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Selective experimental chelation of calcium in the AV node and His bundle
jhrnal
of Molecular
Selective
and Cellular
Cardiology
Experimental
(1976)
Chelation of Calcium His Bundle* THOMAS
Debartment
of Medicine,
University
(Received
8, 361-374
of Alabama
6 May
1975,
in the AV
Node
and
N. JAMES Medical
Center,
accepted in revisedform
Birmingham,
Alabama
22 .j%&
35.294,
U.S.A.
1975)
T. N. JAMES. Selective Experimental Chelation of Calcium in the AVNodeand His Bundle. Journal of Molecular and Cellular Cardiology 8, 361-374. There are P cells in the human and canine AV (atrioventricular) node which are virtually devoid of gap junctions. All other components of myocardial cellular connections are calcium-dependent except the gap junction. Direct perfusion of disodium EDTA through the AV node artery of 16 anaesthetized dogs produced three immediate effects : complete AV block, a rapid irregular atria1 rhythm and a separate rapid irregular ventricular rhythm. The atria1 arrhythmia was short in duration and sinus rhythm resumed, initially with complete AV and VA biock; both waned until normal AV conduction returned in each dog. In 3 of the 16 dogs there was transient complete AV block during which two independent His potentials were separately associated with the atria1 and ventricular complexes. When conducted sinus rhythm resumed, there was initially A-H prolongation (but not H-V). Atropine, propranolol and reserpine had no influence on any electrophysiologic effect of EDTA. Both tachycardias probably originate in P cells of the AV node, the irregularity being attributable to 1 nrying enhancement of automaticity plus functional disaggregation of P cells. AV block is attributed to failure of conduction between disaggregated P cells, which in turn must be an obligatory pathway for normal AV conduction, because of their anatomic interposition. The findings further suggest that the AV nodal P cells are the site of the normal 40 ms delay in AV conduction, and that they may be the site of origin of the His potential. KEY
WORDS:
His bundle
P cells; potential.
Gap junctions;
AV nodal
delay;
AV block;
AV junctional
rhythms;
1. Introduction In addition to its familiar and important role in the electromechanical events of myocardial contraction, calcium ion is also essential for the maintenance of normal intercellular adhesion and electrical propagation within the heart. Bathing myocardial cells in a medium deficient in calcium ion eventually leads to dehiscence of all components of the intercalated disc except the gap junction [Z, 17, 291. The same effect can be achieved more rapidly by chelation of calcium ion with ethylenediamine tetraacetic acid (EDTA). After EDTA the conduction velocity in such is the site preparations remains near normal [I7], suggesting that the gap junction of low electrical resistance between cells of the heart. Since the variety of cell types in the heart includes some which normally have almost no gap junctions [26], it * This work was supported Grant HL 11.310 and MIRU
in part Contract
by the National Heart and Lung 4367-1441) and by the Rast Fund
Institute (Program Project for Medical Research.
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would help in defining the function of those cells to know the electrophysiological effect of selective binding of calcium in their vicinity. Gap junctions are larger and more numerous between Purkinje cells than between working myocardial cells [13, 14, IS] ; this difference may in part explain the faster conduction velocity in Purkinje fibers. Both in the sinus node and in the deep portions of the AV (atrioventricular) node near the His bundle there is a special round or ovoid cell (the P Cell) which contains few myofibrils, randomly scattered mitochondria, and a cytoplasm with little glycogen and only sparse organelles. P cells are more abundant in the sinus node than in the AV node. Indirect evidence suggests that the P cells may be the site of origin of automaticity in the sinus node [25]. P cells are joined only to each other and to slender small transitional cells [I.?, 15, 161, but do not connect directly with either working myocardium or Purkinje cells. Furthermore, intercellular junctions of P cells themselves are comprised primarily of so-called undifferentiated regions in which the two respective plasma membranes are a constant distance from each other without intervening basement membrane. There is a conspicuous paucity of specializations within these junctions, except for occasional desmosomes and even rarer “spot welds” representing small gap junctions. Recent studies with selective chelation of calcium in the canine sinus node demonstrated multiple electrophysiological effects, some of which were attributed to disaggreatation of P cells [7]. While the AV junctional P cells may be the site of automaticity from that region, they are normally suppressed by the intrinsically faster rhythm of the sinus node. In addition to their likely role as subsidiary pacemakers, it is uncertain what other function the P cells may serve in the AV junction. It has been suggested [IO] that slow conduction through P cells could account for the normal 40 ms delay known to occur within the human and canine AV junction, and the paucity of gap junctions between P cells would fit with such a hypothesis. There is evidence that the normal AV junctional delay occurs either at the A-N or N region as defined electrophysiologically, on the basis of microelectrode recording characteristics [3]. But the cytological substrate for these electrophysiologically defined regions may be any of several possible cell groups [ 101. One simple but important question is whether the P cells of the AV junction are an obligatory transit site in the normal transmission of every sinus impulse, or whether they are normally bypassed during sinus rhythm. If the chelation of calcium ion within the AV junction could be selectively produced in the intact canine heart during normal sinus rhythm, one could hypothesize that the only intercellular conduction to be affected would be that occurring between P cells. If the P cells of the AV junction are an obligatory component of normal AV conduction, then their dehiscence should impair conduction. If they are normally bypassed during sinus rhythm, then dehiscence of AV junctional P cells should have no significant influence on AV conduction. The following experiments were conducted to test that hypothesis.
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2. Methods Sixteen dogs were anesthetized with sodium pentobarbital(30 mg/kg intravenously) and the trachea intubated for ventilation with room air. The chest was opened and the heart suspended in the pericardium. In the dog the region of the AV node and His bundle receives a dual arterial supply [5, 81. In each dog the AV node artery was cannulated for selective perfusion of the AV junction. Collateral circulation from the septal artery permits lengthy experiments after such cannulations, during which time the chronotropic and dromotropic functions of the AV node and His bundle remain normal. Details of the method for such cannulation have been published [8]. For the sake of selective perfusion of the sinus node in some dogs the sinus node artery was also cannulated [9] as indicated in special experiments. All injections were delivered from a hand syringe with 2 ml volume in a period of 5 to 10 s. Each injection was routinely compared to a control injection of Ringer’s solution or normal saline, depending on which of these was the diluent for the test substance. To produce chelation of divalent cations the two substances utilized were disodium EDTA (Endrate @‘) and calcium EDTA (Versenate @), each of which was prepared by dilution with 0.9% sodium chloride solution. All other test agents were prepared in Ringer’s solution and included acetylcholine chloride, norepinephrine bitartrate, propranolol hydrochloride, atropine sulfate and calcium chloride. Three dogs were pretreated with reserpine (0.5 mg/kg) administered intramuscularly for 2 days prior to the experiment. Pressures were routinely recorded from the central aorta (cannula in the femoral artery) and the right atrium (cannula in the superior vena cava). Electrical activity of the heart was recorded with a standard ECG lead (II or aVR), simultaneously with local electrograms from the region of sinus node [4], the surface of the right or left ventricle, and from two poles of a five-pole Hoffman-type electrode plaque sutured in the vicinity of the His bundle [.5]. The electrode plaque for His bundle recordings was placed through a right atriotomy during a brief period of caval inflow occlusion which never exceeded 3 min. Interpolar distance for the His bundle electrograms was 5 mm, and the bipolar combination was chosen which provided the maximal amplitude, with filtration of frequencies below 40 or above 500 Hz. A tachogram completed the routine recordings, being derived with a small analog computer from successive R waves of the ventricular complexes.
3. Results DisodiumEDTA had no significant chronotropic or dromotropic action in concentrations less than 100 pg/ml. CalciumEDTA had no significant effect in any tested concentration, including the maximal one of 10 mg/ml. The lack of effect by calcium
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EDTA indicates that general binding of divalent cations other than calcium is of little electrophysiological significance for the present study. The responses to disodium EDTA exhibited several distinct phases during which the effects differed, and these are described below. Control injections of either normal saline or Ringer’s solution directly into the AV node artery had no significant electrophysiological effect. Maximal concentrations of disodium EDTA (10 mg/ml) injected as 2 ml directly into the right atrium had no significant effect. All responses to disodium EDTA in the present experiments were therefore selective local effects within the AV junction and not attributable to recirculation.
i I
t
FIGURE I. These two diagrams illustrate the postulated effects of EDTA on the junctions between Purkinje cells (Purk) and transitional cells (Tr). At the atria1 margin of the AV node conduction would normally be from Purkinje type cells of the internodal pathways into transitional cells, which join to form the slender interweaving fibers histologically characteristic of the AV node. Abbreviations include N for nucleus, Mf myofibrils, BM basement membrane, PM plasma membrane, D desmosome, UR undifferentiated region and GJ gap junction. Chelation of calcium causes separation of all components of the intercalated disc except the gap junctions.
