oTournalof MolecularandCellularCardiolo~ (1977) 9, 921-931
Potassium-related Humoral Transmission o f Overdrive S u p p r e s s i o n * M A R I O V A S S A L L E , D A N I E L J . K R E L L E N S T E I N , t M I C H A E L B. P L I A M ++ AND C H A N D L E R M c C . B R O O K S
Department of Physiology, State University of.New York, Downstate Medical Center, 450 Clarkson Avenue, Brooklyn, aVew York 11203, U.S.A. (Received 1 aTuly 1976, and accepted in revisedform 27 January 1977) M. VASSALLE,D . J . KRELLENSTEIN,M. B. PLIAMAND C. McC. BROOKS.Potassium-related Humoral Transmission of Overdrive Suppression. ffournal of Molecular and CellularCardiology (1977) 9, 921-931. The contribution of potassium to the suppression which follows overdrive (overdrive suppression) was studied in isolated canine hearts perfused in vitro. Complete atrioventricular block was produced by ligafing the His bundle. The electrical activity of the ventricles was recorded with locally implanted electrodes. Coronary sinus potassium concentration [K]es was measured before, during and after 1 to 10 rain drives. The collected coronary sinus effluent was reoxygenated and was used to perfuse in vitro Purldnje fiber strands isolated from the same ventricles. The following results were obtained: (1) during fast drive there was an initial K loss and after the drive (associated with a temporary suppression of pacemaker activity) a net K uptake; (2) Purklnje fiber strands isolated from the ventricle and perfused in vitro in all instances underwent a decrease in the spontaneous rate when exposed to the effluent collected during the first minute of overdrive; (3) when the Purkinje fibers were bathed in the coronary sinus effluent collected after the drive, spontaneous activity usually increased and in no instance decreased; (4) adding atropine to the effluent did not alter the observed changes in automatic discharge; and (5) increasing [K]o in fresh perfusate over the range found in the coronary sinus effluent caused a similar slowing of the Purldnje fiber activity. It is concluded that an increase in [K] o contributes to overdrive suppression which follows short but not long drives. The humoral transmission of overdrive suppression is thus only related to an increase in coronary potassium concentration.
KEY WORDS: Overdrive suppression; Atrio-ventricular block; Potassium; Purkinje fibers; Isolated heart; Acetylcholine; Atropine.
1. Introduction When the ventricles are driven at a fast rate, coronary sinus potassium concentration [K]cs initially increases and then slowly returns toward control [1, 5]. After the end of overdrive, [KJcs usually falls below control value [1, 5]. The temporary * Supported by grants from the New York Heart Association and N.I.H., Heart and Lung Institute. t Present address: Department of Surgery, Mount Sinai School of Medicine of the City University of New York, Fifth Avenue and 100th Street, New York, New York 10029. ++Present address: Department of Cardiothoracic Surgery, The University of U t a h Medical Center, Jesus Christ of the Latter Day Saints Hospital, Salt Lake City, Utah 84143.
922
M. VASSALLE E T A L .
suppression of pacemaker activity which follows a fast drive (overdrive suppression) is present whether [K]~s happens to be high or normal at the end of the drive [1, 5]. This finding indicates that factors other than high [K]o are responsible for overdrive suppression. However, the finding neither rules out a contribution of a high [K]o to the inhibitory process nor indicates the magnitude of the contribution, if any. This point was tested here as follows. Small dog hearts were perfused in vitro and the suppression which follows a fast drive measured. The coronary sinus effluent was collected before, during and after drives of different duration. Once the procedures were completed, strands of free-running Purkinje fibers were removed from the ventricles of the same hearts and were perfused in a tissue bath. T h e fibers were exposed to the oxygenated coronary sinus effluent previously collected from the same perfused heart and changes in spontaneous activity were recorded. I f an increase in potassium level plays a role in overdrive suppression, the coronary sinus effluent should have an inhibitory action on spontaneous activity of Purkinje fibers only when [K]e~ is increased. Also, the magnitude of the inhibition due to the elevated potassium can be determined. The effluent collected after long drives has a [K]cs below control and therefore it should not have an inhibitory effect attributable to potassium. This approach also permits to evaluate other possibilities. For example, if overdrive suppression is due to a release of acetylcholine, the suppression should be transmittable humorally whether the overdrive is short or long. Also, an inhibitory role of acetylcholine should be abolished by atropine. O n the contrary, if overdrive suppression is exclusively due to membrane events (ionic shifts, electrogenic Na extrusion), humoral transmission of suppression would not be expected after either short or long drives.
