J Mol
Cell
Cardiol22,
715-723
(1990)
Functional Interconversion of Rest Decay and Ryanodine Effects Rabbit and Rat Ventricle Depends on Na/Ca Exchange Donald Division
M. Bers
and David
in
M. Christensen
of Biomedical Sciences, University of California,
Riverside, CA 92521-0121,
USA
(Received 8 August 1989, accepted in revisedform 22 January 1990) D. M. BERS AND D. M. CHRISTENSEN. Functional Interconversion of Rest Decay and Ryanodine Effects in Rabbit and Rat Ventricle Depends on Na/Ca Exchange. Journal of Molecular and Cellular Cardiology (1990) 22, 715-723. Rapid cooling contractures were used to assess changes in sarcoplasmic reticulum (SR) Ca content in isolated rabbit and rat ventricular muscle during rest, with altered transsarcolemmal [Na] and [Cal gradients and in the presence and absence of 1OOrm ryanodine. In rabbit there is normally a rest-duration dependent decline in SR Ca content (rest decay), whereas in rat there is a short-term increase in SR Ca content (rest potentiation) and little evidence of rest decay. Ryanodine greatly accelerates the rate of rest decay in rabbit, depleting the SR of Ca in - 1 s, whereas in rat, ryanodine does not appear to drain the SR even after a 10 min rest. Elevation ofintracellular Na activity in rabbit (by Na-pump inhibition) to a level similar to that measured in control rat during rest (Shattock and Bers, Am. J. Physiol., 256: C8134822, 1989) makes rest-dependent changes of SR Ca content in these two tissues similar. The rest decay in rabbit in the presence of ryanodine is also markedly slowed after Na-pump inhibition. In rat, reduction of [Ca]o allows rest decay to occur (+ ryanodine), but this rest decay can be largely prevented by simultaneous reduction of [Na]a (to maintain [Na13/[Ca] constant) which serves to keep the thermodynamic driving force on a 3:l Na/Ca exchange constant. We conclude that the process of rest decay and rest potentiation in both rabbit and rat ventricle depends on the sarcolemmal Na/Ca exchange. Furthermore, these species can be functionally interconverted by manipulation of the [Na] and [Cal gradients. The ability of ryanodine to deplete the SR of Ca also depends critically on other transport systems (p,articularly Na/Ca exchange) to remove Ca from the cytoplasm. KEY
WORDS:
Ryanodine;
Na/Ca
exchange;
Sarcoplasmic
Introduction
Ryanodine has become an invaluable tool in the study of excitation-contraction coupling in striated muscle. In particular, ryanodine binds with high specificity to the protein from the sarcoplasmicreticulum (SR) which is also known as the “foot” protein or spanning protein observed at the SR-transverse tubule junction in skeletal muscle (Inui et al., 1987a,b; Imagawa et al., 1987; Lai et al., 1988; Block et al., 1988). This protein has been isolated and also incorporated into planar lipid bilayers where it appears to function as a calcium channel and may be the SR Ca releasechannel (Lai et al., 1988; Hymel et al., 1988). Ryanodine has been shown to modify the gating of the SR calcium releasechannel incorporated in bilayers by “locking” the channel in an open, but subconducting state (Rousseauet al., 1987). This readily explains someinitial observations with ryanodine, such Please address all correspondence Riverside, CA 92521-0121, USA. 0022-2828/90/060715
+ 09$03.00/O
to: Donald
reticulum;
Rest decay;
Ca flux.
