Delayed depolarization of the cog-wheel valve

The Journal of Experimental Biology 205, 1843–1851 (2002) Printed in Great Britain The Company of Biologists Limited 2002JEB3996 Delayed depolarization of the cog-wheel valve and pulmonary-to-systemic
shunting in alligators
Douglas A. Syme1,*, Kurt Gamperl2,† and David R. Jones2,‡ 1Department of Biological Sciences, 2500 University Drive NW, University of Calgary, Calgary, Alberta, Canada T2N 1N4 and 2Department of Zoology, University of British Columbia, Vancouver, British Columbia, †Present address: Ocean Sciences Centre, Memorial University of Newfoundland, St John’s, Newfoundland, Canada A1C 5S7 ‡Present address: Distinguished Scholar, Peter Walls Institute for Advanced Studies, The University Centre, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z4 Alligators and other crocodilians have a cog-wheel valve
occurred during systole and is likely to strongly influence
located within the subpulmonary conus, and active closure
the amount of blood entering the pulmonary artery and
of this valve during each heart beat can markedly and
thus to directly control the degree of shunting. Left vagal
phasically increase resistance in the pulmonary outflow
stimulation (10–50 Hz) reduced the conduction delay
tract. If this increased resistance causes right ventricular
between the right ventricle and cog-wheel valve by
pressure to rise above that in the systemic circuit, right
approximately 20 % and reduced the integrated cog-wheel
ventricular blood can flow into the left aorta and systemic
ECG by 10–20 %. Direct application of acetylcholine
circulation, an event known as pulmonary-to-systemic
(1–2 mg) also reduced the integrated cog-wheel ECG by
shunting. To understand better how this valve is
10–100 %; however, its effect on the conduction delay was
controlled, anaesthetized American alligators (Alligator
highly variable (–40 to +60 %). When the cog-wheel valve
mississippiensis) were used to examine the relationships
muscle was killed by the application of ethanol, the cog-
between depolarization of the right ventricle,
wheel ECG was absent, right ventricular and pulmonary
depolarization/contraction of the cog-wheel valve muscle
pressures remained low and tracked one another, the
and the resultant right ventricular, pulmonary artery and
secondary rise in right ventricular pressure was abolished
systemic pressures. Depolarization swept across the right
and shunting did not occur. This study provides
ventricle from the apex towards the base (near where
additional, direct evidence that phasic contraction of the
the cog-wheel valve muscle is located) at a velocity of
cog-wheel valve muscle controls shunting, that nervous
91±23 cm s–1 (mean ± S.E.M., N=3). The cog-wheel valve
and cholinergic stimulation can alter the delay and
electrocardiogram (ECG) (and thus contraction of the
strength of valve depolarization and that this can affect
valve) trailed the right ventricular ECG by 248±28 ms
the propensity to shunt.
(N=3), which was equivalent to 6–35 % of a cardiac cycle.
This long interval between right ventricular and valve
depolarization suggests a nodal delay at the junction
Key words: alligator, Alligator mississippiensis, blood pressure, between the base of the right ventricle and the cog-wheel
cardiac muscle, heart, shunt, left aorta, pulmonary artery, right valve. The delay before valve closure determined when the
ventricle, electrocardiogram, cog-wheel valve, conduction velocity, abrupt secondary rise in right ventricular pressure
Introduction
The crocodilian ventricle is unique among reptiles because through the LAo and into the systemic circuit is known as a it is morphologically divided into left and right halves by a pulmonary-to-systemic (P→S) shunt (Jones, 1996). In complete interventricular septum. Although this anatomical addition, blood can be exchanged between the LAo and the design prevents the mixing of systemic and pulmonary venous right aorta (RAo, arising from the left ventricle) through the blood within the ventricle, blood can still cross between the foramen of Panizza, which is located just downstream of the circuits via several routes outside the heart. The right ventricle ventricular valves, and via an anastomosis (JJ) between the two (RV) maintains a connection to the systemic circulation via the left aorta (LAo, Fig. 1), and blood flow from the right ventricle, Crocodilians also possess a well-developed and muscular 1844 D. A. Syme, K. Gamperl and D. R. Jones cog-wheel valve located in the subpulmonary conus just In crocodilians, the merits of P→S shunting and the outside the RV (Fig. 1) (Greenfield and Morrow, 1961; Webb, mechanism(s) by which shunts are controlled are not well 1979; Farrell et al., 1998). This valve consists of connective understood (for reviews, see Jones, 1996; Burggren, 1987).
