Control of interval-force relation in canine ventricularmyocardium studied with ryanodine1D. Bose, 3L.V. Hryshko, 2B.W. King & T. Chau
Cardiovascular Pharmacology Section, Department of Pharmacology and Therapeutics, Anaesthesia &Internal Medicine, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba, Canada R3E OW3
1 The mechanism of post-extrasystolic, rest and frequency potentiation was studied in canineisolated ventricular muscle.2 Ryanodine, which impairs Ca availability from the sarcoplasmic reticulum (SR), reduced theamplitude of the extrasystole less than that of the steady state contraction. Ryanodine also inhibitedpost-extrasystolic potentiation and converted rest-potentiation into rest depression. Rest-potentiation was blocked preferentially by ryanodine compared to post-extrasystolic potentiation.An increase in the contribution of extracellular Ca to the extrasystolic contraction could notentirely account for the post-extrasystolic potentiation.3 Prolonged rest, by itself, also caused depression of the first post-rest contraction. During rest-potentiation, SR Ca seemed to play a greater role in contraction than transmembrane Ca influx.However, the ability of the 'release pool' of Ca in the SR to be reprimed after a contraction wasreduced. This was seen as a decrease in post-extrasystolic potentiation elicited immediately afterrest.4 A decrease in stimulus interval was associated with a transient decrease in contraction ampli-tude followed by an increase. An abrupt increase in stimulus interval had the opposite effect. Ryan-odine blocked the initial transient changes and accelerated the delayed changes. These resultssuggest that the transient changes in contraction after sudden changes in drive interval are depen-dent on the SR.5 Transmembrane Ca entry and the rate of recovery of the Ca release process (repriming) in theSR after a contraction seem to be interval-dependent. The data also indicate that different mecha-nisms are involved in post-extrasystolic and rest-potentiation.6 The results are consistent with a model which proposes 'recirculation' of activator Ca within theSR after a contraction or of the presence of an appreciable amount of inactivation of the SR Carelease process during normal stimulation. An increased pool of releasable Ca due to longer recircu-lation time or a time-dependent decay in the level of inactivation of Ca release from the SR maygive rise to rest-potentiation.
Introduction
Mammalian ventricular myocardium shows complexchanges in contractility when the rate or pattern ofstimulation is changed. These are seen in the form offrequency potentiation (FP), post-extrasystolicpotentiation (PESP) and rest-potentiation (RP). Thelatter is seen in ventricular preparations from somemammalian species (e.g. cat and dog; Koch-Weser &Blinks, 1963; Endoh & Iijima, 1981) but not in
1 Author for correspondence.2 Present address: Hoechst-Roussel Pharmaceuticals, Inc,Sommerville, NJ, U.S.A.I Present address: Div. of Biomed. Sci., Univ. of California,Riverside, CA, U.S.A.
others (rabbit; Edman & Johannsson, 1976). Amodel of excitation-contraction coupling whichwould account for the interval-force relation pro-poses the recycling of activator Ca from an uptakesite to a release site in the sarcoplasmic reticulum(SR), during the interval between contractions (e.g.Wood et al., 1969; Morad & Goldman, 1973; Allenet al., 1976; Edman & Johannsson, 1976, Wohlfart &Noble, 1982; Schouten et al., 1987). The size of thereleasable pool of Ca, at the moment of onset of con-traction, is thought to be a function of the timeelapsed after the last release of Ca and the degree ofrefilling of this pool, due to Ca influx during the pre-ceding beat and by translocation within the SR
(Wohlfart & Noble, 1982). Ca influx across the sar-colemma (SL) during an action potential may con-tribute directly to contraction but the magnitude ofthis is uncertain and may be variable (New & Traut-wein, 1972). It is quite possible that the contributionsmay vary with different physiological situations. Thepresent study examined the relative contributions ofthe SR and extracellular divalent cation to contrac-tion during short-term alteration of contractility dueto the interval-force relation. Ryanodine, an alkaloidwhich impairs Ca release from the SR (Sutko et al.,1979), was used as a tool to provide some insightinto the cation pools involved in the interval-forcerelation.