as induction of contracture in skeletal muscle (Jenden and Fairhurst, 1969). There have also been reports that very high concentrations o’f ryanodine ( > 100~~) can close the calcium releasechannel and inhibit calcium efflux in SR vesicles Uones et al., 1979; Meissner, 1986). The dominant functional effect of ryanodine in cardiac muscle is the depression of twitch force and an especially profound depression of post-rest contraction (Frank and Sleator, 1975; Sutko and Willerson, 1980; Bers, 1985). There is alsogreat species,regional and developmental variation in the effect of ryanodine on steady-state twitches in cardiac muscle (Penefsky, 1974; Sutko and Willerson., 1980; Bers, 1985). For example, contractions in rabbit ventricular muscleare only modestly depressed by ryanodine ( - 30%) whereas contractions in rat ventricle are strongly depressedby this agent ( - 90%). This difference
M. Bers, Division
of Biomedical
Sciences,
University
0 1990 Academic
of California, Press Limited
716
D. M. Bers and D. M. Christensen
in the effect of ryanodine probably reflects a difference in the relative dependence of contraction on SR Ca release (i.e., rabbit ventricle being less SR-dependent than rat) and not a difference in the ryanodine sensitivity of the SR (Shattock and Bers, 1987). This conclusion is supported by the fact that similar results are obtained with caffeine (Bers, 1985) and the similar concentration ( 10-‘“-10-5 M) and temperature dependence (23-37°C) of ryanodine action in both rat and rabbit ventricle (Shattock and Bers, 1987). The halfmaximally effective ryanodine concentrations are exactly the same in these two tissues over a broad temperature range, despite a difference in maximal extent of the effect on twitch amplitude (Shattock and Bers, 1987). Thus, it may be expected that ryanodine will have the same fundamental effect in both species. When rabbit ventricular muscle is rested for increasing periods of time (10 s-5 min), the magnitude of the post-rest twitch or rapid cooling contracture (RCC) progressively decreases. This process is known as rest-decay, and is thought to be due to the gradual loss of SR Ca (e.g. Allen et al., 1976; Bers, 1985). SR calcium loss during rest depends critically on the transmembrane [Na] gradient (Sutko et al., 1986) and rest decay can be virtually abolished by removal of extracellular sodium around isolated myocytes (Bers et al., 1989). Thus, rest-decay is likely to depend on a finite leak of calcium from the SR to the myoplasm, from where some calcium can be extruded via the Na/Ca exchange and some resequestered by the SR. Rapid cooling of mammalian cardiac muscle to -0°C induces the release of SR calcium (while inhibiting other calcium transport systems) and thus induces a contracture (Kurihara and Sakai, 1985; Bridge, 1986; Bers et al., 1989). Thus, RCC amplitude can be used as an index of the quantity of SR calcium available for release. Experiments using extracellular Ca-selective microelectrodes (to assess transsarcolemmal calcium fluxes) and RCCs (to assess the SR calcium content) have confirmed these interpretations and have also demonstrated that ryanodine does not prevent the SR from accumulating calcium (Bridge, 1986; Bers and MacLeod, 1986; MacLeod and Bers, 1987; Bers et al., 1987, 1989; Bers, 1987a; Bers and Bridge, 1989; Hryshko et al., 1989).
However, these studies also demonstrated that when Na/Ca exchange is operational, this SR calcium accumulation with ryanodine is only transient (consistent with a ryanodineinduced leak of calcium from the SR to the cytoplasm). In contrast to the rest-decay process demonstrated in rabbit ventricle, rat ventricle demonstrates a rest potentiation and this is accompanied by an increase in cellular calcium (Shattock and Bers, 1989) and in SR calcium content (Bers, 1989). Shattock and Bers (1989) also found that resting intracellular Na activity in rat ventricular muscle is higher than in rabbit ventricle muscle and high enough that calcium entry via Na/Ca exchange could be slightly favored at rest. The present study was motivated by these findings and the observation that despite the strong depressant effect of ryanodine on rat ventricular twitches, RCCs were not abolished, even after rest periods. This suggests that ryanodine does not empty the SR in rat ventricular muscle. The aims of the present study were to characterize the effect of ryanodine on RCCs in rat ventricle and to evaluate whether these characteristics are consistent with our current understanding of the mechanism of ryanodine action and the process of rest-decay (or rest potentiation). Methods