tissue nodules that fit together to partially or completely Franklin and Axelsson (2000), using an isolated heart model occlude the conus; it is surrounded by a mass of cardiac muscle in which the pulmonary outflow tract had been removed, (van Mierop and Kutsche, 1985). Contraction of this muscle showed that β-adrenergic stimulation reduces resistance in the causes closure of the valve and increased pulmonary input subpulmonary conus, reduces RV pressure development and resistance and would account for the phasic and complex thus inhibits P→S shunting in the crocodile. In addition, alterations in RV pressure that occur with each cardiac cycle Axelsson and Franklin (2001) show that the calibre of the and cause P→S shunting (Greenfield and Morrow, 1961; foramen of Panizza in the aortic outflow tract is variable White, 1969, 1970; Grigg and Johansen, 1987; Axelsson et al., and subject to adrenergic constriction, which will have 1989, 1996; Shelton and Jones, 1991; Jones and Shelton, 1993; consequences for flow patterns in the left and right aortas during shunting. However, sustained adrenergic tonus cannotaccount for the aforementioned phasic and complex changes inRV pressure seen in crocodilians late in the cardiac cycle.
Thus, there must also be large and phasic changes in resistance in the pulmonary outflow tract with each heart beat. Franklin and Axelsson (2000) and Axelsson and Franklin (2001) make no claim that an adrenergically mediated mechanism causesphasic changes in resistance within each cardiac cycle; indeed, such changes occurred with a time course of minutes in their experiments. Hence, some other mechanism(s) must also be responsible for regulating this valve and shunting incrocodilians.
The phasic changes in resistance suggest phasic activity of the cog-wheel valve during each cardiac cycle and stronglysuggest that depolarization of the muscle mass surroundingthe valve is linked to RV contraction. White and Brady(unpublished observation cited in White, 1968) noted a 350 msdelay between depolarization ‘near the pulmonary artery’ andthe adjacent ventricle in alligators, and proposed that this may be the source of the phasic increase in resistance between theRV and pulmonary artery (PA). Further, Burggren’s (1978)work on turtle hearts implies a 200–300 ms delay mechanismlinking depolarization in the main ventricle to that in the bulbussurrounding the PA. There are no published reports thatdescribe a link between depolarization of the RV and of thecog-wheel valve muscle in crocodilians nor the phasicrelationships between valve depolarization and the centralpressure gradients causing P→S shunting. In this paper, wepresent evidence that cog-wheel valve activity is synchronized with RV depolarization through a nodal delay mechanism and that the phasic relationships that exist between RV contraction and valve activity are a key mechanism allowing crocodilians Fig. 1. The crocodilian central circulation, ventral view. The right to regulate shunting on a beat-by-beat basis.
ventricle (RV) maintains connections to both the pulmonary circuitvia the pulmonary artery (PA) and the systemic circuit via the leftaorta (LAo), which continues as the coeliac artery (CA). The Materials and methods
subpulmonary conus contains a muscular cog-wheel valve (CWV, Experiments were conducted at the University of British made of cartilagenous teeth and surrounded by cardiac muscle), the Columbia, and all procedures conformed to UBC animal care contraction of which can occlude the entrance to the pulmonary guidelines. American alligators (Alligator mississippiensis) of artery. The right aorta (RAo) receives blood from the left ventricle both sexes were obtained from commercial farms in Florida, (LV), gives rise to the common carotid artery (CCA) and the USA, and held in the animal care facilities at the university.
right/left subclavian arteries (R/LSA) and then continues as the Animals were housed individually or in small groups in rooms dorsal aorta (DAo). The left and right aortas connect twice, justoutside the ventricles through the foramen of Panizza (FP) and (approximately 15 m2, 30 °C) containing a shallow pool in behind the heart via an anastomosis (JJ).
which they could submerge. They were fed commercial dog Delayed depolarization of the cog-wheel valve 1845 chow ad libitum and given chopped chicken and vitamin inserted into the centre of the mass of muscle surrounding the supplements weekly. All animals were growing and appeared cog-wheel valve. ECG electrodes were connected to Gould healthy before these experiments. Recordings were made on isolated preamplifiers (model 11-5407-58) and Gould universal 11 animals; mass range 8.6–37.5 kg, mean 21.5±2.5 kg (mean amplifiers (model 13-4615-58) with a 3 Hz to 1 kHz band-pass.
