Factors Related to the Initiation of
Ventric-ular Fibrillation in the Isolated Heart:
Effect of Calcium and Potassium
By L. GRUMBACH, J. W. HOWARD, AND V. I. MEKKILLVentricular fibrillation can be initiated in the isolated, perfused heart by injections of calcium chlo-ride into the perfusion stream. It can also be initiated by perfusion with a K+-free perfusion medium. These are related phenomena since the latter type of fibrillation will not occur if Ca++ is also re-moved from the perfusion medium. The mechanism by which Ca++ initiates fibrillation is unknown. The facility with which Ca++ causes fibrillation is increased when the K+ concentration of the tissue fluid is lowered. When it is very low the normal Ca++ content of Krebs solution is sufficient to initiate fibrillation.
T
HE ability of calcium to produce ven-tricular arrhythmias, including fibrilla-tion, in the isolated, perfused heart does not seem to have received much attention hitherto, although other effects of calcium on this type of preparation have been reported. We have been unable to find any complete re-ports on such work other than a statement by Gross1 that large doses of calcium chloride cause fibrillation when injected into the per-fusion cannula of a heart perfused by the Langendorff method. On the other hand, it has long been known that calcium salts can cause ventricular arrhythmias, including fibrillation, when injected into intact animals.2"" A few in-vestigators have attributed this to a direct action of the calcium ions on the myocar-dium,2 •7 but most of the authors quoted have suggested that the arrhythmias are the result of sympathetic stimulation of the heart, the activity of the sympathetic nerves being due in some way to the calcium. More information concerning the conditions under which calcium can initiate fibrillation in the isolated heart should be useful. It is the purpose of this paper to study these conditions.METHODS
Rabbit hearts were perfused by the Langendorff method. The perfusion fluid was maintained at approximately 36 C. and under a pressure of SO
From the Pharmacology Section, Steiling-Win-throp Research Institute, Renssehicr, N. V.
Received for publication June 10, 1954.
mm. Hg. The average volume flow through fresh hearts, perfused with normal Krebs-Henseleit solu-tion, was 25 cc. per minute. Tables 1 and 2 show the K+ and Ca++ content of the various modified solutions employed in this investigation. Rapid injections of small volumes of solutions (1.0 cc. or less) were made directly into the perfusion stream about 7 cm. from the tip of the perfusion cannula. Observations made with trypan blue dye solutions showed that the flow from the reservoir was inter-rupted while the injection was being made and that there was very little dye movement above the point of injection. However, several minutes elapsed before all traces of trypan blue had disappeared from the cannula below the point of injection.
The perfused heart was placed in a double-walled glass chamber in which the perfusate leaving the heart was collected. A constant level was maintained by an outflow tube. Two side-arms, ISO degrees apart, and communicating with the fluid in the chamber, each contained a chlorided silver plate which served to lead off the electrocardiogram. When the heart was properly oriented with respect to the pair of electrodes, a typical Lead II electro-cardiogram was recorded on a Grass Type III D electroencephalograph using paper speeds of 6 and 60 mm. per second.
RESULTS
The Cause of Ventricular Fibrillation Follow-ing Perfusion with K+-Free Solutions. Figure I
illustrates the sequence of electrocardiographic. changes caused by perfusing an isolated rabbit heart with a K+-free Krebs-Henseleit solution. These are virtually identical with those found by Butcher and colleagues12 when the isolated dog heart was perfused with K+-free Tyrode solution. Within approximately 10 seconds there were marked T-wave and Q-T interval 452 Circulation Research. Vol. II, September 1954
L. GRUMBACII, J. W. HOWARD AND V. I. MERRILL 453
TABLE 1.—The Role of K+ in Determining the
Abil-ity of Various Pcrfusion Fluids to Initiate Ventricu-lar Fibrillation in Isolated Rabbit Hearts.