Methods
Preparation
Hearts were removed from mongrel dogs (3-10 kg) ofeither sex, under pentobarbitone anaesthesia(30mgkg-1 i.p.). These were immersed in cold, oxy-genated Krebs-Henseleit (KH) solution. Thin, free-running trabeculae (< 1.5 mm) were tied with 6.0 silkthread at each end and removed from the wall of theventricle. Tissues were usually obtained from theright ventricle where thin trabeculae are more abun-dant.
Recording techniques
The tissue was tied to the base of a stimulating elec-trode and placed in a 20 ml vertical tissue bath con-taining KH solution bubbled with 95% 02 and 5%CO2. The temperature of the bath was maintainedat 37.0 + 0.20C. The free end of the tissue was con-nected with a stainless steel wire to a isometric force-displacement transducer (Grass FT-03C). Astimulator (F. Haer Instruments; Pulsar 6i) con-nected to a custom built computer-controlled prog-rammable pulse generator (Boyechko & Bose, 1984)or to a programmable stimulus parameter incre-mentor (F. Haer Instruments; Pulsar ICR) providedsquare wave stimuli of 3 ms duration to punctateplatinum electrodes. Stimulus amplitude was adjust-ed to about 10-20% above threshold. The restingtension was increased until the maximum twitchtension at a basic stimulus interval (basic cyclelength) of 2000ms was obtained and then the tissueswere equilibrated for 1 h. Contractions and stimuliwere stored on a Hewlett-Packard model 3960instrumentation tape recorder and were simulta-neously displayed on a waveform analyser (DataPrecision; Model Data 6000) and a 4 channel chartrecorder (Grass Model 7; Gould Brush; Model 440)
and photographed on Polaroid film or plotted on adigital plotter (Hewlett Packard; Model 7475B).
Stimulation protocol
A basic cycle length of 2000ms was chosen for mostexperiments. A train of 10 or more stimuli was fol-lowed after a variable interval by another similartrain. The first pulse of the second train was con-sidered the test pulse. The stimulator was prog-rammed to increase the interval between the 2 trainsby a predetermined amount after each train. Thisvaried between 200 ms to 1200s. A beat following acoupling interval of less than the basic cycle lengthwas referred to as an extrasystole and that longerthan the basic cycle length was referred to as a post-rest beat. The interval between the extrasystole andthe subsequent pulse (post-extrasystole) was kept thesame as the basic cycle length. A sufficient number ofconditioning pulses necessary to ensure completerecovery after rest were inserted before the testsequence.
Statistical analysis of data
Each experiment consisted of at least 5 trials, unlessspecifically mentioned. In self-controlled experimentswith single treatment, statistical significance wasmeasured by use of Student's paired t test. When theexperiment involved more than 2 groups, One WayAnalysis of Variance in conjunction with Duncan'sNew Multiple Range Test (Steel & Torrie, 1960) wasemployed. A P value of <0.05 was considered sta-tistically significant.
Solutions and drugs
Krebs-Henseleit solution (KH) had the followingcomposition (mM): NaCl 118.0, KCl 4.7, CaCl2 2.5,MgSO4 1.2, KH2PO4 1.4, Na2HPO4 1.7, NaHCO325, glucose 11.1. Ryanodine was a gift from Dr R.Rogers (Merck Sharp and Dohme) and was dis-solved in 0.1 N HCI.
Results
Effect ofchanges in stimulationfrequency
In KH solution, a decrease in the basic cycle length,in the range of 200 to 600ms, resulted in increasedtension (Figure 1 al, bl, cl and dl). Tension of thefirst contraction at the shorter basic cycle length wassmaller and then gradually increased to a new andhigher steady state.An increase in the coupling interval between beats
led to an increase in the tension of the first contrac-
RYANODINE AND INTERVAL-DEPENDENCE OF VENTRICULAR CONTRACTIONS 813
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Figure 2 Effect of varying the coupling interval on
extrasystolic (ES) and post-extrasystolic (PES) contrac-tions.
tion at the longer interval. The amplitude of the firstcontraction at the longer coupling interval was
inversely proportional to the immediately precedingcoupling interval. The subsequent contractions (notshown in the record) developed decreasing amountsof tension until a new steady state was reached afterabout 10 contractions. This behaviour resemblesrest-potentiation, to be described later.