2. Materials and M e t h o d s T h e details of the methods have been described recently [2]. A general outline follows. Hearts from small dogs were perfused with a modified Tyrode solution in a constant temperature chamber. Complete atrio-ventricular block was produced by His bundle ligation. The ventricular cavities were drained by means of plastic tubes inserted through the apex of each chamber. Electrodes were sutured on the right atrium and both ventricles. T h e perfusate was pumped into the aorta at an average rate of 73ml. rain-1.100 g ventricular weight -x (range 50 to 100 ml). The composition of the perfusate in mmol.1-1 was as follows: NaC1 136.9, KCI 2.7, NaHCO3 11.3, CaCI~ 1.8, MgCI~ 0.5, NaH2PO4 1.51, dextrose 5.5. In some experiments, the potassium content was changed as indicated below. The perfusate was bubbled with 97% 02 and 3% CO2 and the p H was 7.30 to 7.40. Total perfusion time averaged about 2 h. The ventricles were electrically driven at 240. min -1 for 1, 5 and 10 min by means of a bipolar electrode placed on right ventricular outflow tract. T h e total coronary sinus effluent was
H U M O R A L TRANSMISSION OF O V E R D R I V E SUPPRESSION
923
collected over 1 min periods. Between the 25th and 35th of each minute a 5 ml sample was collected for [K]~ determination. All samples were immediately refrigerated. At the end of the above procedures, free-running strands of Purkinje fibers were removed from the ventricles and perfused in a tissue bath with warm (37~ oxygenated Tyrode solution containing 5.4 mEq. I potassium -1. T h e solution was perfused at a rate of 60 to 80 drops.rain -1. After 30 to 60 min of equilibration, the perfusate was changed to Tyrode solution containing 2.7 mEq. 1-1 of potassium to allow the development of spontaneous activity. Membrane potentials were recorded with glass microelectrodes filled with a 3 M KC1 solution. The recording apparatus consisted of a push-pull cathode follower stage, a Tektronix dual beam oscilloscope (RM565) and a Grass Kymograph camera (C4L). After a steady rate was reached in 2.7 mM K Tyrode solution, the perfusate was changed to the control coronary sinus effluent which had been reoxygenated. This effluent had been collected over a 5 rain period prior to the overdriving, while the heart was beating spontaneously. After 10 min (or until a steady rate was reached), the perfusate was changed successively to the 1 rain coronary sinus effluent samples taken during and after overdrive. T h e fiber was perfused with each of these reoxygenated samples for 5 min and the rate of discharge of the Purkinje fibers recorded. Action potentials were photographed during the exposure to each sample. At the end of the procedure the perfusate was changed back to the control coronary sinus effluent and then to normal 2.7 Tyrode solution. Atropine sulfate was added to some effluent samples in a final concentration of 5 rag. 1-1. T o reproduce the inhibition seen with effluent samples, the potassium concentration of Tyrode solution was increased from 2.7 to 2.97, 3.24 and 3.51 mEq.1-1. The fibers were perfused in each of the solutions for 5 rain and the rate recorded. The Tyrode solution containing 2.7 mEq. I-1 potassium was perfused again during the recovery period.
3. R e s u l t s
Humoral transmission of overdrive suppression Nine isolated hearts were studied. T h e Purkinje fibers from two of these hearts failed to develop spontaneous activity when perfused in the tissue bath and were discarded. In one of these two instances, a Purkinje strand from a freshly excised, non-perfused heart was used to test the effect of the effluent on automatic discharge with results similar to those of the other experiments. T h e results from these eight hearts are reported here.
924
ET AL
M, VASSALLE
Z
Z
7,
3.0 2.5
20
F2,7
o fc
~~ -~
~0 i
0
I
i
2 $ Time(rain)
4
FIGURE 1. Effect of perfusing isolated Purkinje fibers with the coronary sinus effluent collected before, during and after overdrive. ( • x ) Ventricular rate in beats.rain-1 ; ( H ) coronary sinus potassium concentration in mEq.l -I, (C) O) Purkinje fiber rate in beats.rain-I. Action potentialsof Purkinjc fiberrecorded during the exposure to the coronary sinus ci~uent arc shown at the bottom of the figure.The six groups of two action potentialswcrc recorded during exposure to the second, third,fourth, sixth,seventh and eighth pcrfusatc sample, respectively.The verticalbar is the voltage calibration(I00 m V ) and the horizontalbar isthe time calibration(I0 s).