Chronotropic
action
of disodium
EDTA
in the AV node artery
At a concentration of 100 pg/ml disodium EDTA produced minor and variable degrees of brief AV junctional tachycardia or premature beats, none lasting more than a few seconds. At 1000 pg/ml disodium EDTA regularly produced AV junc-
I’I.ATl~ 1. The immediate effect following dirwt pcrlilGon of thr 1~1,’ node artery (AWNA! with disodium EDTA (Na-EDTA) is illustrated in this record. There is rapid development of tachycnrdia. deformation of QRS complcxrs and dissociation of atria1 and ventricular rhythms. Probable His potentials during an early phase ofthr tachyrardia arc indicated with arrows but thrsc soon disappcxred. Recording channels from above down are aortic prwurr (Ao). elcctrogram from rrgiorl 01’ sinus [lode (SiYj, His bundle clwtrogram (HBE), right vrntricular clectrogram (RV), surfarc Iwcls aVR and a tachogram. Prrssurc is scaled in mm Hg and heart (HR) in beats/min. PI,.4’I‘E 2. After thr period of maximal acwleration produrcd by disodium EDTA, the rapid irregular rhythm gradually slows as shown hew. At this stage the sinus rhythm has become almost regular fblack dots) while the vcntrirular rhytfim rcmains completely dissociated. chaoticall! irregular and rapid. The original onset of effect 1s Illustrated from this same* dog in Plate 1. PIATE 3. In some dogs after disodium EDTA in thr .4\. nodr artery the rrcovrry period passed throllgh a phase ofAVjunctiona1 rhythm as shcnvn hrrc. Black dot\ mark v~~ntricular conrplew~ an(l thr ~.irclcs mark atrial complexes. P1..2’W 4. Sinus rhythm returnrd to dominatt, the atria rathw wrly and was in thr prows ol rrturning hew. All sinus beats (black dots) arp rrgular in timing, vxccpt for on? prcmaturc bum ibla<,k dot with a vertical line). The tachograph scnsccl vcncrictllar conrplcxcs :~nd illustr.;ttr.\ thrir 1 vry rapid irregularity. Variation of (1RRs configuration is apparrnt. PI.:\‘I‘E 5. For some period after A\’ conduction rrturnccl thrrc was irlcompletc heart block. hcrc. illrlstr,ttc.d as 2: 1 AV block. Atropinc (.4TR) had been sclvctiwly p~rli~srd into the .4\’ node arl,‘r\ 1 IO :Lg ml. 2 ml) 2 min before this rc,vord. and had no cff~t on the AL’ block. 1’1.1\‘I‘1: 6. During periods of2 : 1 block, I : I conduction could by rrctorcd by slowing thr sinus node,. Any injection into thr sinus node artcry (Ringer’s solution h(w) transicwtly slows thr sinus node. l’I,:\TI< 7. Thcsc five polygraphs Ii.om one dog dcrnotla(ratC the transient splitting of the Hi5 potential obscrvcd aftrr srlcctivr~ perftlsion of the AL’ node ;trtwy with disodium EDTA. In (ai thf. cornponcwt of Hia potential associated with each atria1 cornplcx is indicated as H \; most wntricular cot~~~~l~xc~ at this stagr had no associated His potential but IWO which do arc indicated H \.. .4bout one tninutc after (a) the vrntriwlar. complcxcs in (b) now Ix,gin 10 have an assoriatrd His pottntial for nx~rt (onr cxrcption in this scgmrnt) ; a separate His porcntial is WCY) with most atria1 complcxcs although its configuration varies sornc; the> last Tao atria1 c~onlplrxcs in this strip reprrsent retrograde wntlrlction from A\’ junctional beats. In (c) the complctrly dissociated atria1 and wntricular complrxt.\ art’ illustratc~d, caach with its own accompanying His potvn’ial; open circles mark atrial cornplrx~s and black dots ventricular complwes. .4t a late stage !tli thcrc is now a stable .\\’ ,junctirmal rhythln with regular I : I \‘A conduction: thr Gnglc His potential prewdcs each VWtricular cornplrx. The final prriod ol’recovcry in this one cxpcrimrnt is shown by one example in (~1. with L\‘cnckebnch pwiods: ~ww the only Hi\ potcntiah iarro\v\ I whrr~~ there is sinus dominancr occur at rtgular HV but phasic AH intervals. PI..Vl’Ii 8. The chaotic irregular hut rapid atrial rhythni prodrwd by wlcctive perfusion of the Grls nodr with clisodium EDTA wa\ never associated with delbrmation of thr QRS complexes or with ,iny variation of the H\’ interval in thr His rlc(,trogl.Im. .\trixl complrx~~s arc’ marked ivith virc~lv~ :IIKI wntric 11l;lr coml)lrxv\ \virh lllack dot?.
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tional tachycardia at a rate 12 f 8 beats above control sinus rate, lasting for 22 f 9 s. The maximal consistent response occurred with 10 mg/ml of disodium EDTA. This caused the immediate onset of tachycardia reaching a peak level of 41 & 14 beats/min above control sinus rate (Plate 1). The maximal level of acceleration lasted as a regular rhythm for no more than a few beats, however, then being replaced by an equally rapid (net rate) but totally irregular tachycardia. Concomitant important changes affecting AV conduction and QRS configuration are discussed separately below. In 13 of the 16 dogs this irregular tachycardia lasted for 9 & 5 min, gradually slowing in net rate until normal sinus rhythm returned as the dominant mechanism (Plate 2). In the other 3 dogs the initial period ofirregularity for 1 to 3 min was followed by 2 to 5 min of a gradually slowing AV junctional rhythm very regular in nature and more rapid than the original sinus rate, until sinus rhythm was re-established (Plate 3). At about the same time as the rapid irregular rhythm controlling the ventricles began (within the first few beats after injection of disodium EDTA), a separate and totally dissociated irregular tachycardia assumed control of the atria at a rate faster than sinus rhythm (Plates 2 and 4). There were thus separate atria1 and ventricular rhythms, both chaotically rapid, in this initial phase. The dissociated and irregular atria1 rhythm was of relatively short duration, only lasting 1 to 3 min. As the irregular atria1 rhythm terminated, normal sinus rhythm resumed in 13 of the 16 dogs, initially with interference from numerous premature atria1 beats; the
FIGURE cells. Their
2. Most junctions
of the AV node consists of slender interweaving and the effect of EDTA on them are illustrated
fibers composed here.
of transitional
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frequency of this interference progressively waned until steady sinus rhythm at control rate was again established for the atria. At that time the rapid irregular ventricular rhythm was still present, and the absence of interference with sinus rhythm (controlling the atria) indicated a high grade of ventriculoatrial (VA) block. During the period of irregular tachycardia, injections of calcium chloride (1 mg/ml) into the AV node artery had no discernible effect. The chaotically rapid chronotropic response was the same in the 3 dogs pretreated with reserpine as it was in the other 13 dogs. Pretreatment with atropine, 10 pg/ml into the AV node artery (4 dogs) or 1 mg/kg intravenously (3 dogs), failed to alter the chronotropic response, although either of these uses of atropine completely blocks the response to local perfusion with acetylcholine 1 pg/ml or supramaximal vagal stimulation [S, 81. Selective perfusion of the AV node artery with propranolol 10 pg/ml completely blocks the response to local perfusion with norepinephrine 0.1 pg/ml [8, 271, but had no effect in preventing the irregular tachycardia produced by disodium EDTA. Once the tachycardia was produced with EDTA, it could not be suppressed by local perfusion with acetylcholine through the AV node artery (4 dogs). In all these respects the positive chronotropic action of disodium EDTA in the AV junction closely resembled its effect following selective perfusion of the sinus node in previous experiments [7], except that both the maximal rates achieved and the duration of the positive chronotropic action was greater in the sinus node.