1'erfusion - — K'] mEq./L. 5.0 5.0 5.0 2.4 2.4 2.4 .1.1 1.1 0.0 0.0 0.0 0.0 0.0 1.1 1"uid — [CaM] mEq./L. (after K+ Ca+ + = 1.1 (after K+ Ca+ + = 61.2* 20.4* 5.1 5.1 20.4* 30.6* 5.1 20.4* 0.0 2 . 5 5.1 20.4* 30.6* 20.4* = 5.0; 5.1) 20.4* = 1.1; 5.1) Ventricular Fibrillation — — — — — + in 1 out of 4 + — — + -1-+ + + Time of onset, min. 12-20 2-3 — — 6-10 2-3 1 2.5 0.5
* Equivalent, amounts of Na+ removed to preserve osmotic pressure.
Normal Krcbs-Hcnsclcit: [K+l = 5.0 mEq./L. and |Ca++l = 5.1 mEq./L.
changes similar to those seen in hypopotas-semia in humans.13 During the second minute of the perfusioii the P-R interval increased markedly. Soon afterward complete atrioven-tricular dissociation occurred with the ventri-cles beating at a slightly faster rate than before. From the third to the seventh minute of per-fusioii, the ventricular pacemaker wandered until ventricular fibrillation began abruptly 6 minutes and 20 seconds after the start of the perfusioii. The low voltage of the oscillations and their relatively high frequency as seen in this figure are characteristic of ventricular fibrillation caused by low K+ or K+-free solu-tions. If the perfusioii of K+-free solution was continued after fibrillation had begun, the am-plitude declined further until very high ampli-fication was required to reveal it.
In another experiment it was found that restoration of K+ to the perfusioii medium, which was accomplished by changing back to normal Krebs solution, first increased the
am-plitude and reduced the frequency of the fibril-latory oscillations. Two minutes after fibrillation had started due to the perfusioii of K+-free solution, the frequency of the oscilla-tions was 32 per second. This was reduced to 1-i per second .10 minutes after the change back to normal Krebs solution. Records c, d, and e of figure 2 show that reversion to a normal sinus rhythm eventually occurred as the perfusioii with normal Krebs-Henseleit solution was con-tinued. Reversion could also be obtained by using a Krebs solution in which the K+ was replaced by Rb+.
Figure 3 shows that fibrillation did not occur in the absence of K+ from the perfusioii medium if Ca4"*" was also removed. However, as can be seen in record d, injections of Ca4"4' under these circumstances caused fibrillation to occur again. The fibrillation in this case, which was caused by the injection of 0.5 cc. of 0.55 M calcium chloride, lasted nine minutes. It was followed by a period of ventricular tachycardia that lasted 18 more minutes at which time it abruptly stopped. Record b illustrates the marked difference between a previous fibrilla-TABLE 2.—The Role of K+ in Determining
Suscepti-bility to Fibrillation Following Rapid Injections of Calcium Chloride Solutions into the Perfiision Cnnnula of Isolated Rabbit Hearts.
Perfusion Fluid [K+| mEq./L. 5.0 5.0 5.0 5.0 5.0 1.1 0.0 0.0 11.8 11.8 |Ca++| mEq./L. 5.1 5.1 5.1 5.1 5.1 5.1 0.0 0.0 5.1 5.1 CaCU Injection Concen-tration, i\l. 1.10 0.55 0.55 0.45 0.40 0.55 0.55 0.022 0.55 1.10 Volume, cc. 0.4 O.S 0.6 1.0 2.0 0.4 0.5 1.0 1.0 1.0 Ventric-teatr Fibril-lation + + + + + + + Duration Persistent Persistent Persistent Persistent 10* 2f 6
* Followed by a period of ventricular tachycardia lasting 18 minutes.
t Followed by a period of vent.ricu.lar tachycardia lasting four minutes.
Normal Krebs-Henseleit: [K+] = 5.0 mEq./L. and Ca++1 = 5.1 mEq./L.