Short overdrive I n four experiments the hearts were overdriven for 1 min at 24:0 beats.min -1. The results of one of these experiments are illustrated in Figure 1. The top curve shows the ventricular rate of the perfused heart. Before overdrive, the spontaneous idioventricular rate was 7 beats.rain -1. After the drive was stopped, there was a pause of 50 s followed b y a return to the pre-drive rate. T h e middle curve represents the coronary sinus potassium concentration in mEq. 1-1. The control effluent [K]~ was 2.65 m E q . l - 1 and it increased to 3.20 m E q . l - t after 30 s of overdrive. During the first minute of the post-drive period, the [K]~s decreased to 2.75 mEq. 1-1 and then to 2.65 mEq. 1-1 thereafter. There was no undershoot in this particular instance but samples were collected over 1 rain periods and thus a small undershoot may have been missed. A Purkinje strand removed from the ventricles of this heart was perfused in the tissue bath. The spontaneous action potentials recorded from this preparation are shown at the bottom of the figure and their rate of discharge is
HUMORAL
TRANSMISSION O F O V E R D R I V E
925
SUPPRESSION
plotted i n the curve above. T h e s p o n t a n e o u s rate i n 2.7 mM K T y r o d e solution was 16 b e a t s . m i n - t a n d decreased to 14 b e a t s . m i n - t w h e n exposed to the c o n t r o l c o r o n a r y sinus effluent. W h e n the effluent collected d u r i n g the 1 m i n overdrive was perfused, the rate of the p r e p a r a t i o n decreased to 10 b e a t s . m i n - t . T h e rate t h e n increased to 12 b e a t s . m i n -1 w h e n exposed to the sample collected d u r i n g the pause. T h e r e was a further transient increase i n the rate to 15 b e a t s . m i n -1. By the 5th m i n ' s sample, the rate was back to control. T h e r e was no increase i n rate a b o v e control d u r i n g perfusion w i t h the first post-drive sample as seen i n other experiTABLE 1. Humoral transmission of overdrive suppression Drive duration (rain)
Rate [K] 0= 2.7
Control rate 1'
A
B
C
Average :t:s.E.
1 1 1 1 5 5 5 5 5 5 5 10 10 10 10 5 5 5 10
16 21 40 18 I0 40 25 25 44 24 40 45 16 36 50 18 36 25 40 29.9 2.7
14 17 40 16 15 32 23 23 44 24 32 40 14 36 50 18 32 25 38 28.1 2.5
--4 --2 --10 --1 --6 --7 --5 --19 --4 --20 --5 --8 --8 --14 --6 --2 --6 --9 --12 --7.8 1.2
Rate change at 4'
Rate change Recovery in pause rate sample 9'
--1 --1 --3 --I --17 --1 --4 --3 + 1 --2 --1 --6 --2 --10 0 --1 +2 --8 --3 --3.9 ---0.5 1.2 0.6
--2* +3 --5* +5 +4 +3 +2 +9 +1 +13 +6 +9 +1 +9 (+33) +4 +9 +1 (+20) +6.6 1.9
14 13 40 18 10 28 18 16 20 16 26 42 9 30 (70) 20 30 26 (64) 26.8 3.8
Effect ofperfusing isolated Purkinje fibers with coronary sinus effluent collected before, during and after overdrive of the heart. A, 1 min overdrive; B, 5 and 10 min overdrive; C, Perfusion with atropinized effluent. Drive duration (rain) = duration of overdrive of the ventricles. Rate [K]0 = 2.7: spontaneous rate of discharge of Purklnje fibers in Tyrode containing 2.7 m~ K. Control rate: spontaneous rate of discharge of Purkinje fibers when exposed to control coronary sinus effluent. Rate change at 1', 4', 9': change in r.ate beats.rain-t when the fibers were exposed to coronary sinus effluent collected during the first, fourth and ninth rain of perfusion. Rate change in pause sample: change in spontaneous rate when Purkinje fibers were exposed to perfusate collected during the first minute after drive. Recovery rate: spontaneous rate of Purkinje fibers perfused again with 2.7 mMK Tyrode solution. The results in parenthesis are of little significance because of arrhythmia and were not included in the average values. * No undershoot of the [K]eg was seen.
M. VASSALLEETAL.
926
ments, presumably because there was no undershoot in the [K]os. The changes in rate of isolated Purkinje fibers when perfused with the coronary sinus effluent collected before, during and after 1 min overdrive are reported in Table I, section A.
Long drive In three hearts, coronary sinus effluent was collected before, during and after a 10 rain drive in five runs. The results are shown in Figure 2. The top curve shows the ventricular rate in beats.rain -1 and the bottom curve shows the changes in coronary sinus potassium in mEq.1-1. The maximum increase in [K]o, (from 2.58 + 0.05 mEq.1-1 to 3.01 -t- 0.01 mEq.1-1) was attained during the first minute of drive and was followed by a slow return toward control. By the 9 mln, the [K]~ was only 0.06 mEq. 1-1 above the pre-drive value. During the first minute of the post-drive period, [K]ca decreased to 2.31 ! 0.01 m E q . l - I and then increased again toward control. ~ A
2501
:~
3., 9o
2.9
/
+:'ua
X, T
2.7
........................