Dromolropic
action of disodium EDTA
in the AV node artery
As indicated above, complete AV dissociation appeared at about the same time as the positive chronotropic effect. The irregular tachycardia in the atria was of comparatively short duration but was followed by a more prolonged period of AV and VA block before sinus rhythm resumed control of the ventricles. When sinus rhythm was again conducted to the ventricles, after the rapid irregular ventricular rhythm produced by disodium EDTA in the AV node artery had subsided, there were varying degrees of AV block in each of the 16 dogs (Plate 5). In 10 dogs this recovery period was characterized by prolongation of the A-H interval alone, while in 6 dogs there was second degree AV block. Neither of these forms of block could be influenced by either local (6 dogs) or systemic (4 dogs) administration of atropine prior to or during the AV block. The AV block occurred in the same fashion in the reserpine-pretreated dogs and in 4 dogs pretreated by selective perfusion of propranolol into the AV node artery. Both the first and second degree AV block gradually waned but could not be influenced except transiently (for no more than 30 s) by local selective perfusion of the AV node artery with calcium chloride 1 mg/ml. The first degree AV block when it occurred alone (10 dogs) lasted 7 + 3 min, and the second degree AV block
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Normal
FIGURE 3. Deeply placed within the AV node there are groups of P cells which join only with each other and with transitional cells. Their junctions and the effect of EDTA on them are shown here. A major difference between junctions of P cells and of other myocardial cells is the prevalence of undifferentiated regions and paucity of gap junctions in the connections made between P cells.
(6 dogs) waned through Wenckebach periods into first degree block as AV conduction gradually recovered in 13 f 6 min. After both the chronotropic and dromotropic actions of disodium EDTA in the AV node artery were completed, the same effects could be reproduced in 4 dogs with a second perfusion of disodium EDTA 10 mg/ml; this serial reproducibility in the same dog was comparable to that previously reported with sinus node perfusion [ 71. By separate manipulation of sinus rate during the period of incomplete AV block, it was possible to increase or decrease the grade of AV block. For example, selective perfusion of norepinephrine into the sinus node artery during first degree AV block would cause sinus tachycardia with second degree block. During second degree AV block, selective slowing of the sinus node led to I :I conduction (Plate 6). These procedures in 5 dogs did not cause any variation in QRS configuration. During the selective slowing of the sinus node, there was no escape of AV junctional rhythm. Since the later phases of chronotropic effect by disodium EDTA in the sinus node were characterized by prolonged sinus bradycardia [7], this lack of AV junctional escape may represent a similar depression of latent automaticity within the AV junctional region.
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Reversible splitting
of the His potential
During the period of completely dissociated and rapid irregular rhythms in both atria and ventricles, the form and location of the His potential was highly variable and difficult to identify separately from multiple other local electrical potentials. As steady sinus rhythm resumed control of the atria with the irregularly rapid and dissociated ventricular rhythm persisting, in 3 of the 16 dogs a His bundle potential became associated with the A complex at a normal A-H interval of slightly varying duration; however, there was still initially no consistent His bundle potential before the V complex. In 2 of these 3 dogs a separate His bundle potential later appeared just proximal to the V complex with a normal H-V interval, at a time when complete AV dissociation was still present [Plate 7(a)-(e)]. This phenomenon of two separate His bundle electrograms, each respectively related to an A complex and V complex which were independent of each other, lasted for 11 and 18 min, respectively, in these 2 dogs; in both of these dogs there was a later period of AV junctional rhythm driving both the atria and ventricles [Plate 7(d)] shortly before sinus control resumed [Plate 7(e)]. After normal sinus rhythm and AV conduction were restored in one of these dogs, a second injection of disodium EDTA into the AV node artery exactly reproduced the same sequence of split His potentials and subsequent recovery.
Site of dromotropic action of disodium Although various degrees of AV and VA block were consistently observed as indicated previously, there was never any prolongation of the H-V interval of the His electrogram. All the delay in AV conduction when a stable His potential could be clearly identified was either in the A-H interval, or between two split components of the His bundle potential [Plate 7(a)-(e)]. When two components of the His potential were split, the A-H interval was not prolonged. A long A-H interval was observed only during sinus rhythm transmitted to the ventricles and only when a single His potential was present.
QRS conjguration
during and following
the rapid irregular
ventricular rhythm
In every dog there was an immediate change (broadening) and frequent variation in the configuration of the QRS complex during the chaotic tachycardia caused by disodium EDTA in the AV node artery (Plates 2 and 4). For comparative purposes, a similar degree of AV junctional tachycardia was produced in 4 dogs by norepinephrine perfused into the AV node artery and was associated with completely normal QRS complexes, confirming previous similar results [Z]. During the chaotic supraventricular tachycardia caused by disodium EDTA in the sinus node artery,
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the net rate exceeded that of the AV junctional EDTA, yet the configuration of QRS complexes These results illustrate that the QRS deformation node artery cannot be due exclusively to either branch block, or enhanced (latent) automaticity junction, but must be due to something other or
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tachycardia caused by disodium was normally preserved (Plate 8). after disodium EDTA in the AV rate-dependent forms of bundleimpairing conduction in the AV more than these factors.
4. Discussion Selective perfusion of disodium EDTA into the AV node artery rapidly produces a number of electrophysiological responses which significantly alter cardiac rhythm and conduction. Three things occurred almost simultaneously: failure of AV conduction, an independent and rapid but irregular rhythm controlling the ventricles, and a separate rapid and equally irregular rhythm which interfered with the sinus node to dominate atria1 activity. The supraventricular irregularity was relatively short-lived, soon being replaced by the resumption of a steady sinus rhythm at the control rate but which did not conduct to the ventricles, nor was it influenced by retrograde conduction from the rapid irregular ventricular rhythm. To explain the two forms of rapid irregular rhythm and concomitant complete AV block, discussion will be focused on the P cells of the AV junctional region and their postulated function. Selective perfusion of disodium EDTA into the sinus node produces an immediate irregular rhythm at a rate near maximal levels achievable by the normal sinus node [7], and this is best attributed to two local effects : the known positive chronotropic action of lowered extracellular calcium ion, and the concomitant effective separation of multiple independently firing sites of automaticity. Since the junctions between these multiple independent units and the transitional cells, which serve as the normal conduit out of the sinus node, would presumably remain functionally intact for rapid conduction (their gap junctions being preserved), most of the new signals of multifocal origin would have ready egress and conduct to the atrium. An analogous explanation may be made for the results observed in the AV node and His bundle, where the P cells have all the same fine structural characteristics as in the sinus node. However, there is one important difference and that is the fact that under normal circumstances the main function of the AV junction is conduction rather than automaticity. If transnodal conduction was altered within the sinus node, one need not anticipate any effect on AV conduction. On the other hand, if the P cells of the AV node are functionally disaggregated and if they form an essential component of the normal route of AV conduction (the latter until now being unknown), then one would not only anticipate the chaotic positive chronotropic action seen but also disruption of AV conduction. This is exactly what happened. As corollaries to the interpretation that AV nodal P cells are an obligatory transit
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site for normal AV conduction perhaps by their normal anatomic interposition in the AV node, then there are two logical additional considerations. First, the P cells may represent the exact locus for the normal 40 ms (approximate) delay present with every beat during sinus rhythm. Second, any circumventing of conduction through AV nodal P cells would be more rapid than normal AV conduction. The nature of normal connections between P cells themselves (largely devoid of gap junctions) would support the first corollary, while the prevalence of gap junctions between all other types of myocardial cells supports the second corollary. It is the continued AV dissociation during the phases of rapid irregular rhythm, as well as the varying degrees of first and second degree AV block even when sinus control was re-established (Plates 5 and 7), which together serve as the strongest evidence that the P cells are an essential component of normal AV conduction. If this were not so, then there should have been normally preserved AV conduction both during the period of positive chronotropic effect (allowing for interference dissociation) and particularly when sinus rhythm was resumed. The connections between all myocardial cells except P cells contain numerous gap junctions, all of which should have been functioning normally since they are uninfluenced by the lack of local calcium ion. None of this established that the rapid irregular rhythm controlling the ventricles was of AV junctional origin. In fact, the immediate appearance of changed QRS complexes, most being wider than normal, would seem to indicate that the ventricular rhythm originated at multifocal sites beyond the level of the His bundle. The loss of an identifiable His potential preceding these broad ventricular origin. However, in other expericomplexes would also suggest their “ventricular” ments we have shown that the perfusion of substances with marked positive chronotropic action, such as norepinephrine or isoproterenol [Z, 81 or glucagon [26] into the AV node artery consistently produced exclusively a regular AV junctional tachycardia with QRS complexes identical to those during sinus rhythm and with a His bundle potential present at the normal interval before every ventricular complex. If catecholamines or glucagon failed to produce any but a supraventricular rhythm, then it seems more likely that the positive chronotropic effect ofdisodium EDTA given in the same fashion would also have produced a supraventricular rhythm, but one with odd features (broad QRS, absent His potential). If the rapid irregular ventricular rhythm is AV junctional in origin, then why are the QRS compiexes changed and where is the expected His bundle potential which should be present proximal to the ventricular complexes? On the basis of clinical [21, .?2] and anatomical [rl] evidence we have previously suggested that longitudinally dissociated conduction is normally present in the human and canine His bundle. If there is normally partitioned or longitudinally dissociated conduction in the canine His bundle, then the configuration of the QRS complexes will necessarily depend on the pattern of the activation front which arrives at the proximal end of the His bundle. That pattern arriving at the His bundle during