454 VENTRICULAR FIBRILLATION BY CALCIUM AND POTASSIUM 0* 2'15"
4^4
0.1 H C i i 5' 6 ' 3 2 " LOT mV_|_I
200 1.0 sec.FIG. 1. Tho olTect of perfusing an isolated rabbit heart with K+-frec Krcbs-Henseleit solution. The O' record is the control electrocardiogram in normal Krebs solution just before the change. The 200/iV calibration applies to the last record only.
tion caused by K+ and that subsequently caused by Ca4"1" in the same heart (record d). The amplitude of the oscillations was much greater and their frequency was much lower in K+ fibrillation. Further, the duration of K+ fibrillation was brief; in this case it lasted a little over one minute in comparison with the 27 minutes of fibrillation and ventricular tachy-cardia caused by a Ca++ injection that con-tained fewer cations than did the K+ injection.
The Effect of K+ on the Ability of Ca++ to Cause Ventricular Fibrillation in Perfusion Ex-periments. Table 1 shows that when the K+ concentration of the perfusion medium was greater than 2.3 mEq. per liter, fibrillation did not occur even though the perfusion medium contained as much as 61.2 mEq. of Ca"4"4" per liter. The latter solution stopped the heart completely within 30 seconds without causing fibrillation. When the Ca44" concentration was 20.4 mEq. per liter, a high grade A-V block was produced within two minutes but as the per-fusion was continued this wore off. Again there was no fibrillation in any of the 12 cases observed.
The second part of table 1 shows that K+ also had an effect on the ability of Ca44- to in-itiate fibrillation when the concentration of K+ in the perfusion medium was less than 2.3 mEq. per liter. Perfusion with K+-free Krebs solution never failed to produce fibrillation. The
fibril-lation usually occurred in 0 to 10 minutes, the shorter times being obtained when the hearts were perfused with this solution directly after removal from the animal, and the longer ones when the hearts were initially perfused with a normal Krebs solution. This time could be shortened, as table 1 shows, by increasing the Ca44" concentration of the perfusion medium. When the hearts were perfused with a Krebs solution containing 1.1 mEq. per liter fibrilla-tion occurred only in about one out of every four hearts, and the time of onset of fibrillation was about double that when K+ was absent altogether. If the Ca++ concentration of the
per-Control
Normal Krebs-Henseleit solution
13' after change
K*-free Krebs-Henseleit solutior 2 min after start of fibrillation 2I1 after change
1.0
mV
0.1 sec
* I I
Normal Krebs-Henseleit solution 2 min. after end of fibrillation
F I G . 2. Records c, d, and e show the development of K+-free fibrillation and its reversion by peH'usion with normal Krebs solution.
L. GUUMBACH, J. W. HOWARD AND V. I. MERRILL 455
I
1.0 mVO.I sec. 1.0 S0C.
6 a f t e r Co**- and K*-free Krebs-Henseleit solution
I
0.5 mV a i we. 100I1 after 0.5 cc. 0.77 M KCI injection
I i
O.I sec.
1 J
I' after 0.5cc. 0.55M CaC^ injection
FICI. 3. Record a shows the effect of perfusion with n solution from which both K+ and Ca++ are re-moved. Records 6 and d show the differences in amplitude and frequency of fibrilhitory oscillations following injections of potassium chloride and calcium chloride. The fibrillation caused by potassium chloride lasted 1 minute 30 seconds; that caused by calcium chloride. 9 minutes and the ventricular tachycardia following it, ]8 minutes.
fusion medium was raised to 20.4 mEq. per liter when the K+ concentration was LI mEq. per liter, fibrillation again occurred in every case and the time of onset was shortened materially. This time could be further dimin-ished by first equilibrating the heart with a low K+-.Krebs solution (1.1 mEq. per liter) in which the Ca"1"4" concentration was normal. When the perfusion medium was changed to one with a Ca"1"*" concentration of 20.4 mEq. per liter, fibrillation occurred within 30 seconds instead of the two to three minutes required with this solution when the hearts were equili-brated first with a normal Krebs solution.