± S.E.M.). Because not all recordings could be made on every The ECG data were digitally filtered offline using a high-pass animal, the number of alligators used for each set of finite impulse response (FIR) filter (10–30 Hz cut-off) to measurements varied. Data in the text, figures and table are remove movement artifacts. All pressure and ECG data were collected on a computer using LabTech Notebook Pro v9 correlations were used to describe relationships between heart software (Labtech, Andover, Massachusetts, USA).
rate, electrocardiogram (ECG) timing and conduction velocityand delay, with P<0.05 considered significant.
Propagation of the ECG across the RV and into the muscle surrounding the cog-wheel valve was recorded by positioning Animals were initially sedated by injecting 25 mg kg–1 one electrode in the cog-wheel muscle (located Ketamine HCl (Ketalean, Bimeda-MTC, Cambridge, Ontario, approximately 1 cm from the base of the RV) and a second Canada) into the tail musculature, and then placing a mask electrode at different locations on the RV (11–15 different containing a Halothane-wetted cloth over their nostrils (MTC locations in each of three animals). Conduction velocity Pharmaceuticals, Cambridge, Ontario, Canada). When the across the RV was calculated as the slope of the regression animals had been sufficiently sedated, they were weighed and relating the distance between electrodes and conduction time.
placed supine on a surgical table. They were then intubated and The intercept of this relationship was the conduction delay ventilated with a small-animal ventilator, modified for use on that occurred at the junction of the RV and cog-wheel valve alligators, and brought to surgical anaesthesia with 3–4 % Halothane in 1:1 N2O:O2. A catheter was placed in the rightfemoral vein, and infusion of Ketamine (5–15 mg–1 kg–1 h–1) was initiated. Halothane and N2O anaesthesia were suspended After recording the RV conduction velocity and delay, the at this point, but forced ventilation with 100 % oxygen was effects of parasympathetic and cholinergic stimulation on continued. A heating pad was used to maintain core body central pressures, ECGs and cog-wheel valve function were temperature at 30 °C throughout the experiments (monitored studied. ECG electrodes were placed in the middle of the RV and in the muscle mass surrounding the cog-wheel valve.
Xylocaine (2 % lidocaine HCl, Astra Pharma Inc., Pressures from two of the three catheters (RV, subclavian, PA) Mississauga, Ontario, Canada) was then injected and both ECGs were measured under ‘control’ conditions (no subcutaneously along the ventral midline, and an incision was manipulations), when cog-wheel valve contraction and heart made through the skin and ribs to expose the heart and central rate were modified by direct application of acetylcholine (ACh) vasculature. The heart was exposed by slitting the pericardial to the muscle surrounding the cog-wheel valve or to the RV sac along the rostro-caudal midline. Silk sutures were tied to muscle (see Table 1 for details), during vagal stimulation or the cut edges of the pericardium and secured to a ring stand after cog-wheel valve function had been temporarily weakened placed above the heart. This formed a bath around the heart by injecting Xylocaine or the valve had been killed by injection that was filled with mineral oil to prevent desiccation and to of 95 % ethanol into the cog-wheel valve muscle. Vagal reduce the bulk flow of current around the heart. A non- stimulation was successful in two of the three animals in which occlusive pressure catheter (Bolab medical vinyl tubing, it was attempted; we report results from only the two Lake Havasu City, Arizona, USA), pre-treated with an successful experiments. In these animals, a cuff electrode anticoagulating agent (TD-MAC, Polysciences Inc., (custom-made) was placed around the isolated left vagus Warrington, Pennsylvania, USA), was inserted into the right nerve, and the stimulation frequency was set at 10, 20 or 50 Hz subclavian artery. The tip of a 16-gauge hypodermic needle (50 µs pulse duration and 5–7 V amplitude). Changes in was fixed to a second pressure catheter and inserted into the pressure profiles, heart rate, the conduction delay between the pulmonary artery. A third catheter, similarly fashioned, was RV and cog-wheel ECGs, the phase of the cog-wheel ECG (see inserted directly into the right ventricle through the ventricular below) and the integrated cog-wheel ECG (see below) were wall. Catheters were filled with degassed, heparinized measured after these manipulations. These experimental (40 i.u. ml–1), 0.9 % NaCl saline, cleared of bubbles and recordings were bracketed by control recordings, although it connected to Deltran II pressure transducers (Utah Medical was not possible to bracket experiments in which the cog- Products, Midvale, Utah, USA). These transducers were wheel muscle was killed with ethanol.