2.6
2.5 2.42.3
1 0
1 i
I 2
l 3
I 4
I 5
1 I I 6 7 8 Time (rain)
I 9
lxl I0
II
I I 12 13 14-
FIGURE 2. Effect of long overdrive on potassium balance in five experiments in three hearts. The average idioventricular rate was 33.4 4- 4.3 beats.min-1. The average pause was 44.5 4- 14.4 s. Perfusate potassium concentration was 2.58 4- 0.06 mEq.l-1. The results of one experiment in which the coronary sinus effluent collected during and after a 10 rain drive was used to bathe Purkinje fiber are shown in Figure 3. The top curve shows the ventricular rate of the perfused ventricle. Before overdrive, the spontaneous idioventricular rate was 28 beats.min -1. After the ventricles were overdriven at 240 beats.rain -1 for 10 rain, there was a pause of 90 s. The middle curve shows the coronary sinus potassium concentration in mEq. 1-1. The control effluent [K]c~ was 2.85 mEq.1-1 and increased to 3.50 m E q . l - 1
I-IUMORAL TRANSMISSION OF OVERDRIVE SUPPRESSION
~.
260 I' . 240~-
.
.
.
.
~
4Ol- ~_~
9
927
.
I
/
2oi-
/
~
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20f
e-~.
'.~ is
IJ
J
i
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.............
i!
---/~ i
I 4
I 6
'
...... J
~ J
| I ,. 8 I0 Time (rain)
2.0 '-"
:~ I 12
I 14
~. - 16 18
FIGURE 3. Effect of perfusing an isolated Purkinje fiber with the coronary sinus effluent collected before, during and after overdrive. ([-] V-I) Ventricular rate in beats.min-t showing the predrive rate of 28, the overdrive for 10 min at 240, a pause of 90 s and a recovery rate of 22 beats.min -1. ( ~ - - - - O ) Coronary sinus potassium concentration in mEq.1-1. Perfusate [K]= 2.70 mEq.l -t. (9 0) Purkinje fiber rate in beats.min -1. The gradual slowing is indicated by the interrupted line. The lower portions of the action potentials of Purkinje fibers (showing diastolic depolarization) are displayed at the bottom of the figure. after 30 s of driving. After cessation of the overdrive, the [K]e~ decreased to 2.25 m E q . l - 1 a n d then returned toward control. A Purkinje strand was r e m o v e d from the ventricles o f this heart and perfused with fresh T y r o d e solution. T h e spontaneous rate of the Purkinje fibers was 16 beats.min -1 in that solution. T h e m a x i m u m diastolic potential was - - 8 8 m V and the threshold - - 6 6 inV. T h e lower p a r t o f the action potentials recorded during this experiment are shown at the b o t t o m o f the figure. W h e n this fiber was exposed to the control c o r o n a r y sinus effluent collected before overdrive, the rate fell to 14 beats.rain-1 a n d stabilized at that value as shown b y the curve above the action potential traces. W h e n exposed to the effluent collected during the first minute of overdrive, the Purkinje fiber rate fell to 7 beats. m i n -x ( - - 5 0 % ) . T h e rate declined to 6 beats.rain -1 during exposure to the 2nd rnin's effluent a n d then slowly increased toward the control rate with the exception o f the sixth sample. T h e t e m p o r a r y increase in rate over control during the 6th rain's effluent is possibly due to m e c h a n i c a l d a m a g e to the fiber by the microelectrode as at the time the m a x i m u m diastolic potential had decreased a n d as the increase subsided once the electrode was repositioned. W h e n exposed to the effluent collected during the 11 th a n d 12th rain, the preparation accelerated slightly from 10 to 12 beats.min-t a n d then decelerated to 10 beats.rain -~. W h e n the T y r o d e solution was perfused again, the rate was 9 beats.rain -1. It is a p p a r e n t that w h e n the potassium was high in the effluent, the rate slowed; w h e n the potassium was near control, the rate returned to control; and when the potassium was lower, the