The Initiation of Ventricular Fibrillation Caused by the Injection of Calcium Chloride Solutions into the Perfusion Cannida. Ventric-ular fibrillation could be induced by rapid injections of hypertonic calcium chloride solu-tions into the perfusion cannula while normal Krebs-Henseleit solution was being perfused under the conditions previously described. The fibrillation thus initiated invariably began no later than 20 to 30 seconds after the injection. As table 2 shows, the least concentration of calcium chloride which could initiate fibrilla-tion was 0.45 M (four times isotonic). In most experiments 0.55 M calcium chloride (five times
45G VENTRICULAR FIBRILLATION BY CALCIUM AND POTASSIUM
isotonic) was used, and at least 0.8 cc. of this solution had to be injected (see table 2) in order to cause fibrillation. In the winter this dose caused fibrillation in 19 out of 28 cases with the first injection; in eight out of the remaining nine cases fibrillation occurred with a second injection of this dose. In the spring, 1.0 cc. of 0.55 M calcium chloride only caused fibrillation at the first injection 13 out of 51 times. Fifteen of the remaining 38 hearts fibrillated when 1.0 cc. of 1.10 M calcium chloride was injected 10 minutes after the first injection failed. Six more hearts of these 38 fibrillated when this dose was repeated a third time. Seventeen out of the 51 did not ribrillate at all. Control injec-tions of 0.77 M sodium chloride and 1.54 M sucrose solutions which had the same osmotic pressure as 0.55 M calcium chloride did not cause fibrillation. However, they did cause T-wave changes characteristic of hyper-potassemia.
The fibrillation initiated by rapid injections of calcium chloride into the perfusion cannula while normal Krebs-Henseleit solution was being perfused was usually quite persistent. Sponta-neous reversions to a normal sinus rhythm were rarely seen in hearts of rabbits obtained in the winter even though the period of observation was sometimes as long as an hour. Reversions
were more frequent in hearts obtained from rabbits in the spring. Actually about half of the 38 failures to fibrillate mentioned above in spring rabbits were reversions occurring within a few minutes and not absolute failures to fibrillate at all.
Fibrillation induced in this way could be stopped at any time followed by a reversion to a normal sinus rhythm by the injection of adequate amounts of 0.77 M potassium chlo-ride. Changing to a perfusion medium contain-ing twice the normal amount of K+ was also found to be effective in ending the fibrillation with a sinus rhythm following. When 0.2 to 0.4 cc. of 0.77 M potassium chloride was injected, fibrillation stopped promptly and atrioventric-ular dissociation with a wandering ventricatrioventric-ular pacemaker ensued. This period usually ended abruptly four to six minutes later with the appearance of a normal sinus rhythm. Larger amounts of 0.77 M potassium chloride, e.g., 0.8 cc. or more, caused a ventricular standstill whose duration was roughly proportional to the volume of the injection. With 0.8 cc. it some-times was as long as two minutes. The beats occurring after the period of standstill were normal sinus beats. With injection of 0.1 cc. or less of 0.77 M potassium chloride, reversion did not occur. However, the frequency of the
fibril-1.0 sec. 0.2 cc. 0.77 M KCI Control I 0 mV O.I sec. O.I sec.
I
0.5 mV 3' later 2O1 laterFio. 4. Record c shows the effect of potassium chloricle on ventricular fibrillation initiated by the injection of calcium chloricle. Ventricular arrhythmia shown in d lasted two and one-half minutes longer.
L. G RUM BACH, J. W. HOWARD AND V. I. MERRILL 457
latory oscillations was slowed for a short time. Figure 4 illustrates the manner in which re-version to a sinus rhythm is effected by an injection of potassium chloride. In this example the period of ventricular arrhythmia lasted almost six minutes. Injections of 0.77 M ru-bidium chloride acted on fibrillating hearts in the same manner as did injections of potassium chloride.
The Jiffeel of K+ on the Ability of Ca++ to In-itiate Ventricular Fibrillation in Injection Ex-periments. The last two sections of table 2 show that the concentration of K+ in the perfusion medium determines the minimal volume and concentration of the calcium chloride solution required to initiate ventricular fibrillation by injection. Injections of less than 0.8 cc. of 0.55 M calcium chloride, for example, never in-itiated fibrillation when normal Krebs solution was being perfused under the conditions ob-taining in our experiments. However, injections of 0.4 cc. of 0.55 M calcium chloride regularly induced a persistent fibrillation when the per-fusion medium contained only 1.1 mEq. K+ per liter. Injections of 1.0 cc. of 0.55 M calcium chloride rarely failed to cause fibrillation when the perfusion of hearts from winter rabbits with normal Krebs solution appeared to be normal. But when the perfusion medium con-tained K+ at a concentration of 11.8 mEq. per liter, they always failed to initiate fibrillation. At this concentration of K+ fibrillation could frequently by induced by 1.0 cc. of 1.10 M calcium chloride.
