Electrophysiologic effects of exogenous phosphocreatine in cardiac tissue: Potential antiarrhythmic actions

Electrophysiologic effects of exogenous phosphocreatine in cardiac tissue: Potential antiarrhythmic actions The cellular electrophysiologic effects of exogenous phosphocreatine (PCr) were analyzed to ascertain its purported antiarrhythmic properties during myocardial ischemia and reperfusion. Transmembrane potentials were recorded from isolated guinea pig papillary muscles and Purkinje fibers studied in vitro. Under control, normoxic conditions, 10 mmol/L PCr significantly increased the action potential duration (measured at 90% of repolarization) in ventricular muscle by 14.6 k 3.3 msec and the effective refractory period by 11.5 -t 3.8 msec (both p < 0.01). Under ischemic-like conditions (hypoxia, lactic acidosis, elevated [K+],, zero substrate) PCr had no effect. Phosphocreatinine, a related compound that is not a direct substrate in the creatine kinase reaction, acted similarly to PCr suggesting that alterations induced by PCr did not involve a change in the energy state of cells. However, PCr reduced free [Ca*+), by nearly 20%, and its electrical effects under normoxic conditions could be largely reversed by a concomitant 20% increase in [Ca*+],. In Purkinje fibers superfused with low [K+],-Tyrode’s solution to elicit conditions of Ca*+ overload, delayed afterdepolarizations and triggered responses were reversibly inhibited by PCr. These data suggest that the antiarrhythmic effects of PCr in situ may involve prolongation of the effective refractory period in nonischemic tissue or attenuation of membrane changes elicited by Ca*+ overload in ischemic cells. The mechanism by which PCr produces these effects may be related in part to changes in extracellular Ca*+ composition. (AM HEART J 1990;120:1111.)

Leonid V. Rosenshtraukh, PhD, Richard C. Witt, BS, Patrick George J. Rozanski, PhD. Moscow, USSR, and Omaha, Neb.

Phosphocreatine (PC,) is a high-energy phosphate compound that plays an important role in maintaining the normal physiologic state of cardiac cells. This is perhaps most evident during conditions of prolonged hypoxia or ischemia where impairment of myocardial contractility and intracellular ion homeostasis correlate closely with decreased PCr content.le4 Moreover, severe long-term depletion of intracellular high-energy stores has been shown to underlie irreversible cell damage.4 For these reasons attempts have been made to protect the myocardium from the pathophysiologic effects of ischemia and reperfusion by supplementation with high-energy From the Institute of Experimental Cardiology, USSR Cardiology Research Center, and the Departments of Physiology and Biophysics, Internal Medicine, and Anesthesiology, University of Nebraska College of Medicine. Supported by National Institutes in-aid from the American Heart Received

for publication

Nov.

of Health Association, 30, 1989;

grant HL38917 and by a grantNebraska Affiliate. accepted

June

Reprint requests: George J. Rozanski, PhD, Department Biophysics, University of Nebraska College of Medicine, Omaha, NE 68198-4515. 4/l/23477

4, 1990. of Physiology and 600 South 42nd St.,

N. Nance, MD, and

phosphate compounds. Indeed, when added to cardioplegic solutions, PCr improves postischemic recovery of cardiac function5-7 and decreases the incidence of ischemia- and reperfusion-induced arrhythmias in experimental models.69 *Tg Moreover, PCr has been used clinically as an effective antiarrhythmic agent in patients initially seen with acute myocardial infarction. For example, Ruda et al.1° reported that intravenous PCr given within 6 hours of the onset of chest pain reduced the frequency of ventricular premature beats and episodes of ventricular tachycardia. However, despite accumulating experimental and clinical evidence of its antiarrhythmic properties, the mechanisms by which PCr prevents ventricular arrhythmias remain unresolved. The present investigation assessed the cellular electrophysiologic effects of exogenous PCr in ventricular tissues under control, ischemic-like, and Ca2+-overload conditions in an attempt to explain its purported antiarrhythmic action. To learn whether these effects involved a direct improvement in the energy state of cardiac cells, they were compared with the effects of phosphocreatinine (PCr-nine), a struc1111

