 20 mmol/L of the chemical phosphatase 2,3-butanedione monoxime reduced Camax. Therefore, prolonged simulated ischemia increases susceptibility of cardio-myocytes to hypercontracture. This seems to be due to protein dephosphorylation.
Abstract-The aim of this study was to investigate whether treatment with the protein kinase C (PKC) agonist 1,2-dioctanoyl-sn-glycerol (1,2DOG) can protect isolated adult Wistar rat cardiomyocytes against simulated ischemia and reoxygenation. Cytosolic Ca 2ϩ (assessed by fura 2 fluorescence), pH i (assessed by BCECF fluorescence), and cell length were measured during 80 minutes of simulated ischemia (anoxia, pH o 6.4) and 20 minutes of reoxygenation (pH o 7.4) and compared between control cells and cells treated with 20 mol/L 1,2DOG before anoxia (10-minute treatment and 10-minute washout), before and during anoxia (two-step treatment), or only during anoxia. Treatment before anoxia attenuated rigor contracture but did not influence anoxic Ca 2ϩ overload. In contrast, two-step treatment before and during anoxia accelerated rigor contracture but reduced the rate of anoxic Ca 2ϩ accumulation. During reoxygenation, control cells developed irreversible hypercontracture (reduction of cell length to 43Ϯ2% of the initial cell length, nϭ62), which was accompanied by spontaneous oscillations of cytosolic Ca 2ϩ (19.6Ϯ1.6 per minute). Two-step treatment with 1,2DOG before and during anoxia significantly reduced hypercontracture (reduction of cell length to 60Ϯ2%, PϽ.01 versus control, nϭ41) and suppressed spontaneous Ca 2ϩ oscillations (2.8Ϯ0.9 per minute, PϽ.01 versus control). These effects could not be reproduced by treatment with 1,2DOG before anoxia or during anoxia or by a two-step treatment with the PKC-inactive 1,3-dioctanoyl-sn-glycerol and were fully abolished with 1 mol/L bisindolylmaleimide (PKC inhibitor). We conclude that a two-step activation of PKC before and during anoxia is required for effective protection of cardiomyocytes against anoxic Ca 2ϩ overload and reoxygenation-induced hypercontracture. overload. This Ca 2ϩ overload is a determinant not only of cellular injury developing during ischemia but also of reoxygenation-induced injury. One of the important elements of this injury is hypercontracture, caused by the resupply of energy to myofibrils at cytosolic Ca 2ϩ overload. 1,2Recently, it has been shown that myocardial injury induced by ischemia/reperfusion is markedly reduced by ischemic PC, ie, when a prolonged exposure to ischemia is preceded by one or more brief ischemic episodes.3 Activation of PKC has been shown to be an important element in the cardioprotective mechanism of ischemic PC. 4,5 Apart from its involvement in protection by ischemic PC, little is known about the effects of PKC activation on the course of ischemia/reperfusion injury in myocardium. The investigation of this question in whole heart is complicated, because the intact heart is a complex of tissues and because direct effects of PKC stimulation on cardiomyocytes can be masked by side effects on other cells (eg, mast cells, endothelial cells, and neurons), as well as by hemodynamic effects.In the present study, we addressed the question whether direct stimulation of PKC can protect cardiomyocytes against injury induced by si...
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