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normal sinus rhythm and during most forms of supraventricular rhythm will be nearly uniform, because of the normally uniform triage function of the AV node. However, if there is multifocal automaticity within the AV junctional region, then it would be anticipated that a variety of activation fronts would approach the His bundle, each producing its own form of QRS complex and, except on a random basis, none being similar to that seen during sinus rhythm. This brings us to the question of the nature of the normal His bundle potential. If it originates in the His bundle itself, as is generally believed, then there should still be a His bundle potential before every ventricular complex, even if there was a multifocal rhythm originating in the P cells. However, some have suggested that the His bundle potential originates from the “proximal” portion of the His bundle [18], which is very close to the anatomical location of the P cells. If the His bundle potential actually originated in those P cells which are normally present directly in the region of the junction of the AV node and His bundle, then one could readily explain the splitting of the His bundle potential observed in certain of the present experiments [Plate 7 (a)-(e)]. Th us, when a sufficient number of P cells had reaggregated in connection with the internodal pathways, there would be visible a His bundle potential associated with the atria1 complex and regularly controlled by the sinus node. If a sufficient number of P cells further downstream reaggregated to form a separate His bundle potential which represented the site of origin for the ventricular rhythm, and if these latter P cells had not yet reconnected with the upper group of P cells, then one would expect two independent rhythms, each with its own His bundle potential [Plate 7(a)-(c) J. When the two groups of P cells then eventually all reunited, a single His potential would reappear just as it did in the present study, and this was true both for regular AV junctional rhythm [Plate 7(d)] as well as for conducted sinus rhythm [Plate 7(e)]. The fact that neither the A-H nor H-V interval was prolonged when the His potential was split suggests that AV conduction was delayed exclusively within the P cell region when a single His potential returned. This form of reversible splitting of the His potential has not, to my knowledge, been previously demonstrated. Since the rate of local recalcification via the collateral arterial circulation [S] would be focally variable, and re-aggregation of P cells would therefore be of a somewhat random nature, it is not surprising that the degree of heart block, the duration of the irregular rhythms, and the extent of deformation of the QRS complexes would be comparably variable. In particular, the initial rapid disappearance of a recognizable His bundle potential and its variable pattern of return would fit well with this random “t-e-gluing” of P cells. Some would suggest that the AV conduction impairment may in part have been the result of enhanced latent automaticity, lasting beyond the period of overt positive chronotropic effect. Various forms of QRS deformation have been ascribed to such a phenomenon [20, 231, those cells with enhanced but still latent automaticity conducting slower than normal. However, if this were the case, one would
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have anticipated QRS deformation after the selective administration directly into the AV junction of substances with known maximally powerful positive chronotropic influence, and this does not occur, neither in previous experiments with norepinephrine and isoproterenol [Z] or glucagon [26] nor in the present experiments. This calls into question whether latent automaticity is a tenable explanation for impairment of AV conduction in vivo. There is the further possibility that some of the QRS distortion may have been a form of rate-dependent bundle branch block. However, not only was there no such change in the QRS complexes during maximal levels of sinus tachycardia following selective perfusion of norepinephrine into the sinus node, but neither was there any significant variation in QRS configuration during the rapid irregular tachycardia caused by disodium EDTA perfused into the sinus node artery (Plate 8). Finally, the possible neural influence of disodium EDTA [24] either to enhance local vagal effect or to impair local adrenergic p-receptor activity must be considered in the mechanism of production of the AV block. However, the absence of influence by atropine and the occurrence of AV block after pre-treatment with either propranolol or reserpine would suggest that autonomic neural mechanisms did not play a role in the negative dromotropic effect. Conversely, the occurrence of AV junctional tachycardia despite pretreatment with reserpine or propranolol or atropine indicates that the positive chronotropic effect was not mediated via adrenergic P-receptors, nor by release ofvagal tone. Specific chronotropic and dromotropic events may be summarized as follows. The rapid rhythm was due to lowered extracellular calcium concentration around the P cells, and being completely uninfluenced by pretreatment with atropine or propranolol or reserpine, may be presumed to be a direct action having neither anticholinergic or catecholamine-mediated components. The irregular&y of the rhythm was caused by disaggregation (either functional or anatomical) of P cells, with different cell groups having different intrinsic levels of automaticity and therefore different degrees of enhancement by the local calcium deficit. The AL’ block was also due to disaggregation of P cells with failure of conduction between enough of them to preserve communication between upper and lower AV junctional rhythms; this appeared to have no cholinergic or anti-adrenergic component. Since the His bundle pqtential disappeared during these maximal effects, and was transiently split during the recovery phase in some dogs, it is concluded that the His potential actually originates within the P cells and its given magnitude when recorded by bipolar local electrodes would depend on the net vector generated by conduction through the functionally aggregated P cells. The P cells thus appear to be the source of normal sustained automaticity in the AV junction, and they form an essential link in the normal pathway of AV conduction during sinus rhythm, the latter being most likely on the basis of their anatomical location between the main body of the AV node and the His bundle. As an essential link in normal AV conduction, the AV nodal P cells are the probable site of the normal brief delay (about 40 ms) seen
CALCIUM CHELATION IN THE AVJUNCTION
373
with every sinus beat. As a corollary, any route of AV conduction circumventing the AV nodal P cells would be anticipated to occur more rapidly than normal.
REFERENCES 1.
2. 3. 4. 5. 6. 7.
DREIFUSS,J. J., GIRARDIER, L. & FORSSMAN,W. G. Etude de la propagation de l’excitation dam le ventricule de rat au moyen de solutions hypertoniques. Pfriigers Archiu; European Journal of Physiology 292, 13-33 (1966). FRINK, R. J. &JAMES, T. N. Position of the H spike in selective pharmacologic production of AV block or AV junctional rhythm. Journal of Laboratory and Clinical Medicine 81, 506-519 (1973). HOFFMAN, B. F. Physiology of atrioventricular transmission. Circulation 24, 506-517 (1961). JAMES, T. N. Anatomy of the sinus node of the dog. Anatomical Record 143, 251-265 (1962). JAMES, T. N. Anatomy of the AV node of the dog. Anatomical Record 148, 15-27 (1964). JAMES, T. N. Cholinergic mechanisms in the sinus node with particular reference to the actions of hemocholinium. Circulation Research 19,347-357 (1966). JAMES, T. N. Selective experimental chelation of calcium in the sinus node. Journal of Molecular
and Cellular
Cardiology
6,493-504
( 1974).
8. JAMES, T. N., BEAR, E. S., FRINK, R.J., LANG, K. F. & TOMLINSON, J. C. Selective stimulation, suppression or blockade of the AV node and His bundle. Journal of Laboratory and Clinical Medicine 76, 240-256 (1970). 9. .JAMES, T. N. & NADEAU, R. A. Direct perfusion of the sinus node; an experimental model for pharmacologic and electrophysiologic studies of the heart. Henry Ford Hospital Medical Journal 10, 2 l-25 (1962). of the human AV node. Circulation 37, 104910. JAMES, T. N. & SHERF, L. Ultrastructure 1070 (1968). 11. JAMES, T. N. & SHERF, L. Fine structure of the His bundle. Circulation 44, 9-28 (1971). 12. JAMES, T. N., SHERF, L., FINE, G. & MORALES, A. R. Comparative ultrastructure of the sinus node in man and dog. Circulation 34, 139-163 (1966). 13. JAMES, T. N., SHERF, L. & URTHALER, F. Fine structure of the bundle branches. British
14. 15. 16. 17.
18.
Heart
Journal
36,
1-18
(1974).
KAWAMURA, K. Electron microscope studies on the cardiac conduction system of the dog. I. The Purkinje fibers. Japanese Circulation Journal 25, 594-616 (1961). KAWAMURA, K. Electron microscope studies on the cardiac condution system of the dog. II. The sinoatrial and atrioventricular nodes. Japanese CirculationJournal 25, 9731013 (1961). KAWAMURA, K. & JAMES, T. N. Comparative ultrastructure of cellular junctions in working myocardium and the conduction system under normal and pathologic conductions. Journal of Molecular and Cellular Cardiology 3, 3 1-60 ( 197 1) . KAWAMURA, K. & KONISHI, T. Symposium on fuction and structure of cardiac muscle. I. Ultrastructure of the cell junction of heart muscle with special reference to its functional significance in excitation conduction and to the concept of “Disease of the Intercalated Disc”. Japanese Circulation Journal 31, 1533-1543 (1967). KUPERSMITH, J., KRONGRAD, E. & WALDO, A. L. Conduction intervals and conduction velocity in the human cardiac conduction system. Studies during open-heart surgery. Circulation
47, 776-785
(1973).