Isolated ventricular trabeculae (0.1-0.5 mm diameter) were obtained from the hearts of Zealand New White rabbits and Sprague-Dawley rats which had received I.V. (rabbits) or I.P. (rats) injections of pentobarbital sodium ( N 75 mg/kg). The ends of the muscle were tied with fine suture. One end of the muscle was attached to a fixed post and the other to a piezoresistive transducer (AE 875, SensoNor, Horten, Norway) in a 0.15 ml superfusion chamber. The muscles were field stimulated at 0.5 Hz by platinum plates in the chamber walls during equilibration (N 1 h) and between protocols requiring quiescence. The super&sate was a modified normal Tyrode’s (NT) containing (in mM) 140 NaCl, 6 KCI, 1 MgCls, 2 CaClz, 10 glucose, and 5 N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES) at pH 7.40. All solutions were equilibrated with 100% Os, and
Ryanodine
the bath temperature was maintained at 30°C (except during cooling contractures) . The flow rate in the chamber was -30 ml/min. Solenoid pinch valves were situated at the bath inlet, and the perfusion lines leading to these valves were jacketed with either water (at 30°C) or propylene glycol-water (1:3 at - 2°C). At this flow rate, switching to the cold solution cooled the muscle surface to below 3°C in
Cell
Cardiol22,
715-723
(1990)
Functional Interconversion of Rest Decay and Ryanodine Effects Rabbit and Rat Ventricle Depends on Na/Ca Exchange Donald Division
M. Bers
and David
in
M. Christensen
of Biomedical Sciences, University of California,
Riverside, CA 92521-0121,
USA
(Received 8 August 1989, accepted in revisedform 22 January 1990) D. M. BERS AND D. M. CHRISTENSEN. Functional Interconversion of Rest Decay and Ryanodine Effects in Rabbit and Rat Ventricle Depends on Na/Ca Exchange. Journal of Molecular and Cellular Cardiology (1990) 22, 715-723. Rapid cooling contractures were used to assess changes in sarcoplasmic reticulum (SR) Ca content in isolated rabbit and rat ventricular muscle during rest, with altered transsarcolemmal [Na] and [Cal gradients and in the presence and absence of 1OOrm ryanodine. In rabbit there is normally a rest-duration dependent decline in SR Ca content (rest decay), whereas in rat there is a short-term increase in SR Ca content (rest potentiation) and little evidence of rest decay. Ryanodine greatly accelerates the rate of rest decay in rabbit, depleting the SR of Ca in - 1 s, whereas in rat, ryanodine does not appear to drain the SR even after a 10 min rest. Elevation ofintracellular Na activity in rabbit (by Na-pump inhibition) to a level similar to that measured in control rat during rest (Shattock and Bers, Am. J. Physiol., 256: C8134822, 1989) makes rest-dependent changes of SR Ca content in these two tissues similar. The rest decay in rabbit in the presence of ryanodine is also markedly slowed after Na-pump inhibition. In rat, reduction of [Ca]o allows rest decay to occur (+ ryanodine), but this rest decay can be largely prevented by simultaneous reduction of [Na]a (to maintain [Na13/[Ca] constant) which serves to keep the thermodynamic driving force on a 3:l Na/Ca exchange constant. We conclude that the process of rest decay and rest potentiation in both rabbit and rat ventricle depends on the sarcolemmal Na/Ca exchange. Furthermore, these species can be functionally interconverted by manipulation of the [Na] and [Cal gradients. The ability of ryanodine to deplete the SR of Ca also depends critically on other transport systems (p,articularly Na/Ca exchange) to remove Ca from the cytoplasm. KEY
WORDS:
Ryanodine;
Na/Ca
exchange;
Sarcoplasmic
Introduction
Ryanodine has become an invaluable tool in the study of excitation-contraction coupling in striated muscle. In particular, ryanodine binds with high specificity to the protein from the sarcoplasmicreticulum (SR) which is also known as the “foot” protein or spanning protein observed at the SR-transverse tubule junction in skeletal muscle (Inui et al., 1987a,b; Imagawa et al., 1987; Lai et al., 1988; Block et al., 1988). This protein has been isolated and also incorporated into planar lipid bilayers where it appears to function as a calcium channel and may be the SR Ca releasechannel (Lai et al., 1988; Hymel et al., 1988). Ryanodine has been shown to modify the gating of the SR calcium releasechannel incorporated in bilayers by “locking” the channel in an open, but subconducting state (Rousseauet al., 1987). This readily explains someinitial observations with ryanodine, such Please address all correspondence Riverside, CA 92521-0121, USA. 0022-2828/90/060715
+ 09$03.00/O
to: Donald
reticulum;
Rest decay;
Ca flux.