routinely calibrated against a mercury column during Phase was defined as the percentage ratio of the conduction delay to the duration of a cardiac cycle (i.e. the percentage of Bipolar ECG electrodes (1 mm tip spacing) were made from a cardiac cycle that elapsed between the RV ECG and the cog- 44-gauge copper magnet wire and chemically sharpened. One wheel ECG). The integrated cog-wheel ECG was used as a was inserted into the main RV muscle mass, and the other was measure of the strength of the cog-wheel depolarization or the 1846 D. A. Syme, K. Gamperl and D. R. Jones Fig. 2. Electrocardiograms (ECGs) and pressures in the alligator pulmonary circuit. Top traces, ECGs recorded from the middle of the right ventricle (RVECG, red) and the cog-wheel valve muscle (cog- wheel ECG, blue). Bottom traces, right ventricular pressure (RV, red) and pulmonary arterial pressure (PA, green). A and C show results from animals witha functioning cog-wheel valve. B and D show results from the same animals, but after the cog-wheel valve had been inactivated by application of acetylcholine.
muscle’s activity; the filtered cog-wheel ECGs were rectified,and the voltage/time integral was subsequently measured over the period of the ECG. All calculations were made usingAcqKnowledge software (v3.01 BIOPAC Systems, Inc., Santa Barbara, California, USA). At the conclusion of each experiment, the animals were killed with an intracardiac overdose of pentobarbital, and the hearts and central vessels were dissected to confirm the appropriate placement of all ECGs, central blood pressures and elimination of cog-wheel Recordings of RV and cog-wheel valve muscle ECGs, RV pressure, systemic pressure (subclavian) and PA pressure are shown in Figs 2 and 3. The RV ECG was coincident with a rise in RV pressure, and increases in PA pressure wereobserved when RV pressure equalled or exceeded diastolic PA pressure (i.e. the pulmonary valve opened). Coincident with the cog-wheel muscle ECG was a large, secondary risein RV pressure and an uncoupling of PA pressure from that in the RV (Figs 2A,C, 3A). Activation of the cog-wheel valve often caused an almost doubling of the pressure developed by the RV. This marked increase in RV pressure was sufficient to meet or exceed that in the systemic circulation in many instances (Fig. 3B) and would favour the ejection ofRV blood into the LAo and thus the occurrence of a P→S Fig. 3. Effect of cog-wheel valve contraction on pulmonary, right shunt. However, if the muscle surrounding the cog-wheel ventricular and systemic blood pressures in an alligator. (A) Pressurein the right ventricle (RV, red) and pulmonary artery (PA, green) and valve was killed by injection of ethanol directly into the electrocardiograms (ECGs) from the centre of the right ventricle (RV muscle or inhibited with ACh, the cog-wheel ECG and ECG, red) and the cog-wheel valve muscle (cog-wheel ECG, blue) the secondary rise in RV pressure were eliminated when the cog-wheel valve was functioning. (B) Pressures in the right (Figs 2B,D, 3C). In every case where the cog-wheel valve ventricle (red) and systemic circulation (right aorta, RAo, cerise) and was made non-functional by treatment with ethanol, ECGs of the same animal with a functioning cog-wheel valve.