374 19. 20.
21. 22. 23. 24. 25. 26. 27.
T.
N.
JAMES
MUIR, A. R. The effects of divalent cations on the ultrastructure of the perfused rat heart. Journal of Anatomy 101, 239-261 (1967). ROSENBAUM,M. B., ELIZARA, M.V., LAZZARA, J.O., HALPERN, M.S., NAU,G.J.& LEVI, R. J. The mechanism of intermittent bundle branch block: relationship to prolonged recovery, hypolarization and spontaneous diastolic depolarization. Chest 63, 666667 (1973). SHERF, L. & JAMES, T. N. A new electrocardiographic concept. Synchronized sinoventricular conduction. Diseases ofthe Chest 55, 127-140 (1969). SHERF, L. & JAMES, T. N. The mechanism of aberration in late AV junctional beats. American 3ourml of Cardiology 29, 529-539 (1972). SINGER, D. H., LAZZARA, R. & HOFFMAN, B. F. Interrelationships between automaticity and conduction in purkinje fibers. Circulation Research 21, 537-538 (1967). TODA, N. & WEST, T. C. Interaction between Na, Ca, Mg and vagal stimulation in the S-A node of the rabbit. American 3ournd of Physiology 212, 424-430 (1967). TRAUTWEIN,W. & UCHIZONO, K. Electron microscopic and electrophysiologic study of the pacemaker in the sino-atria1 node of the rabbit heart.
of Molecular
Selective
and Cellular
Cardiology
Experimental
(1976)
Chelation of Calcium His Bundle* THOMAS
Debartment
of Medicine,
University
(Received
8, 361-374
of Alabama
6 May
1975,
in the AV
Node
and
N. JAMES Medical
Center,
accepted in revisedform
Birmingham,
Alabama
22 .j%&
35.294,
U.S.A.
1975)
T. N. JAMES. Selective Experimental Chelation of Calcium in the AVNodeand His Bundle. Journal of Molecular and Cellular Cardiology 8, 361-374. There are P cells in the human and canine AV (atrioventricular) node which are virtually devoid of gap junctions. All other components of myocardial cellular connections are calcium-dependent except the gap junction. Direct perfusion of disodium EDTA through the AV node artery of 16 anaesthetized dogs produced three immediate effects : complete AV block, a rapid irregular atria1 rhythm and a separate rapid irregular ventricular rhythm. The atria1 arrhythmia was short in duration and sinus rhythm resumed, initially with complete AV and VA biock; both waned until normal AV conduction returned in each dog. In 3 of the 16 dogs there was transient complete AV block during which two independent His potentials were separately associated with the atria1 and ventricular complexes. When conducted sinus rhythm resumed, there was initially A-H prolongation (but not H-V). Atropine, propranolol and reserpine had no influence on any electrophysiologic effect of EDTA. Both tachycardias probably originate in P cells of the AV node, the irregularity being attributable to 1 nrying enhancement of automaticity plus functional disaggregation of P cells. AV block is attributed to failure of conduction between disaggregated P cells, which in turn must be an obligatory pathway for normal AV conduction, because of their anatomic interposition. The findings further suggest that the AV nodal P cells are the site of the normal 40 ms delay in AV conduction, and that they may be the site of origin of the His potential. KEY
WORDS:
His bundle
P cells; potential.
Gap junctions;
AV nodal
delay;
AV block;
AV junctional
rhythms;
1. Introduction In addition to its familiar and important role in the electromechanical events of myocardial contraction, calcium ion is also essential for the maintenance of normal intercellular adhesion and electrical propagation within the heart. Bathing myocardial cells in a medium deficient in calcium ion eventually leads to dehiscence of all components of the intercalated disc except the gap junction [Z, 17, 291. The same effect can be achieved more rapidly by chelation of calcium ion with ethylenediamine tetraacetic acid (EDTA). After EDTA the conduction velocity in such is the site preparations remains near normal [I7], suggesting that the gap junction of low electrical resistance between cells of the heart. Since the variety of cell types in the heart includes some which normally have almost no gap junctions [26], it * This work was supported Grant HL 11.310 and MIRU
in part Contract
by the National Heart and Lung 4367-1441) and by the Rast Fund
Institute (Program Project for Medical Research.
362
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JAMES
would help in defining the function of those cells to know the electrophysiological effect of selective binding of calcium in their vicinity. Gap junctions are larger and more numerous between Purkinje cells than between working myocardial cells [13, 14, IS] ; this difference may in part explain the faster conduction velocity in Purkinje fibers. Both in the sinus node and in the deep portions of the AV (atrioventricular) node near the His bundle there is a special round or ovoid cell (the P Cell) which contains few myofibrils, randomly scattered mitochondria, and a cytoplasm with little glycogen and only sparse organelles. P cells are more abundant in the sinus node than in the AV node. Indirect evidence suggests that the P cells may be the site of origin of automaticity in the sinus node [25]. P cells are joined only to each other and to slender small transitional cells [I.?, 15, 161, but do not connect directly with either working myocardium or Purkinje cells. Furthermore, intercellular junctions of P cells themselves are comprised primarily of so-called undifferentiated regions in which the two respective plasma membranes are a constant distance from each other without intervening basement membrane. There is a conspicuous paucity of specializations within these junctions, except for occasional desmosomes and even rarer “spot welds” representing small gap junctions. Recent studies with selective chelation of calcium in the canine sinus node demonstrated multiple electrophysiological effects, some of which were attributed to disaggreatation of P cells [7]. While the AV junctional P cells may be the site of automaticity from that region, they are normally suppressed by the intrinsically faster rhythm of the sinus node. In addition to their likely role as subsidiary pacemakers, it is uncertain what other function the P cells may serve in the AV junction. It has been suggested [IO] that slow conduction through P cells could account for the normal 40 ms delay known to occur within the human and canine AV junction, and the paucity of gap junctions between P cells would fit with such a hypothesis. There is evidence that the normal AV junctional delay occurs either at the A-N or N region as defined electrophysiologically, on the basis of microelectrode recording characteristics [3]. But the cytological substrate for these electrophysiologically defined regions may be any of several possible cell groups [ 101. One simple but important question is whether the P cells of the AV junction are an obligatory transit site in the normal transmission of every sinus impulse, or whether they are normally bypassed during sinus rhythm. If the chelation of calcium ion within the AV junction could be selectively produced in the intact canine heart during normal sinus rhythm, one could hypothesize that the only intercellular conduction to be affected would be that occurring between P cells. If the P cells of the AV junction are an obligatory component of normal AV conduction, then their dehiscence should impair conduction. If they are normally bypassed during sinus rhythm, then dehiscence of AV junctional P cells should have no significant influence on AV conduction. The following experiments were conducted to test that hypothesis.
CALCIUM
CHELATION
IN THE
AV
JUNCTION
363
2. Methods Sixteen dogs were anesthetized with sodium pentobarbital(30 mg/kg intravenously) and the trachea intubated for ventilation with room air. The chest was opened and the heart suspended in the pericardium. In the dog the region of the AV node and His bundle receives a dual arterial supply [5, 81. In each dog the AV node artery was cannulated for selective perfusion of the AV junction. Collateral circulation from the septal artery permits lengthy experiments after such cannulations, during which time the chronotropic and dromotropic functions of the AV node and His bundle remain normal. Details of the method for such cannulation have been published [8]. For the sake of selective perfusion of the sinus node in some dogs the sinus node artery was also cannulated [9] as indicated in special experiments. All injections were delivered from a hand syringe with 2 ml volume in a period of 5 to 10 s. Each injection was routinely compared to a control injection of Ringer’s solution or normal saline, depending on which of these was the diluent for the test substance. To produce chelation of divalent cations the two substances utilized were disodium EDTA (Endrate @‘) and calcium EDTA (Versenate @), each of which was prepared by dilution with 0.9% sodium chloride solution. All other test agents were prepared in Ringer’s solution and included acetylcholine chloride, norepinephrine bitartrate, propranolol hydrochloride, atropine sulfate and calcium chloride. Three dogs were pretreated with reserpine (0.5 mg/kg) administered intramuscularly for 2 days prior to the experiment. Pressures were routinely recorded from the central aorta (cannula in the femoral artery) and the right atrium (cannula in the superior vena cava). Electrical activity of the heart was recorded with a standard ECG lead (II or aVR), simultaneously with local electrograms from the region of sinus node [4], the surface of the right or left ventricle, and from two poles of a five-pole Hoffman-type electrode plaque sutured in the vicinity of the His bundle [.5]. The electrode plaque for His bundle recordings was placed through a right atriotomy during a brief period of caval inflow occlusion which never exceeded 3 min. Interpolar distance for the His bundle electrograms was 5 mm, and the bipolar combination was chosen which provided the maximal amplitude, with filtration of frequencies below 40 or above 500 Hz. A tachogram completed the routine recordings, being derived with a small analog computer from successive R waves of the ventricular complexes.