928
M. VASSALLEETAL.
fiber accelerated somewhat. A gradual slow decline in the Purkinje fiber rate appears to have taken place during the perfusion as found in the majority of the fibers when returned to the fresh Tyrode solution (see below). It should be noted that while the Purkinje fiber was exposed to the post-drive effluent (i.e. when the ventricles were suppressed), the fiber did not slow and actually accelerated somewhat. The results of 11 experiments in which 5 or I0 rain overdrive periods were carried out are reported in Table t, section B. In four experiments (Table 1, section C), atropine sulfate was added to the effluent before bathing the Purkinje fibers, in order to block the effect of acetylcholine, if any. No differences were noted between atropinized and the nonatropinized perfusate and therefore all the results were pooled together. The average results of all the experiments are given at the bottom of Table I. In 19 experiments, the spontaneous rate of the preparations when perfused with 2.7 K Tyrode solution was 29.9 4- 2.7 beats.min -1 (range I0 to 50). When the Tyrode solution was changed to the effluent collected during the pre-drive period, the rate increased in one, remained the same in seven and decreased by 2 to 8 beats in eleven experiments. When perfused with the effluent collected during the first minute of overdrive, all fibers slowed. The average rate decline was 7.84-1.2 beats.rain - t (--27.8%, range 1 to 20 P ~ 0.001). T h e average decline in the 4th rain's effluent was 3.9 zkl.2 beats.rain -t (--13.1%, P
Potassium-related Humoral Transmission o f Overdrive S u p p r e s s i o n * M A R I O V A S S A L L E , D A N I E L J . K R E L L E N S T E I N , t M I C H A E L B. P L I A M ++ AND C H A N D L E R M c C . B R O O K S
Department of Physiology, State University of.New York, Downstate Medical Center, 450 Clarkson Avenue, Brooklyn, aVew York 11203, U.S.A. (Received 1 aTuly 1976, and accepted in revisedform 27 January 1977) M. VASSALLE,D . J . KRELLENSTEIN,M. B. PLIAMAND C. McC. BROOKS.Potassium-related Humoral Transmission of Overdrive Suppression. ffournal of Molecular and CellularCardiology (1977) 9, 921-931. The contribution of potassium to the suppression which follows overdrive (overdrive suppression) was studied in isolated canine hearts perfused in vitro. Complete atrioventricular block was produced by ligafing the His bundle. The electrical activity of the ventricles was recorded with locally implanted electrodes. Coronary sinus potassium concentration [K]es was measured before, during and after 1 to 10 rain drives. The collected coronary sinus effluent was reoxygenated and was used to perfuse in vitro Purldnje fiber strands isolated from the same ventricles. The following results were obtained: (1) during fast drive there was an initial K loss and after the drive (associated with a temporary suppression of pacemaker activity) a net K uptake; (2) Purklnje fiber strands isolated from the ventricle and perfused in vitro in all instances underwent a decrease in the spontaneous rate when exposed to the effluent collected during the first minute of overdrive; (3) when the Purkinje fibers were bathed in the coronary sinus effluent collected after the drive, spontaneous activity usually increased and in no instance decreased; (4) adding atropine to the effluent did not alter the observed changes in automatic discharge; and (5) increasing [K]o in fresh perfusate over the range found in the coronary sinus effluent caused a similar slowing of the Purldnje fiber activity. It is concluded that an increase in [K] o contributes to overdrive suppression which follows short but not long drives. The humoral transmission of overdrive suppression is thus only related to an increase in coronary potassium concentration.
KEY WORDS: Overdrive suppression; Atrio-ventricular block; Potassium; Purkinje fibers; Isolated heart; Acetylcholine; Atropine.
1. Introduction When the ventricles are driven at a fast rate, coronary sinus potassium concentration [K]cs initially increases and then slowly returns toward control [1, 5]. After the end of overdrive, [KJcs usually falls below control value [1, 5]. The temporary * Supported by grants from the New York Heart Association and N.I.H., Heart and Lung Institute. t Present address: Department of Surgery, Mount Sinai School of Medicine of the City University of New York, Fifth Avenue and 100th Street, New York, New York 10029. ++Present address: Department of Cardiothoracic Surgery, The University of U t a h Medical Center, Jesus Christ of the Latter Day Saints Hospital, Salt Lake City, Utah 84143.
922
M. VASSALLE E T A L .
suppression of pacemaker activity which follows a fast drive (overdrive suppression) is present whether [K]~s happens to be high or normal at the end of the drive [1, 5]. This finding indicates that factors other than high [K]o are responsible for overdrive suppression. However, the finding neither rules out a contribution of a high [K]o to the inhibitory process nor indicates the magnitude of the contribution, if any. This point was tested here as follows. Small dog hearts were perfused in vitro and the suppression which follows a fast drive measured. The coronary sinus effluent was collected before, during and after drives of different duration. Once the procedures were completed, strands of free-running Purkinje fibers were removed from the ventricles of the same hearts and were perfused in a tissue bath. T h e fibers were exposed to the oxygenated coronary sinus effluent previously collected from the same perfused heart and changes in spontaneous activity were recorded. I f an increase in potassium level plays a role in overdrive suppression, the coronary sinus effluent should have an inhibitory action on spontaneous activity of Purkinje fibers only when [K]e~ is increased. Also, the magnitude of the inhibition due to the elevated potassium can be determined. The effluent collected after long drives has a [K]cs below control and therefore it should not have an inhibitory effect attributable to potassium. This approach also permits to evaluate other possibilities. For example, if overdrive suppression is due to a release of acetylcholine, the suppression should be transmittable humorally whether the overdrive is short or long. Also, an inhibitory role of acetylcholine should be abolished by atropine. O n the contrary, if overdrive suppression is exclusively due to membrane events (ionic shifts, electrogenic Na extrusion), humoral transmission of suppression would not be expected after either short or long drives.