After equilibration for 6 to 10 minutes with a solution devoid of both Ca4"*" and K+, fibrilla-tion could be induced by injecfibrilla-tion of 0.5 to 1.0 cc. of relatively dilute solutions of calcium chloride, 0.022 M being the lowest that was found to be effective. The fibrillation caused by calcium chloride under these circumstances was not persistent like that produced by calcium chloride when perfusion solutions containing K+ in a concentration greater than 2.3 mEq. per liter were used. The duration of the fibril-lation caused by Ca4"4" injections when K+ was absent from the perfusion medium was roughly proportional to the concentration of the cal-cium chloride used for the injection. Also, a period of ventricular tachycardia always fol-lowed the end of the fibrillation. The fibrillation
caused by calcium chloride when the K+ con-centration was greater than normal was also of relatively short duration. However, it was not followed by a period of tachycardia.
DISCUSSION
These experiments indicate that the ventric-ular fibrillation which can be initiated in iso-lated perfused hearts by rapid injections of calcium chloride and the fibrillation which occurs spontaneously when Iv+-free solutions are perfused are apparently related phenomena. Fibrillation of the latter type does not occur when Ca++ is also omitted from the perfusion medium, and its replacement promptly restores the fibrillation. The fibrillation caused in this way lasts until the Ca4"4" is again washed out. Spontaneous fibrillation does not occur when the K+ concentration of the perfusate is 2.3 mEq. per liter or greater, even when the Ca++ concentration is also increased to the point where the heart is stopped completely in a short time. However, fibrillation can be initiated at these K+ levels if Ivypertonic solutions of cal-cium chloride are injected rapidly into the perfusion cannula. It always occurs within a few seconds of the injection or not at all. Since most of the latent period is probably the time required for the transportation of the Cii4"*" ions from the point of injection to the capillaries and across the capillary walls, fibrillation prob-ably begins very shortly after the Ca4"*" concen-tration of the tissue fluid is suddenly elevated. Judged by the minimal concentrations of Ca4" in the perfusion media and injections required for the initiation of fibrillation at the various K+ levels employed, the K+ concentration of the tissue fluid also determines a "threshold" for the initiation of fibrillation both by low K+ solutions and by Ca4"1" injections.
Moe and Mendez,14 in a recent study of the action of cardiac glycosides on conduction and excitability in the dog heart, concluded that the impairment of conduction caused by these glycosides was principally, if not exclusively, in the conducting tissues of the ventricle, the ven-tricular muscle cells showing little, if any, effect. This is probably also true of Ca++. They also showed that the ventricular automaticity produced by the glycosides was independent of the effect of these substances on ventricular
458 VENTRICULAR FIBRILLATION BY CALCIUM AND POTASSIUM excitability as measured by the threshold for
electrical stimulation. The ability of a sub-stance to cause certain cells to fire impulses spontaneously is apparently unrelated to its effect on their excitability. This seems to apply to Ca4"4" as well,2 since Ca++ above the physiologic range of concentrations only de-presses excitability.10' u
Our experiments on the effects of perfusing with low K+ media suggest, therefore, that the removal of K+ from the tissue fluid leads to a failure of these centers to resist the stimulating action of the Ca4"4" present in normal Krebs solution, with the result that they begin to discharge impulses spontaneously when the K+ concentration falls below a certain level. Above this K+ level, however, the resistance of the centers to the stimulating action of Ca4"4" is such that they do not respond to any amount of Ca4"*" in the tissue fluid when the change to the new concentration of Ca4"4" is made slowly, as by changing perfusion media. On the other hand, if the Ca44" concentration is elevated rapidly to a sufficiently high level, as happens when rapid injections of hypertonic calcium chloride solutions are made, fibrillation de-velops. And it occurs at the moment of injec-tion under these circumstances, which suggests that after this moment the situation again resembles that which obtains when the Ca++ level is raised slowly.