3. Results DisodiumEDTA had no significant chronotropic or dromotropic action in concentrations less than 100 pg/ml. CalciumEDTA had no significant effect in any tested concentration, including the maximal one of 10 mg/ml. The lack of effect by calcium
364
T. N. JAMES
EDTA indicates that general binding of divalent cations other than calcium is of little electrophysiological significance for the present study. The responses to disodium EDTA exhibited several distinct phases during which the effects differed, and these are described below. Control injections of either normal saline or Ringer’s solution directly into the AV node artery had no significant electrophysiological effect. Maximal concentrations of disodium EDTA (10 mg/ml) injected as 2 ml directly into the right atrium had no significant effect. All responses to disodium EDTA in the present experiments were therefore selective local effects within the AV junction and not attributable to recirculation.
i I
t
FIGURE I. These two diagrams illustrate the postulated effects of EDTA on the junctions between Purkinje cells (Purk) and transitional cells (Tr). At the atria1 margin of the AV node conduction would normally be from Purkinje type cells of the internodal pathways into transitional cells, which join to form the slender interweaving fibers histologically characteristic of the AV node. Abbreviations include N for nucleus, Mf myofibrils, BM basement membrane, PM plasma membrane, D desmosome, UR undifferentiated region and GJ gap junction. Chelation of calcium causes separation of all components of the intercalated disc except the gap junctions.
Chronotropic
action
of disodium
EDTA
in the AV node artery
At a concentration of 100 pg/ml disodium EDTA produced minor and variable degrees of brief AV junctional tachycardia or premature beats, none lasting more than a few seconds. At 1000 pg/ml disodium EDTA regularly produced AV junc-
I’I.ATl~ 1. The immediate effect following dirwt pcrlilGon of thr 1~1,’ node artery (AWNA! with disodium EDTA (Na-EDTA) is illustrated in this record. There is rapid development of tachycnrdia. deformation of QRS complcxrs and dissociation of atria1 and ventricular rhythms. Probable His potentials during an early phase ofthr tachyrardia arc indicated with arrows but thrsc soon disappcxred. Recording channels from above down are aortic prwurr (Ao). elcctrogram from rrgiorl 01’ sinus [lode (SiYj, His bundle clwtrogram (HBE), right vrntricular clectrogram (RV), surfarc Iwcls aVR and a tachogram. Prrssurc is scaled in mm Hg and heart (HR) in beats/min. PI,.4’I‘E 2. After thr period of maximal acwleration produrcd by disodium EDTA, the rapid irregular rhythm gradually slows as shown hew. At this stage the sinus rhythm has become almost regular fblack dots) while the vcntrirular rhytfim rcmains completely dissociated. chaoticall! irregular and rapid. The original onset of effect 1s Illustrated from this same* dog in Plate 1. PIATE 3. In some dogs after disodium EDTA in thr .4\. nodr artery the rrcovrry period passed throllgh a phase ofAVjunctiona1 rhythm as shcnvn hrrc. Black dot\ mark v~~ntricular conrplew~ an(l thr ~.irclcs mark atrial complexes. P1..2’W 4. Sinus rhythm returnrd to dominatt, the atria rathw wrly and was in thr prows ol rrturning hew. All sinus beats (black dots) arp rrgular in timing, vxccpt for on? prcmaturc bum ibla<,k dot with a vertical line). The tachograph scnsccl vcncrictllar conrplcxcs :~nd illustr.;ttr.\ thrir 1 vry rapid irregularity. Variation of (1RRs configuration is apparrnt. PI.:\‘I‘E 5. For some period after A\’ conduction rrturnccl thrrc was irlcompletc heart block. hcrc. illrlstr,ttc.d as 2: 1 AV block. Atropinc (.4TR) had been sclvctiwly p~rli~srd into the .4\’ node arl,‘r\ 1 IO :Lg ml. 2 ml) 2 min before this rc,vord. and had no cff~t on the AL’ block. 1’1.1\‘I‘1: 6. During periods of2 : 1 block, I : I conduction could by rrctorcd by slowing thr sinus node,. Any injection into thr sinus node artcry (Ringer’s solution h(w) transicwtly slows thr sinus node. l’I,:\TI< 7. Thcsc five polygraphs Ii.om one dog dcrnotla(ratC the transient splitting of the Hi5 potential obscrvcd aftrr srlcctivr~ perftlsion of the AL’ node ;trtwy with disodium EDTA. In (ai thf. cornponcwt of Hia potential associated with each atria1 cornplcx is indicated as H \; most wntricular cot~~~~l~xc~ at this stagr had no associated His potential but IWO which do arc indicated H \.. .4bout one tninutc after (a) the vrntriwlar. complcxcs in (b) now Ix,gin 10 have an assoriatrd His pottntial for nx~rt (onr cxrcption in this scgmrnt) ; a separate His porcntial is WCY) with most atria1 complcxcs although its configuration varies sornc; the> last Tao atria1 c~onlplrxcs in this strip reprrsent retrograde wntlrlction from A\’ junctional beats. In (c) the complctrly dissociated atria1 and wntricular complrxt.\ art’ illustratc~d, caach with its own accompanying His potvn’ial; open circles mark atrial cornplrx~s and black dots ventricular complwes. .4t a late stage !tli thcrc is now a stable .\\’ ,junctirmal rhythln with regular I : I \‘A conduction: thr Gnglc His potential prewdcs each VWtricular cornplrx. The final prriod ol’recovcry in this one cxpcrimrnt is shown by one example in (~1. with L\‘cnckebnch pwiods: ~ww the only Hi\ potcntiah iarro\v\ I whrr~~ there is sinus dominancr occur at rtgular HV but phasic AH intervals. PI..Vl’Ii 8. The chaotic irregular hut rapid atrial rhythni prodrwd by wlcctive perfusion of the Grls nodr with clisodium EDTA wa\ never associated with delbrmation of thr QRS complexes or with ,iny variation of the H\’ interval in thr His rlc(,trogl.Im. .\trixl complrx~~s arc’ marked ivith virc~lv~ :IIKI wntric 11l;lr coml)lrxv\ \virh lllack dot?.
CALCIUM
CHELATION
IN
THE
AV
JUNCTION
365
tional tachycardia at a rate 12 f 8 beats above control sinus rate, lasting for 22 f 9 s. The maximal consistent response occurred with 10 mg/ml of disodium EDTA. This caused the immediate onset of tachycardia reaching a peak level of 41 & 14 beats/min above control sinus rate (Plate 1). The maximal level of acceleration lasted as a regular rhythm for no more than a few beats, however, then being replaced by an equally rapid (net rate) but totally irregular tachycardia. Concomitant important changes affecting AV conduction and QRS configuration are discussed separately below. In 13 of the 16 dogs this irregular tachycardia lasted for 9 & 5 min, gradually slowing in net rate until normal sinus rhythm returned as the dominant mechanism (Plate 2). In the other 3 dogs the initial period ofirregularity for 1 to 3 min was followed by 2 to 5 min of a gradually slowing AV junctional rhythm very regular in nature and more rapid than the original sinus rate, until sinus rhythm was re-established (Plate 3). At about the same time as the rapid irregular rhythm controlling the ventricles began (within the first few beats after injection of disodium EDTA), a separate and totally dissociated irregular tachycardia assumed control of the atria at a rate faster than sinus rhythm (Plates 2 and 4). There were thus separate atria1 and ventricular rhythms, both chaotically rapid, in this initial phase. The dissociated and irregular atria1 rhythm was of relatively short duration, only lasting 1 to 3 min. As the irregular atria1 rhythm terminated, normal sinus rhythm resumed in 13 of the 16 dogs, initially with interference from numerous premature atria1 beats; the
FIGURE cells. Their
2. Most junctions
of the AV node consists of slender interweaving and the effect of EDTA on them are illustrated
fibers composed here.
of transitional
366
T . N. JAMES
frequency of this interference progressively waned until steady sinus rhythm at control rate was again established for the atria. At that time the rapid irregular ventricular rhythm was still present, and the absence of interference with sinus rhythm (controlling the atria) indicated a high grade of ventriculoatrial (VA) block. During the period of irregular tachycardia, injections of calcium chloride (1 mg/ml) into the AV node artery had no discernible effect. The chaotically rapid chronotropic response was the same in the 3 dogs pretreated with reserpine as it was in the other 13 dogs. Pretreatment with atropine, 10 pg/ml into the AV node artery (4 dogs) or 1 mg/kg intravenously (3 dogs), failed to alter the chronotropic response, although either of these uses of atropine completely blocks the response to local perfusion with acetylcholine 1 pg/ml or supramaximal vagal stimulation [S, 81. Selective perfusion of the AV node artery with propranolol 10 pg/ml completely blocks the response to local perfusion with norepinephrine 0.1 pg/ml [8, 271, but had no effect in preventing the irregular tachycardia produced by disodium EDTA. Once the tachycardia was produced with EDTA, it could not be suppressed by local perfusion with acetylcholine through the AV node artery (4 dogs). In all these respects the positive chronotropic action of disodium EDTA in the AV junction closely resembled its effect following selective perfusion of the sinus node in previous experiments [7], except that both the maximal rates achieved and the duration of the positive chronotropic action was greater in the sinus node.