Post-extrasystolic potentiation
A single premature extrasystole caused the poten-tiation of the next normal beat (post-extrasystolicpotentiation). The extrasystolic contraction wassmaller than the steady state contraction at all testpulse intervals between 150-800ms (Figures 2 and3).The post-extrasystolic contraction was potentiated
when the extrasystolic interval was less than1000ms. The magnitude of potentiation increasedwith shortening of the test pulse interval to 200ms,but decreased again at a coupling interval of 150 ms.
3-Figure 1 Effect of reduction of the basic cycle length(BCL) from 2000ms to (a) 200, (b) 300, (c) 400 and (d)600ms followed by resumption of normal BCL. Thefirst two beats in each group are at the normal (longer)driving interval. Panel 1 is the response in the untreatedtissue. The potentiation of the first beat on resuming thenormal coupling interval is inversely proportional tothe change in interval. Panels 2 and 3 are 30min and60min after 10- m ryanodine. Note higher recordinggain. Positive staircase is maintained but potentiationdue to change from a short to a long basic cycle lengthis markedly reduced.
Rest-potentiation
The first contraction after an interruption of stimu-lation produced more tension than steady state con-tractions (Figure 3). This will be referred to as'rest-potentiation'. The effects of interruptions lastingbetween 5s to 1200s were studied at a basic cyclelength of 2000ms. After a pause longer than 240-1200s the post-rest beat was usually depressed (post-rest depression). This effect was seen at shorter restintervals in the young dogs (<6 months old) but atlonger intervals in adult dogs. Maximum poten-tiation of the post-rest beat usually occurred after arest period of 60-120 s (longer period applies to older
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Figure 3 Effects of varying rest intervals on post-restcontractions. The basic cycle length was 20ooms. (i)Represent the first beat after rest while (A) denote thesmallest beat after rest (tension undershoot). This hasbeen shown in the inset. The figure shows that post-restundershoot in tension is related to rest duration.
dogs). Potentiation of the initial few beats followingrest, was followed by a decrease in amplitude belowthe pre-rest level and a final gradual return to thecontrol level. The magnitude of this tension under-shoot was proportional to the rest duration (Figure3).
Effects ofryanodine
The SR has been postulated to play an importantrole in interval-related changes in cardiac contrac-
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tility (Johnson & Kuohung, 1968; Wood et al., 1969;Manring & Hollander, 1971; Bassingthwaighte &Reuter, 1972; Morad & Goldman, 1973; Forrester &Mainwood, 1974; Kaufmann et al., 1974; Allen et al.,1976; Chapman & Leoty, 1976). Since ryanodine hasbeen postulated to impair the release of Ca (Sutko etal., 1979), increase its leakage from the SR(Hilgeman, 1982; Hunter et al., 1983) and also causestructural changes in the vicinity of the t-tubule andlateral cisternae (Penefsky, 1974), its effect on thevarious potentiation phenomena was examined.Steady state contractions, obtained in the presence ofnormal Ca (2.5 mM) at a basic cycle length of2000ms, were reduced to about 5-10% of control inthe presence of ryanodine (10-7M-105 M). Thereduction in contraction amplitude was irreversibleand time-dependent.
Frequency potentiation In the presence of ryan-odine (10-7M) an abrupt change in basic cyclelength from 2000ms to 200, 300, 400 or 600msresulted in a progressive increase in the peak level ofcontractions without the initial decrease seen innormal tissues (Figure 1, panels 2 of a, b, c and d).The increase in contraction amplitude occurred intwo phases, an initial fast one followed by a smallerslow increase. The opposite was seen when the inter-val was abruptly increased. The initial rapid phase ofincrease in contraction amplitude on shortening thebasic cycle length occurred faster in the presence ofryanodine than in the untreated tissue (Figure 4).