2. Materials and M e t h o d s T h e details of the methods have been described recently [2]. A general outline follows. Hearts from small dogs were perfused with a modified Tyrode solution in a constant temperature chamber. Complete atrio-ventricular block was produced by His bundle ligation. The ventricular cavities were drained by means of plastic tubes inserted through the apex of each chamber. Electrodes were sutured on the right atrium and both ventricles. T h e perfusate was pumped into the aorta at an average rate of 73ml. rain-1.100 g ventricular weight -x (range 50 to 100 ml). The composition of the perfusate in mmol.1-1 was as follows: NaC1 136.9, KCI 2.7, NaHCO3 11.3, CaCI~ 1.8, MgCI~ 0.5, NaH2PO4 1.51, dextrose 5.5. In some experiments, the potassium content was changed as indicated below. The perfusate was bubbled with 97% 02 and 3% CO2 and the p H was 7.30 to 7.40. Total perfusion time averaged about 2 h. The ventricles were electrically driven at 240. min -1 for 1, 5 and 10 min by means of a bipolar electrode placed on right ventricular outflow tract. T h e total coronary sinus effluent was
H U M O R A L TRANSMISSION OF O V E R D R I V E SUPPRESSION
923
collected over 1 min periods. Between the 25th and 35th of each minute a 5 ml sample was collected for [K]~ determination. All samples were immediately refrigerated. At the end of the above procedures, free-running strands of Purkinje fibers were removed from the ventricles and perfused in a tissue bath with warm (37~ oxygenated Tyrode solution containing 5.4 mEq. I potassium -1. T h e solution was perfused at a rate of 60 to 80 drops.rain -1. After 30 to 60 min of equilibration, the perfusate was changed to Tyrode solution containing 2.7 mEq. 1-1 of potassium to allow the development of spontaneous activity. Membrane potentials were recorded with glass microelectrodes filled with a 3 M KC1 solution. The recording apparatus consisted of a push-pull cathode follower stage, a Tektronix dual beam oscilloscope (RM565) and a Grass Kymograph camera (C4L). After a steady rate was reached in 2.7 mM K Tyrode solution, the perfusate was changed to the control coronary sinus effluent which had been reoxygenated. This effluent had been collected over a 5 rain period prior to the overdriving, while the heart was beating spontaneously. After 10 min (or until a steady rate was reached), the perfusate was changed successively to the 1 rain coronary sinus effluent samples taken during and after overdrive. T h e fiber was perfused with each of these reoxygenated samples for 5 min and the rate of discharge of the Purkinje fibers recorded. Action potentials were photographed during the exposure to each sample. At the end of the procedure the perfusate was changed back to the control coronary sinus effluent and then to normal 2.7 Tyrode solution. Atropine sulfate was added to some effluent samples in a final concentration of 5 rag. 1-1. T o reproduce the inhibition seen with effluent samples, the potassium concentration of Tyrode solution was increased from 2.7 to 2.97, 3.24 and 3.51 mEq.1-1. The fibers were perfused in each of the solutions for 5 rain and the rate recorded. The Tyrode solution containing 2.7 mEq. I-1 potassium was perfused again during the recovery period.
3. R e s u l t s
Humoral transmission of overdrive suppression Nine isolated hearts were studied. T h e Purkinje fibers from two of these hearts failed to develop spontaneous activity when perfused in the tissue bath and were discarded. In one of these two instances, a Purkinje strand from a freshly excised, non-perfused heart was used to test the effect of the effluent on automatic discharge with results similar to those of the other experiments. T h e results from these eight hearts are reported here.
924
ET AL
M, VASSALLE
Z
Z
7,
3.0 2.5
20
F2,7
o fc
~~ -~
~0 i
0
I
i
2 $ Time(rain)
4
FIGURE 1. Effect of perfusing isolated Purkinje fibers with the coronary sinus effluent collected before, during and after overdrive. ( • x ) Ventricular rate in beats.rain-1 ; ( H ) coronary sinus potassium concentration in mEq.l -I, (C) O) Purkinje fiber rate in beats.rain-I. Action potentialsof Purkinjc fiberrecorded during the exposure to the coronary sinus ci~uent arc shown at the bottom of the figure.The six groups of two action potentialswcrc recorded during exposure to the second, third,fourth, sixth,seventh and eighth pcrfusatc sample, respectively.The verticalbar is the voltage calibration(I00 m V ) and the horizontalbar isthe time calibration(I0 s).