Dromolropic
action of disodium EDTA
in the AV node artery
As indicated above, complete AV dissociation appeared at about the same time as the positive chronotropic effect. The irregular tachycardia in the atria was of comparatively short duration but was followed by a more prolonged period of AV and VA block before sinus rhythm resumed control of the ventricles. When sinus rhythm was again conducted to the ventricles, after the rapid irregular ventricular rhythm produced by disodium EDTA in the AV node artery had subsided, there were varying degrees of AV block in each of the 16 dogs (Plate 5). In 10 dogs this recovery period was characterized by prolongation of the A-H interval alone, while in 6 dogs there was second degree AV block. Neither of these forms of block could be influenced by either local (6 dogs) or systemic (4 dogs) administration of atropine prior to or during the AV block. The AV block occurred in the same fashion in the reserpine-pretreated dogs and in 4 dogs pretreated by selective perfusion of propranolol into the AV node artery. Both the first and second degree AV block gradually waned but could not be influenced except transiently (for no more than 30 s) by local selective perfusion of the AV node artery with calcium chloride 1 mg/ml. The first degree AV block when it occurred alone (10 dogs) lasted 7 + 3 min, and the second degree AV block
CALCIUM
CHELATION
IN
THE
AV
JUNCTION
367
Normal
FIGURE 3. Deeply placed within the AV node there are groups of P cells which join only with each other and with transitional cells. Their junctions and the effect of EDTA on them are shown here. A major difference between junctions of P cells and of other myocardial cells is the prevalence of undifferentiated regions and paucity of gap junctions in the connections made between P cells.
(6 dogs) waned through Wenckebach periods into first degree block as AV conduction gradually recovered in 13 f 6 min. After both the chronotropic and dromotropic actions of disodium EDTA in the AV node artery were completed, the same effects could be reproduced in 4 dogs with a second perfusion of disodium EDTA 10 mg/ml; this serial reproducibility in the same dog was comparable to that previously reported with sinus node perfusion [ 71. By separate manipulation of sinus rate during the period of incomplete AV block, it was possible to increase or decrease the grade of AV block. For example, selective perfusion of norepinephrine into the sinus node artery during first degree AV block would cause sinus tachycardia with second degree block. During second degree AV block, selective slowing of the sinus node led to I :I conduction (Plate 6). These procedures in 5 dogs did not cause any variation in QRS configuration. During the selective slowing of the sinus node, there was no escape of AV junctional rhythm. Since the later phases of chronotropic effect by disodium EDTA in the sinus node were characterized by prolonged sinus bradycardia [7], this lack of AV junctional escape may represent a similar depression of latent automaticity within the AV junctional region.
368
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N. JAMES
Reversible splitting
of the His potential
During the period of completely dissociated and rapid irregular rhythms in both atria and ventricles, the form and location of the His potential was highly variable and difficult to identify separately from multiple other local electrical potentials. As steady sinus rhythm resumed control of the atria with the irregularly rapid and dissociated ventricular rhythm persisting, in 3 of the 16 dogs a His bundle potential became associated with the A complex at a normal A-H interval of slightly varying duration; however, there was still initially no consistent His bundle potential before the V complex. In 2 of these 3 dogs a separate His bundle potential later appeared just proximal to the V complex with a normal H-V interval, at a time when complete AV dissociation was still present [Plate 7(a)-(e)]. This phenomenon of two separate His bundle electrograms, each respectively related to an A complex and V complex which were independent of each other, lasted for 11 and 18 min, respectively, in these 2 dogs; in both of these dogs there was a later period of AV junctional rhythm driving both the atria and ventricles [Plate 7(d)] shortly before sinus control resumed [Plate 7(e)]. After normal sinus rhythm and AV conduction were restored in one of these dogs, a second injection of disodium EDTA into the AV node artery exactly reproduced the same sequence of split His potentials and subsequent recovery.
Site of dromotropic action of disodium Although various degrees of AV and VA block were consistently observed as indicated previously, there was never any prolongation of the H-V interval of the His electrogram. All the delay in AV conduction when a stable His potential could be clearly identified was either in the A-H interval, or between two split components of the His bundle potential [Plate 7(a)-(e)]. When two components of the His potential were split, the A-H interval was not prolonged. A long A-H interval was observed only during sinus rhythm transmitted to the ventricles and only when a single His potential was present.
QRS conjguration
during and following
the rapid irregular
ventricular rhythm
In every dog there was an immediate change (broadening) and frequent variation in the configuration of the QRS complex during the chaotic tachycardia caused by disodium EDTA in the AV node artery (Plates 2 and 4). For comparative purposes, a similar degree of AV junctional tachycardia was produced in 4 dogs by norepinephrine perfused into the AV node artery and was associated with completely normal QRS complexes, confirming previous similar results [Z]. During the chaotic supraventricular tachycardia caused by disodium EDTA in the sinus node artery,
CALCIUM
CHELATION
IN
THE
the net rate exceeded that of the AV junctional EDTA, yet the configuration of QRS complexes These results illustrate that the QRS deformation node artery cannot be due exclusively to either branch block, or enhanced (latent) automaticity junction, but must be due to something other or
AV
JUNCTION
369
tachycardia caused by disodium was normally preserved (Plate 8). after disodium EDTA in the AV rate-dependent forms of bundleimpairing conduction in the AV more than these factors.