30 40
Number of contractionsFigure 4 Effect of a decrease in the basic cycle length from 2000ms to 300ms (C], *) or 200ms (A, A) in control(EJ. A) and ryanodine (10- IM)-treated trabecula (I, A). Note that in contrast to the control preparations (also Figure 1al, bI and cl) there is an absence of the transient negative inotropic effect of the shorter coupling interval and also afaster rate of attaining peak amplitude in the ryanodine-treated tissue. The data are from 4 experiments. All experimentalpoints in the ryanodine-treated tissue except the first one are significantly different (P < 0.05 ANOVA/Duncan's multiplerange test) from the corresponding ones in the control tissue.
RYANODINE AND INTERVAL-DEPENDENCE OF VENTRICULAR CONTRACTIONS 815
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Figure 5 Effect of different durations of treatment withryanodine (3 x 108M) on post-extrasystolic poten-tiation (PESP, after a 300ms extrasystole) and rest-potentiation (RP, after 120s rest). Basic cycle length ofnormal conditioning beats was 2000ms. Note the pref-erential depression of the post-rest beat.
Ryanodine also markedly reduced the potentiationseen immediately after returning to the initial basiccycle length (Figure 1, panel 2 of a, b, c and d). Theeffects of ryanodine increased over a period of time(i.e., 30 vs 60min; Figure 1, panels 2 vs 3).Post-extrasystolic potentiation Unlike control prep-arations, in the presence of ryanodine, extrasystolicbeats were larger than the steady state beats. Thesize of this extrasystolic contraction increased atshorter coupling intervals. This was in contrast tothe behaviour of the extrasystole in the untreatedmuscle. SR plays a relatively insignificant role in thepresence of ryanodine (Sutko et al., 1979). Hencecontractions after treatment with ryanodine aremainly dependent on extracellular Ca. The post-extrasystolic potentiation was not diminished in thepresence of 3 x 10 8M ryanodine (Figure 5) and wasonly partially blocked with a concentration of10- 7M. A larger concentration of the alkaloid(10-6M-10- M) completely abolished post-extrasystolic potentiation.
It has been proposed that activator Ca enteringduring the extrasystole may recirculate back to therelease site in the SR and cause potentiation of thepost-extrasystolic beat. To examine this 5 trials ofthe type shown in Figure 6 were performed.
In this experiment the basic cycle length was2000ms and the extrasystolic interval was 300ms.Ryanodine blocked the steady-state contractions to15.3% of that in the normal muscle. Hence Ca fromthe SR supported roughly 84.7% of the contractionin the normal muscle. Extrasystoles were 19.1% ofthe steady state beats in the control muscle. Thesewere decreased after ryanodine (10-6 M) to only18.4% of the control SS beats without ryanodine. In
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Figure 6 Contribution of Ca from the SR and extra-cellular space to normal (SS), extrasystolic (ES) andpost-extrasystolic (PES) contractions, in the presence ofnormal extracellular Ca, in control (stippled columns)and ryanodine-treated (hatched columns) tissues. Thebasic cycle length was 2000ms and extrasystolic coup-ling interval was 300ms. Numbers above the columnsrefer to contraction amplitude expressed as a % ofsteady state contraction in the normal muscle. Forexplanation see Results and Discussion.
other words the post-ryanodine extrasystoles were3.7% smaller than those before adding the drug.This difference was statistically not significant;P > 0.05 (n = 4). Hence the SR appeared to play aninsignificant role in the extrasystolic contraction. Inthe presumed absence of contribution of Ca from theSR in the ryanodine-treated tissue, the extrasystoleswere about 25% larger than the steady state beats inthe ryanodine-treated tissue. When expressed as a %of the steady state amplitude of the contractionin the absence of ryanodine, this difference was 3.1%.It is not clear whether this increase in the size of theextrasystole in the ryanodine-treated muscle isentirely due to increased extracellular Ca influx ordue to reduced buffering of Ca, by the SR. However,this is an estimation of the maximum increase incontraction possible through enhanced trans-membrane Ca entry. It seems unlikely that this smallincrease in Ca influx or increased intracellular avail-ability can fully account for the observed post-extrasystolic potentiation of 60%.
Rest-potentiation Ryanodine (10-6 M) completelyabolished rest-potentiation at all intervals between5s-1200s. The characteristic response in the pre-sence of ryanodine was a reduction in the size of thefirst post-rest beat to below the level of the precedingsteady-state contraction followed by a gradualrecovery (Figure 7 inset).