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below) returned to baselinevalues. High-energy phosphate solutions were not corrected for possiblechangesin osmolarity. Control Tyrode’s Ischemic Tyrode’s The effects of PCr and PCr-nine were alsocomparedin a secondgroup of experiments under depressedconditions 137.0 127.0 NaCl by exposingpreparations to an ischemic-like Tyrode’s soKC1 4.0 10.0 lution the compositionof which is given in Table I. The PO? 0.9 NaH@& 0.9 NaHC03 20.0 10.0 of this solution was lowered by equilibration with a gas 2.; 5 CaC12 2.5 mixture of 95% Nz, 5% CO2sothat comparedwith normal 0.5 0.5 MS& Tyrode’s solution, the “ischemic” solution was hypoxic, 5.5 0 Glucose hyperkalemic, acidic, and glucose-free.As in control ex0 20.0 Na lactate periments, successiveexposures to PCr and PCr-nine PH 7.4 6.8 solutionswere separatedby a period of drug-free ischemic 594.2 + 29.4* 47.3 I 1.5* POZ (mm W Tyrode’s solution to allow recovery of electrophysiologic ‘n = 5. parameters.However, it is important to keep in mind that the “ischemic” Tyrode’s solution usedin theseexperiments only approximated conditions likely to exist in the extracellular milieu of ischemicmyocardium in situ. Thus our turally related high-energy phosphate compound approach is limited in that other ischemia-derived substancesnot included in our solutions might further augthat is not a direct substrate in the creatine kinase ment or attenuate the electrical changesreported in the reaction.ll A preliminary report of this work has apResults section. peared in abstract form.12 A third seriesof experimentswasconducted in Purkinje METHODS fibers that were isolated along with a small amount of atTissue isolation. Papillary muscles(II = 16) and Purktached ventricular muscle. These preparations were exposed to normal Tyrode’s solution containing 1 mmol/L inje fibers (n = 5) were isolated from the hearts of guinea [K+10 to elicit conditions of Ca”+ overload and the develpigs put to death with sodium pentobarbital, 0.2 mg/gm opment of delayed afterdepolarizations (DADS) and trigintraperitoneally and pinned to the floor of a tissue bath gered activity. To reveal the peak amplitude and time perfused with oxygenated (95% Oz,5 % CO*) Tyrode’s socourseof DADS, trains of 10 stimuli were applied at BCLs lution heated to 37’ C. Isolated tissueswere paced (Model between 150 and 1000msec separated by l- to 2-second 1830, World Precision Instruments, New Haven, Corm.) through bipolar electrodesat a basiccycle length (BCL) of pauses.When changing the BCL, at least 30 secondswas allowed for equilibration before data were recorded. 1000msecwith 3 to 5 msecduration pulsesat 1.5to 2 times diastolic threshold voltage. Transmembrane potentials Measured parameters and statistical analysis. The measurementsmadefrom transmembranerecordingswere were recorded (Model KS 700, World Precision Instruas follows: resting membrane potential, action potential ments) with microelectrodeshaving resistancesof 10 to 20 MO and were simultaneouslydisplayed on an oscilloscope amplitude, action potential duration at 50% and 90”;. repolarization (APD5cand APDse),and maximum rate of rise (Tektronix model 5lllA, Tektronix, Inc, Beaverton, Ore.) of the action potential upstroke (v,,,), which was meaand recorded on FM tape (TEAC model XR-310, TEAC Corporation of America, Montebello, Calif.) for later analsuredfrom the output signalof an electronic differentiator ysis on a strip-chart recorder (Gould model 26OOS,Gould (Caltronics, Indianapolis, Ind.). Effective refractory period Inc, Cleveland, Ohio). Oxygen tension of control and (ERP) was determined with decremental stimuli (Sz) apischemic-like solutions (see below) was measuredwith a plied after every tenth basicstimulus(Si) through the same blood gas analyzer (Instrumentation Laboratory model electrode. ERP was measuredasthe shortest SI-Sz inter1302,Instrumentation Laboratory/Fisher Medica, Lexingval at which Sz elicited a regenerative responsehaving an ton, Mass.), and the concentration of free ionized extraamplitude of at least 50% of the basicaction potential. For experiments assessing the effects of PCr on oscillat,ory afcellular Ca2+ was determined by ion-selective electrode terpotentials in Purkinje fibers, the amplitude of generated (ICA-1, Radiometer, Copenhagen,Denmark). Tissueswere equilibrated for 1 hour while being superDADSwasmeasuredfrom the maximum diastolic potential fused with control, normoxic Tyrode’s solution (Table I). of the last driven action potential of a train to the peak of Under normoxic conditions PCr (Sigma-Aldrich Corp, St. the DAD. The timing of DADS was measuredas the couLouis, MO.) or PCr-nine (Sigma-Aldrich Corp, St. Louis, pling interval from the midpoint of the upstroke of the last MO.) was superfusedat a concentration of 10 mmol/L for action potential of a train to the peak of the ensuingDAD. 10 to 20 minutes. Inasmuch asPCr and PCr-nine are disResults are expressedas mean i standard error of the odium and monosodiumsalts,respectively, the solutionsto mean. Comparisonof two groups was made by meansof which these compounds were added were appropriately Student’s t test,l” whereas simultaneous comparison of formulated to maintain [Na+], constant. Normal Tyrode’s more than two groups wascarried out by analysisof varisolution wasreturned betweensuccessiveexposuresto PCr ance. When a significant difference amonggroups was inand PCr-nine solutions until measured parameters (see dicated by the initial analysis, individual paired compariTable 1.Composition of control and ischemicTyrode’s so-