4. Discussion Selective perfusion of disodium EDTA into the AV node artery rapidly produces a number of electrophysiological responses which significantly alter cardiac rhythm and conduction. Three things occurred almost simultaneously: failure of AV conduction, an independent and rapid but irregular rhythm controlling the ventricles, and a separate rapid and equally irregular rhythm which interfered with the sinus node to dominate atria1 activity. The supraventricular irregularity was relatively short-lived, soon being replaced by the resumption of a steady sinus rhythm at the control rate but which did not conduct to the ventricles, nor was it influenced by retrograde conduction from the rapid irregular ventricular rhythm. To explain the two forms of rapid irregular rhythm and concomitant complete AV block, discussion will be focused on the P cells of the AV junctional region and their postulated function. Selective perfusion of disodium EDTA into the sinus node produces an immediate irregular rhythm at a rate near maximal levels achievable by the normal sinus node [7], and this is best attributed to two local effects : the known positive chronotropic action of lowered extracellular calcium ion, and the concomitant effective separation of multiple independently firing sites of automaticity. Since the junctions between these multiple independent units and the transitional cells, which serve as the normal conduit out of the sinus node, would presumably remain functionally intact for rapid conduction (their gap junctions being preserved), most of the new signals of multifocal origin would have ready egress and conduct to the atrium. An analogous explanation may be made for the results observed in the AV node and His bundle, where the P cells have all the same fine structural characteristics as in the sinus node. However, there is one important difference and that is the fact that under normal circumstances the main function of the AV junction is conduction rather than automaticity. If transnodal conduction was altered within the sinus node, one need not anticipate any effect on AV conduction. On the other hand, if the P cells of the AV node are functionally disaggregated and if they form an essential component of the normal route of AV conduction (the latter until now being unknown), then one would not only anticipate the chaotic positive chronotropic action seen but also disruption of AV conduction. This is exactly what happened. As corollaries to the interpretation that AV nodal P cells are an obligatory transit
370
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site for normal AV conduction perhaps by their normal anatomic interposition in the AV node, then there are two logical additional considerations. First, the P cells may represent the exact locus for the normal 40 ms (approximate) delay present with every beat during sinus rhythm. Second, any circumventing of conduction through AV nodal P cells would be more rapid than normal AV conduction. The nature of normal connections between P cells themselves (largely devoid of gap junctions) would support the first corollary, while the prevalence of gap junctions between all other types of myocardial cells supports the second corollary. It is the continued AV dissociation during the phases of rapid irregular rhythm, as well as the varying degrees of first and second degree AV block even when sinus control was re-established (Plates 5 and 7), which together serve as the strongest evidence that the P cells are an essential component of normal AV conduction. If this were not so, then there should have been normally preserved AV conduction both during the period of positive chronotropic effect (allowing for interference dissociation) and particularly when sinus rhythm was resumed. The connections between all myocardial cells except P cells contain numerous gap junctions, all of which should have been functioning normally since they are uninfluenced by the lack of local calcium ion. None of this established that the rapid irregular rhythm controlling the ventricles was of AV junctional origin. In fact, the immediate appearance of changed QRS complexes, most being wider than normal, would seem to indicate that the ventricular rhythm originated at multifocal sites beyond the level of the His bundle. The loss of an identifiable His potential preceding these broad ventricular origin. However, in other expericomplexes would also suggest their “ventricular” ments we have shown that the perfusion of substances with marked positive chronotropic action, such as norepinephrine or isoproterenol [Z, 81 or glucagon [26] into the AV node artery consistently produced exclusively a regular AV junctional tachycardia with QRS complexes identical to those during sinus rhythm and with a His bundle potential present at the normal interval before every ventricular complex. If catecholamines or glucagon failed to produce any but a supraventricular rhythm, then it seems more likely that the positive chronotropic effect ofdisodium EDTA given in the same fashion would also have produced a supraventricular rhythm, but one with odd features (broad QRS, absent His potential). If the rapid irregular ventricular rhythm is AV junctional in origin, then why are the QRS compiexes changed and where is the expected His bundle potential which should be present proximal to the ventricular complexes? On the basis of clinical [21, .?2] and anatomical [rl] evidence we have previously suggested that longitudinally dissociated conduction is normally present in the human and canine His bundle. If there is normally partitioned or longitudinally dissociated conduction in the canine His bundle, then the configuration of the QRS complexes will necessarily depend on the pattern of the activation front which arrives at the proximal end of the His bundle. That pattern arriving at the His bundle during
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normal sinus rhythm and during most forms of supraventricular rhythm will be nearly uniform, because of the normally uniform triage function of the AV node. However, if there is multifocal automaticity within the AV junctional region, then it would be anticipated that a variety of activation fronts would approach the His bundle, each producing its own form of QRS complex and, except on a random basis, none being similar to that seen during sinus rhythm. This brings us to the question of the nature of the normal His bundle potential. If it originates in the His bundle itself, as is generally believed, then there should still be a His bundle potential before every ventricular complex, even if there was a multifocal rhythm originating in the P cells. However, some have suggested that the His bundle potential originates from the “proximal” portion of the His bundle [18], which is very close to the anatomical location of the P cells. If the His bundle potential actually originated in those P cells which are normally present directly in the region of the junction of the AV node and His bundle, then one could readily explain the splitting of the His bundle potential observed in certain of the present experiments [Plate 7 (a)-(e)]. Th us, when a sufficient number of P cells had reaggregated in connection with the internodal pathways, there would be visible a His bundle potential associated with the atria1 complex and regularly controlled by the sinus node. If a sufficient number of P cells further downstream reaggregated to form a separate His bundle potential which represented the site of origin for the ventricular rhythm, and if these latter P cells had not yet reconnected with the upper group of P cells, then one would expect two independent rhythms, each with its own His bundle potential [Plate 7(a)-(c) J. When the two groups of P cells then eventually all reunited, a single His potential would reappear just as it did in the present study, and this was true both for regular AV junctional rhythm [Plate 7(d)] as well as for conducted sinus rhythm [Plate 7(e)]. The fact that neither the A-H nor H-V interval was prolonged when the His potential was split suggests that AV conduction was delayed exclusively within the P cell region when a single His potential returned. This form of reversible splitting of the His potential has not, to my knowledge, been previously demonstrated. Since the rate of local recalcification via the collateral arterial circulation [S] would be focally variable, and re-aggregation of P cells would therefore be of a somewhat random nature, it is not surprising that the degree of heart block, the duration of the irregular rhythms, and the extent of deformation of the QRS complexes would be comparably variable. In particular, the initial rapid disappearance of a recognizable His bundle potential and its variable pattern of return would fit well with this random “t-e-gluing” of P cells. Some would suggest that the AV conduction impairment may in part have been the result of enhanced latent automaticity, lasting beyond the period of overt positive chronotropic effect. Various forms of QRS deformation have been ascribed to such a phenomenon [20, 231, those cells with enhanced but still latent automaticity conducting slower than normal. However, if this were the case, one would
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have anticipated QRS deformation after the selective administration directly into the AV junction of substances with known maximally powerful positive chronotropic influence, and this does not occur, neither in previous experiments with norepinephrine and isoproterenol [Z] or glucagon [26] nor in the present experiments. This calls into question whether latent automaticity is a tenable explanation for impairment of AV conduction in vivo. There is the further possibility that some of the QRS distortion may have been a form of rate-dependent bundle branch block. However, not only was there no such change in the QRS complexes during maximal levels of sinus tachycardia following selective perfusion of norepinephrine into the sinus node, but neither was there any significant variation in QRS configuration during the rapid irregular tachycardia caused by disodium EDTA perfused into the sinus node artery (Plate 8). Finally, the possible neural influence of disodium EDTA [24] either to enhance local vagal effect or to impair local adrenergic p-receptor activity must be considered in the mechanism of production of the AV block. However, the absence of influence by atropine and the occurrence of AV block after pre-treatment with either propranolol or reserpine would suggest that autonomic neural mechanisms did not play a role in the negative dromotropic effect. Conversely, the occurrence of AV junctional tachycardia despite pretreatment with reserpine or propranolol or atropine indicates that the positive chronotropic effect was not mediated via adrenergic P-receptors, nor by release ofvagal tone. Specific chronotropic and dromotropic events may be summarized as follows. The rapid rhythm was due to lowered extracellular calcium concentration around the P cells, and being completely uninfluenced by pretreatment with atropine or propranolol or reserpine, may be presumed to be a direct action having neither anticholinergic or catecholamine-mediated components. The irregular&y of the rhythm was caused by disaggregation (either functional or anatomical) of P cells, with different cell groups having different intrinsic levels of automaticity and therefore different degrees of enhancement by the local calcium deficit. The AL’ block was also due to disaggregation of P cells with failure of conduction between enough of them to preserve communication between upper and lower AV junctional rhythms; this appeared to have no cholinergic or anti-adrenergic component. Since the His bundle pqtential disappeared during these maximal effects, and was transiently split during the recovery phase in some dogs, it is concluded that the His potential actually originates within the P cells and its given magnitude when recorded by bipolar local electrodes would depend on the net vector generated by conduction through the functionally aggregated P cells. The P cells thus appear to be the source of normal sustained automaticity in the AV junction, and they form an essential link in the normal pathway of AV conduction during sinus rhythm, the latter being most likely on the basis of their anatomical location between the main body of the AV node and the His bundle. As an essential link in normal AV conduction, the AV nodal P cells are the probable site of the normal brief delay (about 40 ms) seen
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with every sinus beat. As a corollary, any route of AV conduction circumventing the AV nodal P cells would be anticipated to occur more rapidly than normal.
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MUIR, A. R. The effects of divalent cations on the ultrastructure of the perfused rat heart. Journal of Anatomy 101, 239-261 (1967). ROSENBAUM,M. B., ELIZARA, M.V., LAZZARA, J.O., HALPERN, M.S., NAU,G.J.& LEVI, R. J. The mechanism of intermittent bundle branch block: relationship to prolonged recovery, hypolarization and spontaneous diastolic depolarization. Chest 63, 666667 (1973). SHERF, L. & JAMES, T. N. A new electrocardiographic concept. Synchronized sinoventricular conduction. Diseases ofthe Chest 55, 127-140 (1969). SHERF, L. & JAMES, T. N. The mechanism of aberration in late AV junctional beats. American 3ourml of Cardiology 29, 529-539 (1972). SINGER, D. H., LAZZARA, R. & HOFFMAN, B. F. Interrelationships between automaticity and conduction in purkinje fibers. Circulation Research 21, 537-538 (1967). TODA, N. & WEST, T. C. Interaction between Na, Ca, Mg and vagal stimulation in the S-A node of the rabbit. American 3ournd of Physiology 212, 424-430 (1967). TRAUTWEIN,W. & UCHIZONO, K. Electron microscopic and electrophysiologic study of the pacemaker in the sino-atria1 node of the rabbit heart.