816 D. BOSE et al.
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Figure 7 Comparison of the effect of rest in thenormal muscle (C) and ryanodine (10-6M, 45min)-treated muscle (R). Inset shows that the post-rest poten-tiation after a 30s rest is converted to a transientdepression. This effect is similar to that seen in theuntreated muscle after a very long rest. Each columndepicts the mean of 5 experiments. Post-rest contrac-tions after ryanodine are significantly different from thecorresponding contractions in the control, P < 0.01level (paired t test).
Comparison ofpost-extrasystolic and rest-potentiation
Even though the experiments with ryanodine sug-gested that both post-extrasystolic and rest-potentiations may depend on the integrity of the SR,the quantitative differences in the effect of ryanodineon the two types of potentiation and their differenttime courses suggested that the mechanism control-ling post-extrasystolic and rest-potentiation may bedifferent. Rest-potentiation was more sensitive to theblocking action of ryanodine than was post-extrasystolic potentiation. This was most clearly seenat very low concentrations of ryanodine shown inFigure 5. Rest-potentiation was depressed within10 min with 30nm ryanodine and was reduced from373% of steady state beat to 25% in 70min. In con-trast the post-extrasystolic beat was virtuallyunchanged at this concentration of ryanodinealthough, as mentioned earlier, it was blocked athigher concentrations of the alkaloid.
In order to test the difference between rest andpost-extrasystolic potentiation further, the experi-mental protocol shown in Figure 8 was employed.The purpose of this experiment was to see whetherafter a period of rest, which resulted in potentiation,an extrasystole could also elicit potentiation. Fourmuscles from different animals were stimulated at a
Stimulus protocol
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Figure 8 Effect of rest on post-extrasystolic poten-tiation. The inset illustrates the two experimental proto-cols. In the top procotol two extrasystoles and theircorresponding post-extrasystolic potentiated beats areshown, one before (ESI; PESI) and one after a periodof rest (ES2; PES2). The second type of protocol wasused to correct for the contribution of residual restpotentiation to the amplitude of PES2. The differencebetween the observed PES2 and the contribution ofdecaying rest-potentiation (PR + 1) gives the true mag-nitude of post-extrasystolic potentiation after rest. Stip-pled columns, post-rest potentiation; hatched columns,PES2 (corrected). The steady state contraction isreferred to as SS. The amplitude of PES1 (dashed line)has been shown for reference. For more details see text.
basic cycle length of 2000ms. After being condi-tioned with a train of 50 normal stimuli they wereprovided with an extrasystole (ESI) of varyingprematurity. A normally spaced post-extrasystolicbeat was produced after the extrasystole (PES1).This was followed by another period of conditioningwith a steady train of pulses having a basic cyclelength of 2000ms. This was succeeded by anotherrest period which varied from 5 s to 25 min. The firstpost-rest beat (PR) was followed by an extrasystole(ES2) which had an interval exactly equal to ES1preceding the rest period. The next beat (PES2) hadthe same relation to ES2 as PES1 had to ES1. Oneof the problems that this experimental design had tocircumvent was that any observed increase in PES2could be due to the residual potentiation from thepost-rest beat (as it takes several beats for rest poten-tiation to decay). To correct for this a second stimu-lus protocol was adopted in parallel experiments.After obtaining ES1 different durations of rest wereimposed as was done previously. After this thestimulation was resumed but no premature stimuluswas presented. The second beat after resumption ofstimulation (PR + 1) was indicative of the residual
RYANODINE AND INTERVAL-DEPENDENCE OF VENTRICULAR CONTRACTIONS 817
rest-potentiation remaining after the first post-restbeat. When the potentiation seen in the post-extrasystolic beat (PES2) of the previous stimulusprotocol was corrected for the potentiation ofPR + 1, a clearer picture of the effect of rest on sub-sequent post-extrasystolic potentiation was obtained.This was done by using the following formula:
Corrected PES2 = [PES2 - (PR + 1)]/SS
In Figure 8 the post-rest potentiation, PESI andthe corrected PES2 values following different dura-tions of rest are compared. The values shown areexpressed as % potentiation. It is shown that theeffect of rest on the potentiation of a subsequent beatvaried with the rest interval. The small differencebetween the potentiated beat at 60 and 120s was notstatistically significant (P > 0.05). Potentiation felloff significantly at rest intervals shorter than 60s orlonger than 120 s. On the contrary, post-extrasystolicpotentiation ((PES2), corrected for rest-potentiation(PR + 1)), was significantly reduced after rest periodsof 30 s. It seems therefore that conditions whichincrease rest-potentiation impair post-extrasystolicpotentiation.