Short overdrive I n four experiments the hearts were overdriven for 1 min at 24:0 beats.min -1. The results of one of these experiments are illustrated in Figure 1. The top curve shows the ventricular rate of the perfused heart. Before overdrive, the spontaneous idioventricular rate was 7 beats.rain -1. After the drive was stopped, there was a pause of 50 s followed b y a return to the pre-drive rate. T h e middle curve represents the coronary sinus potassium concentration in mEq. 1-1. The control effluent [K]~ was 2.65 m E q . l - 1 and it increased to 3.20 m E q . l - t after 30 s of overdrive. During the first minute of the post-drive period, the [K]~s decreased to 2.75 mEq. 1-1 and then to 2.65 mEq. 1-1 thereafter. There was no undershoot in this particular instance but samples were collected over 1 rain periods and thus a small undershoot may have been missed. A Purkinje strand removed from the ventricles of this heart was perfused in the tissue bath. The spontaneous action potentials recorded from this preparation are shown at the bottom of the figure and their rate of discharge is
HUMORAL
TRANSMISSION O F O V E R D R I V E
925
SUPPRESSION
plotted i n the curve above. T h e s p o n t a n e o u s rate i n 2.7 mM K T y r o d e solution was 16 b e a t s . m i n - t a n d decreased to 14 b e a t s . m i n - t w h e n exposed to the c o n t r o l c o r o n a r y sinus effluent. W h e n the effluent collected d u r i n g the 1 m i n overdrive was perfused, the rate of the p r e p a r a t i o n decreased to 10 b e a t s . m i n - t . T h e rate t h e n increased to 12 b e a t s . m i n -1 w h e n exposed to the sample collected d u r i n g the pause. T h e r e was a further transient increase i n the rate to 15 b e a t s . m i n -1. By the 5th m i n ' s sample, the rate was back to control. T h e r e was no increase i n rate a b o v e control d u r i n g perfusion w i t h the first post-drive sample as seen i n other experiTABLE 1. Humoral transmission of overdrive suppression Drive duration (rain)
Rate [K] 0= 2.7
Control rate 1'
A
B
C
Average :t:s.E.
1 1 1 1 5 5 5 5 5 5 5 10 10 10 10 5 5 5 10
16 21 40 18 I0 40 25 25 44 24 40 45 16 36 50 18 36 25 40 29.9 2.7
14 17 40 16 15 32 23 23 44 24 32 40 14 36 50 18 32 25 38 28.1 2.5
--4 --2 --10 --1 --6 --7 --5 --19 --4 --20 --5 --8 --8 --14 --6 --2 --6 --9 --12 --7.8 1.2
Rate change at 4'
Rate change Recovery in pause rate sample 9'
--1 --1 --3 --I --17 --1 --4 --3 + 1 --2 --1 --6 --2 --10 0 --1 +2 --8 --3 --3.9 ---0.5 1.2 0.6
--2* +3 --5* +5 +4 +3 +2 +9 +1 +13 +6 +9 +1 +9 (+33) +4 +9 +1 (+20) +6.6 1.9
14 13 40 18 10 28 18 16 20 16 26 42 9 30 (70) 20 30 26 (64) 26.8 3.8
Effect ofperfusing isolated Purkinje fibers with coronary sinus effluent collected before, during and after overdrive of the heart. A, 1 min overdrive; B, 5 and 10 min overdrive; C, Perfusion with atropinized effluent. Drive duration (rain) = duration of overdrive of the ventricles. Rate [K]0 = 2.7: spontaneous rate of discharge of Purklnje fibers in Tyrode containing 2.7 m~ K. Control rate: spontaneous rate of discharge of Purkinje fibers when exposed to control coronary sinus effluent. Rate change at 1', 4', 9': change in r.ate beats.rain-t when the fibers were exposed to coronary sinus effluent collected during the first, fourth and ninth rain of perfusion. Rate change in pause sample: change in spontaneous rate when Purkinje fibers were exposed to perfusate collected during the first minute after drive. Recovery rate: spontaneous rate of Purkinje fibers perfused again with 2.7 mMK Tyrode solution. The results in parenthesis are of little significance because of arrhythmia and were not included in the average values. * No undershoot of the [K]eg was seen.
M. VASSALLEETAL.
926
ments, presumably because there was no undershoot in the [K]os. The changes in rate of isolated Purkinje fibers when perfused with the coronary sinus effluent collected before, during and after 1 min overdrive are reported in Table I, section A.
Long drive In three hearts, coronary sinus effluent was collected before, during and after a 10 rain drive in five runs. The results are shown in Figure 2. The top curve shows the ventricular rate in beats.rain -1 and the bottom curve shows the changes in coronary sinus potassium in mEq.1-1. The maximum increase in [K]o, (from 2.58 + 0.05 mEq.1-1 to 3.01 -t- 0.01 mEq.1-1) was attained during the first minute of drive and was followed by a slow return toward control. By the 9 mln, the [K]~ was only 0.06 mEq. 1-1 above the pre-drive value. During the first minute of the post-drive period, [K]ca decreased to 2.31 ! 0.01 m E q . l - I and then increased again toward control. ~ A
2501
:~
3., 9o
2.9
/
+:'ua
X, T
2.7
........................