lution (in mmol/L)

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effects of phosphocreatine

1113

sons were made with a modified t test.14Differences were considered significant at p < 0.05. RESULTS Effects of PCr and ischemic-like conditions.

PCr-nine

under

normoxic

and

The effects of exogenous PCr and P&nine were tested under control, normoxic conditions in six ventricular muscle preparations and under ischemic-like conditions in 10. Typical effects of PCr on a ventricular muscle fiber are shown in Fig. 1. Fig. 1, A shows two superimposed action potentials and their respective differentiated upstrokes (lower tracing) recorded from a fiber under normoxic conditions before and 10 minutes after exposure to 10 mmol/L PCr. Note that PCr primarily depressed the plateau phase of the action potential and prolonged its duration (o,,, was identical for both tracings in this experiment). Although prolongation of the APD elicited by PCr under normoxic conditions was modest, it was nevertheless significant (Table II) and was accompanied by a significant increase in the ERP. In contrast PCr exerted relatively little effect when tested under ischemic-like conditions. This is illustrated in Fig. 1, B, which shows superimposed action potentials recorded in a different preparation from that shown in Fig. 1, A. In the presence of ischemic Tyrode’s solution alone there was marked abbreviation in the APD compared to control conditions but unlike the response in normal tissue, PCr did not prolong the APD (Table II, bottom portion). Although addition of PCr to the ischemic Tyrode’s solution was associated with a small decrease in APD and ERP, these changes were not statistically significant. To assess whether the changes elicited by PCr possibly involved an alteration in the energy state of cardiac cells, they were compared with the effects of PCr-nine, a structurally related compound that is not a direct substrate in the creatine kinase reaction.ll As shown in Fig. 2, PCr-nine produced changes under normoxic (A) and ischemic-like (B) conditions that were qualitatively similar to those observed with PCr. A summary of the effects of PCr and PCr-nine under normoxic and ischemic-like conditions is given in Table II. Even though the changes in APD and ERP elicited by PCr-nine were generally greater in magnitude than those produced by PCr, the differences were not statistically significant. Alterations in extracellular ion composition. The similarity between the effects of PCr and PCr-nine on cardiac cells suggests that the former may act by mechanisms other than those involving the energy state of the cell. An alternative mechanism for the actions of PCr (and possibly PCr-nine) is suggested by the work of Fabiato and Fabiato,15 who have

B

O-

60 mV

60 msec 1. Electrophysiologic effects of 10 mmol/L PCr under normoxic (A) and ischemic-like (B) conditions. Each

Fig.

panel shows two superimposed action potentials and their respective differentiated upstrokes (V,,,) before and 10 minutes after administration of PCr. V,,, signals are offset in time from recorded action potentials for purposes of clarity. Calibrations shown are for recorded action potentials. For V,,, signalsin A (lower tracing), x and y calibra-

tion bars are equivalent to 15 msec and 1.20V/set, respectively. In B, x and y calibration bars for V,, msecand 150 V/set, respectively.