Discussion
The present experiments were performed to elucidatethe mechanisms underlying the interval-force rela-tion. Of special interest to us was the relative impor-tance of the SL and the SR in frequency,post-extrasystolic and rest potentiation. Theseexperiments made use of ryanodine, an alkaloidwhich impairs release of Ca from the SR eitherdirectly or indirectly as a result of depletion. Persist-ence of the staircase in the presence of ryanodinegives further support to the importance of Ca influxthrough the SL in causing the phenomenon. Ourconclusions regarding the importance of the SR indetermining the amplitude of the first beat after restare similar to those obtained in the rabbit and rat byseveral investigators (Bers, 1985; Lukas & Bose,1986).
Role ofsarcolemma and sarcoplasmic reticulum ininterval-force relationship studied with ryanodine
Experiments were done with ryanodine, whichimpairs SR function, to investigate the role of SR incausing twitch potentiation due to alterations in thebasic cycle length. The overall purpose of theseexperiments was to test the contribution of a pos-sible increase in Ca influx from the extracellularspace during the extrasystolic beat, its accumulationin the SR and its subsequent contribution to the
potentiation of the post-extrasystolic beat. Based onthe finding that the extrasystole was larger than thesteady state contraction by 3.1% in the presumedabsence of SR function (in the presence ofryanodine), it can be said that there is extra calciumentry into the cell which may add to the calciumavailable for the next (post-extrasystolic) contraction.A rough estimate of this extra amount, based on thepCa-tension curve in skinned cardiac muscle byFabiato (1985a; Figure 2), is less than 11Mm. Theamount of calcium needed for a normal contraction,which is about 40% of a maximally potentiatedpost-extrasystolic beat, is 5,pM. Had the amount ofcalcium entering the cell during an extrasystoleremained the same as the amount entering during asteady state contraction, the post-extrasystolic con-traction should have theoretically remainedunchanged. This is because the interval between theextrasystole and the post-extrasystolic contractionremained the same as the interval between two regu-larly stimulated contractions. However, the addi-tional calcium entering during the extrasystole canaccount for only 25% of the potentiation, which isless than that observed during post-extrasystolicpotentiation. Our calculation of the extra calciumentering the cell during an extrasystole may be anoverestimate. It is possible that ryanodine may affectthe Ca buffering ability of the SR by impairing thefunction of the SR. As a result, a greater fraction ofthe calcium coming from the transmembrane routecould participate in the contraction. It seems reason-able to speculate that the extra calcium for extra-systolic potentiation may result from an increase inthe mobilization of Ca from an intracellular pool, inaddition to the priming of the intracellular pool bythe Ca entering the cell during the extrasystole. Twopossible explanations may be considered. There maybe a fraction of Ca entering the cell during an extra-systole which does not contribute to the immediatelyoccurring beat but is stored in the SR and releasedduring post-extrasystolic potentiation. Another pos-sible explanation is that the rate of recycling of Cafrom the uptake to the release site of the SR isinterval-dependent. Our data show that mechanicalrestitution is faster at a shorter basic cycle length(unpublished observations). This is somewhat differ-ent from the results of Edman & Johannsson (1976)who showed that, in rabbit papillary muscle, tensionrecovery after a preceding contraction was maximumafter about 800ms regardless of the 'priming' fre-quency preceding the test pulse. This points to afrequency-independence of the recycling mechanism.This difference may arise from the fact that the SR isfunctionally deficient in the rabbit (Bers, 1985).Hence one will see a lesser dependence of mechanicalrestitution on the previous driving frequency in thisspecies. The present data strongly suggest a
818 D. BOSE et al.
frequency-dependence of Ca influx as a primaryevent in the interval-force relationship in the dog fol-lowed by increased release of Ca from the SR.
