The consequences of simulated ischaemia on intracellular Ca2+ and tension in isolated ferret ventricular muscle.

Abstract

SUMMARY1. In order to study cellular events occurring in ischaemia, we have developed a method for simulating ischaemia in an isolated papillary muscle. Muscles were suspended in a chamber and changed from conventional superfusion with Tyrode solution to gas perfusion with 95% N2/5 % CO2 (N2 gas perfusion), thus simultaneously stopping oxygenation and flow. Surface cells of the preparation were injected with the photoprotein aequorin in order to monitor intracellular free calcium2. Gas perfusion with 95% 02/5% CO2 (02 gas perfusion) had little effect on the tension or Ca2+ transients. Superfusion with Tyrode solution equilibrated with 95% N2/5% CO2 (N2 Tyrode) caused tension to decline to 30-40% of control, but had little effect on the amplitude of the Ca2+ transients. N2 gas perfusion caused tension to fall more rapidly and to a lower level than superfusion with N2 Tyrode. Ca2+ transients showed a small initial decline followed by a slowly developing increase in magnitude and duration.3. Long exposures to N2 gas perfusion caused tension to decline to very low levels and Ca2+ transients to increase to a maximum. After a variable length of time, resting tension began to increase. At approximately the same time, Ca2+ transients began to decrease and eventually disappeared. Resting Ca2+ increased during N2 gas perfusion and remained elevated when the Ca2+ transients had declined. These changes could be reversed by restarting superfusion with standard Tyrode or by perfusion with 02 gas.4. N2 gas perfusion caused a depolarization of the resting potential and an abbreviation of the action potential. In a long exposure the action potential eventually failed. These changes could be reversed by restarting superfusion with standard Tyrode or by perfusion with 02 gas.5. Many of the effects of N2 gas perfusion could be mimicked by the addition of 20 mM-lactic acid to the superfusing solution, which caused a profound reduction of tension and also an increase in the amplitude and duration of the Ca21 transients. Calculation of the changes in intracellular pH caused by the addition of lactic acid suggest that the fall in intracellular pH produced by lactic acid was similar to that occurring in ischaemia. D. G. ALLEN, J. A. LEE AND G. L. SMITH6. Repeated exposures to N2 gas perfusion caused tension to fall more rapidly and an increased resting tension to develop more rapidly. The slowly developing rise in Ca2+ transients was abolished and a rise in resting Ca2+ occurred more quickly.7. When muscles were quiescent, exposure to N2 gas perfusion caused an increase in resting light. Low-frequency, spontaneous oscillations of [Ca2+]i appeared which increased in magnitude and frequency with increasing duration of exposure. Such oscillations were suppressed by stimulation during exposure to N2 gas perfusion but appeared when stimulation was stopped. 8. Addition of 20 mM-lactic acid to the solution superfusing a quiescent muscle caused an increased resting Ca2+. Large, low-frequency oscillations of [Ca2+]i appeared, which increased in ...

Metabolic changes during ischaemia and their role in contractile failure in isolated ferret hearts.

“…Allen et al 1989;Lee & Allen, 1991). However, this approach is complicated by the need to estimate the degree of activation of the intact preparations (see Eisner et al 1987b, andAllen et al 1989, for discussion). An 470 CAUSES OF ISCHAEMIC CONTRACTILE FAILURE alternative is to try to mimic the metabolic changes accompanying ischaemia in a model with continued perfusion.…”

“…This is supported by the finding that the acidosis which accompanies anoxia is increased in size in the presence of extracellular lactate (Ellis & Noireaud, 1987) or in the presence of a-cyano-4-hydroxycinnamate. However, a further mechanism which may contribute to the intracellular acidosis is the accumulation of CO2 within the cell (see Allen et al 1989, for a discussion of this effect).…”

“…It is known that intracellular free calcium concentration ([Ca2+] Porterfield, Yue & Chacko, 1987;Allen. Lee & Smith, 1989).…”

Changes in myoplasmic pH and calcium concentration during exposure to lactate in isolated rat ventricular myocytes.

“…Secondly, a proton-induced slowing of Ca2" uptake by the sarcoplasmic reticulum (Mandel et al 1982) (Earm & Irisawa, 1986) and since Ca2" is normally extruded by the Na'-Ca2+ exchanger in resting muscle (for review see Eisner & Lederer, 1989) (Kleber, 1983). It is also likely that changes in the action potential which become prominent after 10-15 min are involved in the subsequent decline in Ca2`transients which occurs later in ischaemia (Allen et al 1989). (ii) A number of studies have suggested that operation of the Na+-H+ exchanger leading to Na+ and Ca2`loading is the mechanism by which acidosis causes increased [Ca2+], during ischaemia (Tani & Neely, 1989;Pike, Kitakaze & Marban, 1990 (Kleber, 1983;Vanheel et al 1990) and, as noted above, Vanheel et al suggested that extracellular acidosis was inhibiting the exchanger. This was also the basis for the hypothesis of Lazdunski et al (1985) in which it was proposed that reactivation of the Na+-H+ exchanger on reperfusion led to Na+ and therefore Ca2`loading.…”

“…However, our result is close to the predictions of data of Vaughan-Jones and colleagues. They showed that a reduction in pH0 from 7-4 to 6-4 caused approximately a 5-fold fall in proton efflux (Vaughan-Jones & Wu, 1990b) while a decrease in pHi from 7-15 to 6-68 caused approximately a 5-to 10-fold increase in proton efflux (Kaila & Vaughan-Jones, 1987 Allen et al 1989). Since fura-2 was found to be sensitive to changes of pH it was necessary to correct the [Ca2+]i using the changes of pH, measured during lactate exposure.…”