signalsare 6

shown that high-energy phosphate compounds bind divalent cations. Inasmuch as Ca2+ was the major extracellular divalent ion in the superfusates used in these studies, free [Ca2+], of otherwise normal Tyrode’s solution was measured with and without PCr and PCr-nine. In six experiments, control [Ca2+], (2.53 t 0.01 mmol/L) was reduced approximately 20% (to 2.06 -t 0.01 mmol/L; p < 0.001) by PCr and nearly 60% (to 0.97 -t 0.01; p < 0.001) by PCr-nine. Moreover, [Ca2+], was significantly lower with PCr-nine (p < 0.001) than with PCr, suggesting that the former was a more avid chelator of Ca2+. To further test the hypothesis that PCr acted in part by altering [Ca2+],, additional experiments were conducted to determine (1) whether the effects of PCr could be reversed by

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1114

Table

al.

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1990 Journal

II. Effects of PCr and Per-nine on ventricular musclefibers under control and ischemic conditions RMP CmVi

APA CmV)

V”,, Wlsec)

APDso

APDSo

(msec)

(msec)

C

-92.7 + 1.5

209.8

+ 23.2

129.3

+ 3.1

119.8

PCr P&-nine

-96.5 -95.7

f f

1.9 1.8

230.4 211.4

t 26.7 of- 27.6

125.8 126.2

i 3.6 k 2.6

131.6 160.1

I

-70.1

i

1.0

109.4

i

8.2

94.3

I

3.6

27.3

PCr P&-nine

-69.8 -70.1

zk 1.8 f 1.2

106.9 121.5

i 12.1 ?- 12.1

92.9 92.1

I 3.8 + 4.6

25.6 20.7

+ 4.8 I 8.1*

145.0 157.2

5 5.2 i 8.9*

139.4 150.7

z 5.6 f 10.5*

_t 8.7*

182.3

i- 9.0’

171.2

+

8.7*

_t 4.4 + 4.3

43.0 43.5

Z!I 4.8 -+ 5.2

57.1 55.8

_+ i

5.7 6.2

t- 4.2

33.6

+ 5.2

46.9

i

6.6

PCr and PCr-nine were each given at a concentration of 10 mmol/L; n = 6 for control and n = Ill for ischemic-like RMP, resting membrane potential; V,,,, maximum rate of rise of act,ion potential; APA, action potential amplitude; sured to 50’, of repolarization; APDso, action potential duration measured to 90 ‘<, of repolarization; ERP, effective conditions; I, ischemic-like conditions. ‘p < 0.01.

elevating [Ca2+lo 20% and (2) whether a simple 20%

reduction in [Ca2+], of normal Tyrode’s solution could mimic the effects of PCr alone. Examples of these experiments are given in Fig. 3. Fig. 3, A, shows two superimposed action potentials illustrating the typical effects of PCr when added to normal Tyrode’s solution. Similar to the results of previous experiments (Table II, upper portion), PCr prolonged APDso and ERP from control values in this example by 14.5 and 12 msec, respectively. In Fig. 3, B, which shows the same fiber exposed to a solution containing PCr plus 3.0 mmol/ L [Ca2+], (equivalent to a 20% elevation in [Ca2+],), APDeo and ERP were only slightly prolonged from control values by 9.8 and 5.0 msec, respectively. However, as shown in Fig. 3, C, exposure to an altered Tyrode’s solution containing 20% less [Ca2+], (2.0 mmol/L) failed to fully mimic the effects of PCr alone. Data from these experiments (n = 6), which are summarized in Table III, show that the effects of PCr plus 3.0 mmol/L [Ca2+], and altered Tyrode’s solution were not significantly different from control values. Thus the lack of a significant change in APD and ERP by altered Tyrode’s solution suggests that the full effect of PCr may only partly be mediated by a simple change in [Ca2+],. Effects

of PCr on delayed

oscillatory

afterpotentials.