Differences between post-extrasystolic andrest-potentiation
Rest potentiation is associated with conditions whichare different from those necessary for post-extrasystolic potentiation or frequency potentiation.Some of the experiments described in this papersuggest that the mechanisms of these two types ofpotentiation are different and the results are consis-tent with different speeds of 'repriming' of the'release pool' of Ca in the SR during these two typesof potentiation. Post-extrasystolic potentiation isassociated with a faster 'repriming' of the releasepool in the SR. Consequently if this process isimpaired there will be a decrease in the post-extrasystolic potentiation. After rest for 120s, rest-potentiation is maximal while post-extrasystolicpotentiation, elicited immediately after this restperiod is at a minimum. This suggests slowing of'repriming' during rest. Another difference betweenthe two types of potentiation is their response toryanodine. This can be explained on the basis of thepostulated ability of ryanodine to increase 'leak' ofCa from the SR (Hilgeman, 1982). Since there ismore time for this process during rest as comparedto the post-extrasystolic period, a greater decrease inthe size of the post-rest beat is expected. Indeed, theexperimental observations support such a prediction.
Model
Our data are consistent with the 'recirculationmodels' mentioned in the Introduction (Wood et al.,1969; Morad & Goldman, 1973; Kaufmann et al.,1974; Allen et al., 1976; Edman & Johannsson, 1976;Wohlfart & Noble, 1982; Schouten et al., 1987). Theaction potential causes the release of Ca from anintracellular release site in the SR to cause contrac-tion. Ca entering the cell through both the slowinward current and possibly through the Na-Caexchange (Coraboeuf, 1974; Horackova & Vassort,1976) may also be taken up by the SR for releaseduring a subsequent beat. The ability of Ca, comingfrom these two sources to activate the contractilemechanism is doubtful during normal contractionsbut may become increasingly important when theaction potential is prolonged. Membrane repolariza-tion causes a decrease in Ca entry from the extra-cellular space. Uptake of Ca by the SR Ca pumpcauses relaxation. Ca in the SR uptake site may betranslocated to the release site physically or func-tionally, possibly in a frequency-dependent fashion.
Ca bound to the inside of the SL is eliminated fromthe cell by Na-Ca exchange (Glitsch et al., 1970) or aCa pump, and the process is ready to be repeated.According to this model, frequency potentiationresults from (i) an interval-dependent increase in themagnitude of the slow inward current during eachbeat (Noble & Shimoni, 1981), (ii) an increase intotal cellular Ca content as a result of increased Cainflux due to the average increase in the time the SLis depolarized (Braveny & Sumbera, 1970) and (iii)an interval-dependent increase in the rate of recy-cling of Ca from the uptake to the release site in theSR (Morad & Goldman, 1973) or in the ability of aCa pool to be released in the SR (Fabiato, 1985a). Itwill be appropriate to use the term 'repriming' todescribe the process governing recovery of tensiondependent upon refilling of the release compartmentof the SR, either by Ca entering the cell or recirculat-ing back from the uptake sites in the SR and theterm 'restitution' to describe recovery of inactivationof the Ca release process after previous activation.Fabiato (1985b) has argued against the postulationof anatomically distinct sites in the SR for Ca uptakeand release. This argument is supported by a lack ofdata consistent with Ca redistribution as seen in theskeletal muscle SR using electron-probe microanaly-sis (Somlyo et al., 1981). Based on these reasons oneought to propose a functional rather than structuralcompartmentalization of SR Ca, with the differencebetween uptake and release based on time- andconcentration-dependent activation and inactivationof Ca release from the SR and of Ca accumulation inthe SR. The presence of a slow component of Caincrease around the rat cardiac SR results in a posi-tive staircase phenomenon with a decrease in stimu-lus interval, whereas an absence of this componentcauses a negative staircase (Fabiato, 1985b). Whilethese results can easily explain frequency poten-tiation and to some extent post-extrasystolic poten-tiation, it is difficult to explain rest potentiation inthe dog because releasable Ca in the SR continues tobuild up for 60-120s during the rest period resultingin an increase in the amplitude of the post-rest con-traction. As no action potentials occur during thisperiod, the slow component of Ca rise, necessary forrefilling of the SR, is absent. To explain satisfactorilythe rest potentiation one will have to invoke a slowprocess of 'recycling' of Ca from the uptake to therelease site or a slow process of 'repriming' of arelease pool or 'restitution' of a release mechanism.According to calculations made by Fabiato (1985a)the Ca release process in the rat has an absoluterefractory period of 0.8s and a relative refractoryperiod of 0.8-3.5 s. These time periods are too fast tobe compatible with the actual time course of restpotentiation we have demonstrated in the dog. Fur-thermore, recovery from such an inactivation process