2.6
2.5 2.42.3
1 0
1 i
I 2
l 3
I 4
I 5
1 I I 6 7 8 Time (rain)
I 9
lxl I0
II
I I 12 13 14-
FIGURE 2. Effect of long overdrive on potassium balance in five experiments in three hearts. The average idioventricular rate was 33.4 4- 4.3 beats.min-1. The average pause was 44.5 4- 14.4 s. Perfusate potassium concentration was 2.58 4- 0.06 mEq.l-1. The results of one experiment in which the coronary sinus effluent collected during and after a 10 rain drive was used to bathe Purkinje fiber are shown in Figure 3. The top curve shows the ventricular rate of the perfused ventricle. Before overdrive, the spontaneous idioventricular rate was 28 beats.min -1. After the ventricles were overdriven at 240 beats.rain -1 for 10 rain, there was a pause of 90 s. The middle curve shows the coronary sinus potassium concentration in mEq. 1-1. The control effluent [K]c~ was 2.85 mEq.1-1 and increased to 3.50 m E q . l - 1
I-IUMORAL TRANSMISSION OF OVERDRIVE SUPPRESSION
~.
260 I' . 240~-
.
.
.
.
~
4Ol- ~_~
9
927
.
I
/
2oi-
/
~
3.0 ~ .E 2.5 " ~ _-'-
20f
e-~.
'.~ is
IJ
J
i
L 9 I 0 2
.............
i!
---/~ i
I 4
I 6
'
...... J
~ J
| I ,. 8 I0 Time (rain)
2.0 '-"
:~ I 12
I 14
~. - 16 18
FIGURE 3. Effect of perfusing an isolated Purkinje fiber with the coronary sinus effluent collected before, during and after overdrive. ([-] V-I) Ventricular rate in beats.min-t showing the predrive rate of 28, the overdrive for 10 min at 240, a pause of 90 s and a recovery rate of 22 beats.min -1. ( ~ - - - - O ) Coronary sinus potassium concentration in mEq.1-1. Perfusate [K]= 2.70 mEq.l -t. (9 0) Purkinje fiber rate in beats.min -1. The gradual slowing is indicated by the interrupted line. The lower portions of the action potentials of Purkinje fibers (showing diastolic depolarization) are displayed at the bottom of the figure. after 30 s of driving. After cessation of the overdrive, the [K]e~ decreased to 2.25 m E q . l - 1 a n d then returned toward control. A Purkinje strand was r e m o v e d from the ventricles o f this heart and perfused with fresh T y r o d e solution. T h e spontaneous rate of the Purkinje fibers was 16 beats.min -1 in that solution. T h e m a x i m u m diastolic potential was - - 8 8 m V and the threshold - - 6 6 inV. T h e lower p a r t o f the action potentials recorded during this experiment are shown at the b o t t o m o f the figure. W h e n this fiber was exposed to the control c o r o n a r y sinus effluent collected before overdrive, the rate fell to 14 beats.rain-1 a n d stabilized at that value as shown b y the curve above the action potential traces. W h e n exposed to the effluent collected during the first minute of overdrive, the Purkinje fiber rate fell to 7 beats. m i n -x ( - - 5 0 % ) . T h e rate declined to 6 beats.rain -1 during exposure to the 2nd rnin's effluent a n d then slowly increased toward the control rate with the exception o f the sixth sample. T h e t e m p o r a r y increase in rate over control during the 6th rain's effluent is possibly due to m e c h a n i c a l d a m a g e to the fiber by the microelectrode as at the time the m a x i m u m diastolic potential had decreased a n d as the increase subsided once the electrode was repositioned. W h e n exposed to the effluent collected during the 11 th a n d 12th rain, the preparation accelerated slightly from 10 to 12 beats.min-t a n d then decelerated to 10 beats.rain -~. W h e n the T y r o d e solution was perfused again, the rate was 9 beats.rain -1. It is a p p a r e n t that w h e n the potassium was high in the effluent, the rate slowed; w h e n the potassium was near control, the rate returned to control; and when the potassium was lower, the
928
M. VASSALLEETAL.
fiber accelerated somewhat. A gradual slow decline in the Purkinje fiber rate appears to have taken place during the perfusion as found in the majority of the fibers when returned to the fresh Tyrode solution (see below). It should be noted that while the Purkinje fiber was exposed to the post-drive effluent (i.e. when the ventricles were suppressed), the fiber did not slow and actually accelerated somewhat. The results of 11 experiments in which 5 or I0 rain overdrive periods were carried out are reported in Table t, section B. In four experiments (Table 1, section C), atropine sulfate was added to the effluent before bathing the Purkinje fibers, in order to block the effect of acetylcholine, if any. No differences were noted between atropinized and the nonatropinized perfusate and therefore all the results were pooled together. The average results of all the experiments are given at the bottom of Table I. In 19 experiments, the spontaneous rate of the preparations when perfused with 2.7 K Tyrode solution was 29.9 4- 2.7 beats.min -1 (range I0 to 50). When the Tyrode solution was changed to the effluent collected during the pre-drive period, the rate increased in one, remained the same in seven and decreased by 2 to 8 beats in eleven experiments. When perfused with the effluent collected during the first minute of overdrive, all fibers slowed. The average rate decline was 7.84-1.2 beats.rain - t (--27.8%, range 1 to 20 P ~ 0.001). T h e average decline in the 4th rain's effluent was 3.9 zkl.2 beats.rain -t (--13.1%, P