as induction of contracture in skeletal muscle (Jenden and Fairhurst, 1969). There have also been reports that very high concentrations o’f ryanodine ( > 100~~) can close the calcium releasechannel and inhibit calcium efflux in SR vesicles Uones et al., 1979; Meissner, 1986). The dominant functional effect of ryanodine in cardiac muscle is the depression of twitch force and an especially profound depression of post-rest contraction (Frank and Sleator, 1975; Sutko and Willerson, 1980; Bers, 1985). There is alsogreat species,regional and developmental variation in the effect of ryanodine on steady-state twitches in cardiac muscle (Penefsky, 1974; Sutko and Willerson., 1980; Bers, 1985). For example, contractions in rabbit ventricular muscleare only modestly depressed by ryanodine ( - 30%) whereas contractions in rat ventricle are strongly depressedby this agent ( - 90%). This difference
M. Bers, Division
of Biomedical
Sciences,
University
0 1990 Academic
of California, Press Limited
716
D. M. Bers and D. M. Christensen
in the effect of ryanodine probably reflects a difference in the relative dependence of contraction on SR Ca release (i.e., rabbit ventricle being less SR-dependent than rat) and not a difference in the ryanodine sensitivity of the SR (Shattock and Bers, 1987). This conclusion is supported by the fact that similar results are obtained with caffeine (Bers, 1985) and the similar concentration ( 10-‘“-10-5 M) and temperature dependence (23-37°C) of ryanodine action in both rat and rabbit ventricle (Shattock and Bers, 1987). The halfmaximally effective ryanodine concentrations are exactly the same in these two tissues over a broad temperature range, despite a difference in maximal extent of the effect on twitch amplitude (Shattock and Bers, 1987). Thus, it may be expected that ryanodine will have the same fundamental effect in both species. When rabbit ventricular muscle is rested for increasing periods of time (10 s-5 min), the magnitude of the post-rest twitch or rapid cooling contracture (RCC) progressively decreases. This process is known as rest-decay, and is thought to be due to the gradual loss of SR Ca (e.g. Allen et al., 1976; Bers, 1985). SR calcium loss during rest depends critically on the transmembrane [Na] gradient (Sutko et al., 1986) and rest decay can be virtually abolished by removal of extracellular sodium around isolated myocytes (Bers et al., 1989). Thus, rest-decay is likely to depend on a finite leak of calcium from the SR to the myoplasm, from where some calcium can be extruded via the Na/Ca exchange and some resequestered by the SR. Rapid cooling of mammalian cardiac muscle to -0°C induces the release of SR calcium (while inhibiting other calcium transport systems) and thus induces a contracture (Kurihara and Sakai, 1985; Bridge, 1986; Bers et al., 1989). Thus, RCC amplitude can be used as an index of the quantity of SR calcium available for release. Experiments using extracellular Ca-selective microelectrodes (to assess transsarcolemmal calcium fluxes) and RCCs (to assess the SR calcium content) have confirmed these interpretations and have also demonstrated that ryanodine does not prevent the SR from accumulating calcium (Bridge, 1986; Bers and MacLeod, 1986; MacLeod and Bers, 1987; Bers et al., 1987, 1989; Bers, 1987a; Bers and Bridge, 1989; Hryshko et al., 1989).
However, these studies also demonstrated that when Na/Ca exchange is operational, this SR calcium accumulation with ryanodine is only transient (consistent with a ryanodineinduced leak of calcium from the SR to the cytoplasm). In contrast to the rest-decay process demonstrated in rabbit ventricle, rat ventricle demonstrates a rest potentiation and this is accompanied by an increase in cellular calcium (Shattock and Bers, 1989) and in SR calcium content (Bers, 1989). Shattock and Bers (1989) also found that resting intracellular Na activity in rat ventricular muscle is higher than in rabbit ventricle muscle and high enough that calcium entry via Na/Ca exchange could be slightly favored at rest. The present study was motivated by these findings and the observation that despite the strong depressant effect of ryanodine on rat ventricular twitches, RCCs were not abolished, even after rest periods. This suggests that ryanodine does not empty the SR in rat ventricular muscle. The aims of the present study were to characterize the effect of ryanodine on RCCs in rat ventricle and to evaluate whether these characteristics are consistent with our current understanding of the mechanism of ryanodine action and the process of rest-decay (or rest potentiation). Methods Isolated ventricular trabeculae (0.1-0.5 mm diameter) were obtained from the hearts of Zealand New White rabbits and Sprague-Dawley rats which had received I.V. (rabbits) or I.P. (rats) injections of pentobarbital sodium ( N 75 mg/kg). The ends of the muscle were tied with fine suture. One end of the muscle was attached to a fixed post and the other to a piezoresistive transducer (AE 875, SensoNor, Horten, Norway) in a 0.15 ml superfusion chamber. The muscles were field stimulated at 0.5 Hz by platinum plates in the chamber walls during equilibration (N 1 h) and between protocols requiring quiescence. The super&sate was a modified normal Tyrode’s (NT) containing (in mM) 140 NaCl, 6 KCI, 1 MgCls, 2 CaClz, 10 glucose, and 5 N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES) at pH 7.40. All solutions were equilibrated with 100% Os, and
Ryanodine
the bath temperature was maintained at 30°C (except during cooling contractures) . The flow rate in the chamber was -30 ml/min. Solenoid pinch valves were situated at the bath inlet, and the perfusion lines leading to these valves were jacketed with either water (at 30°C) or propylene glycol-water (1:3 at - 2°C). At this flow rate, switching to the cold solution cooled the muscle surface to below 3°C in