The potential for exogenous PCr to alter [Ca2+], suggests that it may effectively attenuate the electrophysiologic effects of Ca2+ overload that often accompany myocardial ischemia and reperfusio&uid that may mediate cardiac rhythm disturbances.r7 To test this hypothesis conditions of Ca2+ overload were produced in five isolated Purkinje fibers by superfusing normal Tyrode’s solution containing 1 mmol/ L [K+],. On switching from 4 to 1 mmol/L K+Tyrode’s solution, DADS and triggered responses developed within 10 to 15 minutes when the BCL was shortened to less than 1000 msec. Fig. 4 gives an ex-

ERP imsec)

conditions. APDbo, action potential duration mearefractory period; C. control, normoxic

ample of triggered activity generated in the presence of low K+-Tyrode’s solution and its suppression by PCr. In Fig. 4, A, recorded in the presence of 1 mmol/L K+-Tyrode’s solution, the last three driven action potentials in a train of 10 (BCL = 300 msec) are shown followed by five nondriven, triggered responses that continued after the pace was stopped. Note that this activity terminated when the DAD after the fifth nondriven response failed to reach threshold. When the same fiber was superfused with 1 mmol/L K+-Tyrode’s solution plus 10 mmol/L PCr (Fig. 4, B), DADS and triggered activity were suppressed within 10 minutes. On washout of PCr (Fig. 4, C), triggered activity returned. Fig. 5 illustrates a more complete analysis of the BCL dependence of DAD amplitude and the inhibitory effects of PCr in a different preparation from that shown in Fig. 4. The top, middle, and bottom rows of this figure each show the lower portion of action potentials from the same fiber in response to a train of 10 stimuli given at BCLs of 400,300, and 200 msec, respectively. In the left column with 1 mmol/L K+-Tyrode’s solution but before PCr superfusion, DAD amplitude increased as BCL was progressively shortened. At a BCL of 200 msec (lower left panel) the last driven action potential in the train was followed by a nondriven, triggered response and subthreshold DAD. The middle column of panels shows that after 10 minutes of 10 mmol/L PCr, DAD amplitude at each BCL tested was reduced and triggered responses were abolished. Finally after 10 minutes of PCr washout with 1 mmol/L K+-Tyrode’s solution (right-hand column), DAD amplitude at each BCL returned to predrug values and triggered responses returned at a BCL of 200 msec (lower right panel). Data from all five Purkinje fiber preparations tested in the preceding manner are summarized in Fig. 6. Fig. 6, A, represents the peak amplitude of

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O-

o-

0

O-

I

60

mV

1 60 msec

Fig. 2. Effects of 10 mmol/L PCr-nine under normoxic (A) and ischemic-like (B) conditions. Format for this figure is sameas that in Fig. 1. For V,,, signalsin A, x and y cal-

ibration bars are equivalent to 3 msec and .120 Vlsec, respectively. In B, x and y calibration bars for V,,, signals are 6 msecand 150 V/set, respectively. 60 mV (120

generated DADS (ordinate) as a function of train BCL in the presence of 1 mmol/L K+-Tyrode’s solution (filled circles) and 10 to 20 minutes after the addition of 10 mmol/L PCr (open circles). At each BCL tested, PCr significantly reduced the amplitude of DADS and suppressed triggered activity. Fig. 6, B plots the coupling interval of generated DADS (ordinate) as a function of BCL. Although the coupling intervals of DADS varied considerably from preparation to preparation, PCr in all experiments delayed the development of DADS and reduced their amplitude. DISCUSSION Potential antiarrhythmic

effects of PCr. Several electrophysiologic mechanisms have been invoked to explain the genesis of ischemia- and reperfusion-related ventricular arrhythmias in humans.17 From the present experiments in ventricular muscle preparations the changes in APD and ERP produced by PCr under normoxic conditions suggest that it might ex-

Vlsec)

60 rnsec (15 msec)

Fig. 3. A, Superimposed signalsbefore and 10 minutes

after superfusion of 10 mmol/L PCr in normal Tyrode’s solution. B, Reversal of the effects of PCr (as in A) by elevation of [Ca2+10to 3 mmol/L. C, Effects of lowering [Ca2+], of normal Tyrode’s solution by 20% to 2.0 mmol/ L. Calibrations shownin parenthesesare for V,, signals in all panels. Sameimpalement was maintained throughout this experiment.