The mechanism by which Ca4"4" stimulates the cells of the stimulus-formation centers to fire impulses, either spontaneously when the K+ concentration is low or when the Ca4"4" con-centration is rapidly elevated in the presence of larger amounts of K+, is at present unknown. However, the recent observations of Hall and Knox15 may offer a clue to the solution of this problem. These authors showed that the full development of fibrillation can be precipitated in a recently denervated skeletal muscle that shows little if any fibrillatory activity in normal Krebs solution by lowering the K+ content of the solution bathing the muscle from 5.88 mM per liter to 1.17 mM per liter. They also found that the effect of lowering the K+ con-centration could not be enhanced by lowering the Ca4"*" concentration. However, they appar-ently did not remove the Ca4"4" completely.
According to Hunt and KufHer,16 the old view that the fibrillation of denervated skeletal muscle is caused by circulating acetylcholine reaching the muscle end-plates can no longer be supported by experimental evidence. Jarcho and associates17 demonstrated that the fibrilla-tion potentials of partially denervated skeletal muscle arise in the zone of the denervated end-plates. Since the latter region is known to con-tain relatively large amounts of aeetylcholin-esterase, it is of interest that, according to Mommaerts and co-workers, in the ventricle only the Purkinje tissue contains this enzyme. The end-plates from the motor nerve fibers to the skeletal muscle fibers and Purkinje tissue are functionally analogous in that their chief roles are to transmit excitation. It seems sig-nificant to us that both systems fibrillate when the transmitting and effector elements arc decentralized.
As was mentioned in the introduction, the ventricular arrhythmias and fibrillation caused by the injection of Ca++ salts into intact ani-mals have recently been explained as resulting solely from the sympathetic stimulation evoked by the action of the calcium. If the conclusion that Ca4"4" acts in intact animals only through the sympathetic nervous system should prove correct, our experiments would suggest that Ca++ must be able to stimulate peripheral adrenergic structures directly as well as being able to activate them rerlexly. Lissak9 showed that perfusion of the frog heart with a high Ca4-1" solution causes the release of a sympathin-like substance. Thus far, this has not been demonstrated to occur in the mammalian heart. However, Hoffman and associates19 demonstrated that there is adrenergic tissue in the mammalian heart which can be stimulated to release an epinephrine-like substance by chemical means. It is, therefore, not impossible that the stimulating action of Ca++ by virtue of which ventricular automaticity is enhanced is exerted through the release of epinephrine.
SUMMARY
The factors concerned in the initiation of ventricular fibrillation in isolated rabbit hearts by the perfusion of low K+ Krebs-Henseleit solutions and by the injection of hypertonic
L. GRUMBACH, J. W. HOWARD AND V. I. MERRILL 459
calcium chloride solutions into the perfusion caimula while normal Krebs solution was being perfused were studied by altering the electro-lyte content of the perfusion medium.
These two types of fibrillation are related phenomena; fibrillation is initiated only by the perfusion of K+-free solutions when Ca"1"*" is present in a concentration of 3.5 mEq. per liter or greater.
Fibrillation cannot be initiated by steady state perfusion with high Ca++-solutions when the K+ concentration of the perfusion medium is 2.3 mEq. per liter or greater.
Fibrillation can be initiated immediately by rapid injections of hypertonic calcium chloride solutions into the perfusion cannula when the K+ concentration of the perfusion medium is greater than 2.3 mEq. per liter. The minimal effective volumes and concentrations of the calcium chloride solutions varied directly with the K+ concentration in the perfusate.
These data indicate that the initiation of fibrillation by calcium chloride injections de-pends upon a sufficiently rapid elevation of the Cix++ concentration in the tissue fluid to a
critical level. The rapidity with which the elevation must occur and the concentration which must be reached appear to be determined by the K+ concentration of the tissue fluid. Spontaneous fibrillation occurs when K+-free solutions are perfused because of a failure to resist the stimulating action of the Ca4"1" present in the perfusion medium.
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