RYANODINE AND INTERVAL-DEPENDENCE OF VENTRICULAR CONTRACTIONS 819
does not explain the large increase in potentiationafter rest in the absence of Ca influx from the intra-cellular space during the preceding rest period,unless a large amount of inactivation is present evenin muscles stimulated at a normal rate. The modelproposed by Adler et al. (1985) attempts to describea variety of potentiation phenomena dependent onalterations in stimulus intervals. This model is essen-tially a refinement of older models based on recircu-lation of Ca in the SR. However, rest potentiation isnot explained by this model. The more recent modelof Schouten et al. (1987) explains rest potentiation inthe rat on the basis of Ca translocation from anintermediate compartment in the SR between theuptake and release compartments. This model ismore appropriate for explaining our results. Wepropose that an extrasystole would augment bothcellular Ca content and the 'repriming' (recirculationof Ca taken up by the SR) or 'restitution' rate(recovery of inactivation of the Ca release processwith time; and assuming that during normal stimu-lation there is a large amount of inactivation capableof being lost during rest). Finally, the degree ofpotentiation after rest would depend upon thebalance between three factors: (1) the extent of 're-priming' of the SR with Ca or 'restitution' of the Carelease process continuing beyond the previous inter-stimulus interval, (2) the rate of Ca loss from therelease site to some other pool from which it cannotbe readily utilized (e.g. the extracellular space), and(3) the rate of decline of the late component of con-traction due to the absence of stimulation. One mayspeculate that the slow decline in tension withincreasing duration of rest, which ultimately leads toa rested-state contraction, indicates the decay in thesize of the releasable pool in the SR. This situationmay be brought about because of a lack of beat-dependent replenishment of the pool and also by areduction in the frequency-dependent Ca influxprocess.
It should be noted that the changes in contrac-
tions during alteration of the basic cycle length arefaster initially in the ryanodine-treated trabecula.This suggests that the interval-dependent sarco-lemmal processes reach a new steady state morerapidly than those involving the SR. The latter there-fore behaves like a 'flywheel', giving some stability tothe rapid changes in Ca movement across the sarco-lemma, during changes in heart rate. Indeed,amphibian hearts having relatively sparse SR behavein a manner similar to the ryanodine-treated mam-malian heart (Morad & Goldman, 1973; Bose,unpublished observations).
In conclusion, the present work shows that thecontribution of extracellular and intracellular Ca tocontractile tension varies with the rate and patternof stimulation. Rapid stimulation as well as prema-ture extrasystole stimulates transmembrane Cainflux which may directly increase the size of the re-leasable Ca pool in the SR and, in addition, mayindirectly increase the release of Ca from the SReither by increasing recycling between the uptakeand release site or by some other mechanism whichmay govern the restoration of the ability of the Capool in the SR to be released during excitation. Rest-potentiation occurs because of the continuation of'repriming' for some time during the initial phase ofthe rest period, despite the gradual slowing of thisprocess and the lack of action potential mediated Cainflux during the rest period. Replenishment of therelease site occurs at a rate faster than the rate ofsimultaneous loss of Ca to a non-releasable site.Eventually the latter process catches up due to thefinite amount of Ca available for 'repriming'. Thisresults in the rested state contraction (Reiter et al.,1978).
This work was funded by grants from the MRC of Canada,Manitoba Heart Foundation and the St. Boniface GeneralHospital Research Foundation. L. V. H. and B. W. K. wereCanadian Heart Foundation students.
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