ert its antiarrhythmic effects in situ at least in part by preventing reentry. For this to occur it is necessary to consider that reentrant circuits may be prevented from forming by converting unidirectional to bidirectional block via (1) prolongation of the refractory period of cells proximal to the site of unidirectional block, (2) accelerated conduction via an alternate pathway, or (3) further slowing and block of conduction in depressed fibers.ls I9

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C I -0

1

1

80 mV I

2 set Fig. 4. Suppression of triggered responses by 10 mmol/L PCr. In presence of 1 mmol/L K+-Tyrode’s solution, nondriven, triggered responses ensued when external stimuli (BCL = 300 msec) were stopped. Each panel shows the last three driven action potentials in a train of 10 stimuli followed by short pause during which external stimuli were not applied. A, Before PCr. Note series of five triggered responses during pause that spontaneously terminated with subthreshold DAD. B, Ten minutes after addition of 10 mmol/L PCr to superfusate. C, Ten minutes of PCr washout.

In this investigation PCr prolonged the ERP in normal fibers (Table II) while eliciting little change under depressed, ischemic-like conditions. Thus the major effect of PCr on normal ventricular muscle fi-

November 1990 Heart Journal

bers was similar to that produced by class III antiarrhythmic agents, 2o although further studies are required to determine whether the magnitude of change in the ERP elicited by PCr would be functionally effective in preventing reentrant circuits from forming. Nevertheless, our data differ from those of Hearse et al9 who found that PCr in hypoxic rat ventricular muscle significantly prolongs APD whiIe exerting little effect under normoxic conditions. The basis for this discrepancy is unclear and could be the result of differences in species (rat vs guinea pig) or conditions used to depress the myocardium (hypoxia alone vs hypoxia + hyperkalemia + acidosis + zero substrate), More direct information concerning the potential antiarrhythmic properties of PCr was derived from experiments in Purkinje fibers superfused with low Kt-Tyrode’s solution (Figs. 4 to 6). It is known that exposure to low ]K+], solutions,21* 22 high concentrations of cardiac glycosides,23 or catecholaminesz4 elicits a marked increase in intracellular [Ca2+] ( [Ca2+]J that precipitates the development of oscillatory afterpotentials (DADs)17 through Ca2+-mediated activation of nonspecific inward current channels.“1-24 From the experiments in Figs. 4 to 6 it is clear that PCr exerted a rapid and potent inhibitory effect on development of DADS and triggered activity. Inasmuch as triggered activity has been documented in several experimental studies under ischemia- and reperfusion-like conditions,25T 26it may be hypothesized that at least some of the antiarrhythmic properties of PCr in situ may be related to an attenuation of membrane electrical changes produced by ischemia-related accumulation of intracellular Ca*+. Mechanism of action. Several ionic currents in cardiac cell membranes are modulated by metabolic processes to the extent that electrophysiologic properties can be significantly altered when the availability of intracellular ATP and PCr is limited.27, 28Thus it has been proposed that when ATP content is reduced under ischemic conditions, exogenous PCr may replenish intracellular ATP level& 6and thereby restore those energy-dependent mechanisms that maintain transmembrane ion gradients and active membrane properties. However, our data imply that the electrophysiologic effects of PCr do not directly involve changes in the energy state of the cell. This is based on the finding that PCr-nine’l elicited changes similar to those of PCr. Of course we cannot rule out the possibility that Per-nine was spontaneously converted to PC@ or that PCr freely crossed

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1 mMKi

1 mM Ki

electrical

+ PCr

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1 mMKi

BCL 400

J

I 1111111u-

1 80 mV

4 set

Fig. 5. Suppressionof DADS elicited in low K+-Tyrode’s solution by 10 mmol/L PCr. Top, middle, and bottom rows showsamefiber in responseto train of 10 stimuli at BCLs of 400,300, and 200 msec,respectively. Only lower portion of action potentials are shown.Left column illustrates BCL dependenceof DAD development before PCr. Last driven action potential at BCL = 200 msec(lower left panel) wasfollowed by triggered responseand subthreshold DAD. Middle column illustrates reduction in DAD amplitude and elimination of triggered responsesby PCr. After 10 minutes of PCr washout with 1 mmol/L K+-Tyrode’s solution (right-hand column) DAD amplitude returned to predrug values.

Table

III. Alteration in [Ca2+], in the presenceand absenceof PCr Vmax Wlsec)

RMP frnv) C PCr C PCr + 3 mmol/L Ca*+ C AT (2 mmol/L Ca2+) Data are summarized AT, altered Tyrode’s

-92.7 -96.5 -92.2 -95.6 -95.7 -95.9

for six preparations. solution containing

+ zt * j: * *

2.2 1.9 2.8 3.4 0.7 1.5

0.5 mmol/L

230.0 230.4 222.2 217.7 208.9 206.0

+ -t -+ -t k t

26.7 26.7 28.2 28.1 24.2 24.1

APA fmV) 127.4 125.8 127.2 127.4 132.7 132.6

k ck ? f + +

less CaC12; other abbreviations

APDm (msec) 3.9 3.6 3.9 3.9 0.8 0.9

117.2 f 5.9 131.6 k 8.1** 121.6 k 6.6 126.3 + 7.5 126.1 c 5.6 132.8 t 7.0

APDB (msec) 142.6 f 6.5 157.2 + 8.9** 147.7 k 6.5 153.5 + 7.5 151.8 k 7.9 158.6 t 7.9

ERP (msec) 139.2 150.7 141.5 144.0 145.8 155.5

+ 6.9 + 10.5* * 7.4 + 9.0 k 7.3 * 10.5

as in Table II.

*p < 0.05. tp < 0.005 compared with control conditions.

the cell membrane.2g The latter possibility, however, seems unlikely because of the low lipophilicity of PCr at physiologic PH.~O Alternatively the actions of PCr may be mediated

in an indirect manner by a reduction in free [Ca2+],.12 This possibility was supported by the fact that PCrinduced changes in normal fibers were largely but not completely reversed by a concomitant increase in

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252015105-

** ot; 200

-

800 :

300

400

1

** T

E z 5 E .-F E 2 u 2 n

*

600

T

0 400 i

500

l 1

I 0-O

Ly o------ +---o 1 1

1

I

1

200

0

L

200

300

---iz

500

-

BCL (msec) Fig. 6. Data are summarizedfrom five Purkinje fiber preparations illustrating BCL dependenceof DAD amplitude (A) and coupling interval (6) before (filled circles) and after (open circles) exposureto 10mmol/L

PCr.

[Ca2+], (Fig. 3 and Table III). Indeed the effects of PCr on normoxic ventricular muscle cells are consistent with the hypothesized modulation of APD by Ca2+. Specifically, previous studies have proposed that reduction in [Ca2+], prolongs APD primarily through a decrease in [Ca2+]i, which regulates steadystate K+ conductance (gkr)31j 32 and the rate of inactivation of the slow inward Ca2+ current (i,,).33 Moreover, the oscillatory increase in [Ca2+]i that occurs in states of Ca2+ overload and underlies development of DADS is attenuated when [Ca2+], is reduced.23 Thus it is possible that a PCr-mediated decrease in [Ca2+], and consequently [Ca2+]i34 may reduce the amplitude of DADS by decreasing the

magnitude of inward current carried by the Ca2+gated channels. Although the effects of PCr in ventricular muscle and Purkinje fibers in the present experiments are consistent with these proposed mechanisms, further experimentation is required to test these hypotheses in a more quantitative manner. We also cannot rule out the possibility that PCr acted directly on the sarcolemma independent of a change in Ca2+ composition. This is suggested by previous investigations that have shown that PCr reduces the extracellular accumulation of arrhythmogenic lysophosphoglycerides35 and release of intracellular enzymes in ischemic hearts.6 Moreover, the experiments illustrated in Fig. 3 and summarized

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5

in Table III of the present investigation showed that a 20 % increase in [Ca2+], in the presence of PCr did not completely reverse the effects of the latter, whereas a simple reduction of [Ca2+], by 20% failed to produce the same degree of APD and ERP prolongation elicited by PCr alone. It is therefore likely that PCr had a direct influence on membrane electrical properties, although the magnitude of this effect in normoxic ventricular muscle, at least at the concentration used, was small. REFERENCES

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