Xylocaine or ACh, neither the secondary rise in RV pressure (C) Same as A, except that the cog-wheel valve has been inactivated nor P→S shunting was seen. PA pressure during systole was Delayed depolarization of the cog-wheel valve 1847 notably lower than RV pressure, even when the cog-wheel There was no relationship between the absolute ECG delay valve was inactivated by ACh or Xylocaine or killed by ethanol injection (see Figs 2, 3); obviously, the pulmonary Because of the slow heart rates exhibited by these animals outflow tract itself presented a significant resistance to blood (28.6±2.2 beats min–1), it was possible to confirm visually that contraction of the muscle surrounding the cog-wheel valvecoincided with the cog-wheel muscle ECG. Further evidence that the cog-wheel muscle ECG coincided with valve The delay between the RV ECG and the ECG in the cog- contraction comes from the changes in RV and PA pressures wheel valve muscle decreased as the electrode in the RV was that followed the cog-wheel muscle ECG (see above). We did moved towards the base of the heart (i.e. closer to the cog- not detect a deflection in the cog-wheel ECG that might wheel valve) (Fig. 4), suggesting that the RV ECG spread signify termination of the action potential (equivalent to a across the RV surface from the apex towards the base. The ventricular ‘T’ wave). However, relaxation of the cog-wheel slope of the relationship between electrode separation and valve muscle preceded relaxation of the ventricle by a period conduction delay is the conduction velocity across the RV great enough that it could easily be observed; the duration of and averaged 91±23 cm s–1 (Fig. 4). The intercept of this the cog-wheel contraction was considerably shorter than that relationship is the nodal delay that occurred at the junction of the RV and cog-wheel muscle and averaged 248±28 ms inthe three animals studied (Fig. 4). This delay was only slightly shorter than the total conduction delay measured from the centre of the RV to the cog-wheel valve muscle(267±21 ms, N=9), which includes both the time for the action potential to sweep over the RV and the delay associated with its passage across the RV/cog-wheel junction. Thus, approximately 90 % of the total conduction delay was due to a nodal delay at the RV/cog-wheel junction.
The mean phase of the cog-wheel muscle ECG was 13.2±1.89 % of a cardiac cycle. There was a highly significant relationship between phase and heart rate (Fig. 5A) and between phase and absolute delay (Fig. 5B).
Fig. 4. Delay between the electrocardiogram (ECG) in the right ventricle and the ECG in the cog-wheel valve muscle as a function ofthe distance between the two recording sites. One ECG electrode was Fig. 5. (A) Relationships between the delay separating the left fixed in the middle of the cog-wheel valve muscle, while the electrocardiogram (ECG) in the middle of the right ventricle from electrode in the right ventricle was moved to different locations.
that in the cog-wheel valve muscle and heart rate (open circles) Results from three animals are shown. Inverse slopes give the and between the phase of the cog-wheel muscle ECG and heart conduction velocity in the right ventricle, and are 0.43 m s–1 rate (filled circles): delay versus heart rate was not significant (P<0.001, r2=0.449), 1.41 m s–1 (P=0.14, r2=0.037) and 0.85 m s–1 (P=0.24); phase versus heart rate slope=0.67 (P<0.001, r2=0.63).
(P<0.001, r2=0.152) from top to bottom, respectively. The intercept (B) Relationship between phase and the delay between the right is the ‘nodal’ delay at the junction of the right ventricle and cog- ventricle and cog-wheel valve muscle ECGs: slope=0.069 (P<0.001, wheel valve muscle. Values are means ± S.E.M.
1848 D. A. Syme, K. Gamperl and D. R. Jones Table 1. Effects of left vagal stimulation, acetylcholine application and Xylocaine application on heart rate, the delay between the right ventricular and cog-wheel electrocardiograms (ECGs), the phase of the cog-wheel ECG and the integrated cog-wheel Values are expressed as a percentage of the control measurements that bracketed each experimental trial. Phase is the ECG delay as a fraction of the heart beat duration. The pulse frequency during vagal stimulation is shown in parentheses (0.05 ms pulse duration, 5–7 V). Spaces separate results for individual experimental animals.
10.5 ml of 2 mg ml–1 acetylcholine (ACh) on the right ventricle. 2One drop of 10 mg ml–1 ACh on the cog-wheel valve muscle. 3Two drops of 10 mg ml–1 ACh on the cog-wheel valve muscle. 40.2 ml of 10 mg ml–1 ACh injected into the cog-wheel valve muscle. 52 ml of 2 % lidocaine HCl (Xylocaine) injected into the cog-wheel valve muscle. 6Xylocaine dripped onto the cog-wheel valve muscle. (Table 1). In one case, the cog-wheel muscle was inactivated Conduction delay was quite constant for a given heart and in the other case there was an increase in conduction delay working at a particular heart rate. However, it changed when but no effect on the integrated cog-wheel ECG.
the heart was vagally stimulated or when ACh was applied tothe cog-wheel valve muscle. In the two animals in which vagalnerve stimulation was successful, heart rate, conduction delay Discussion
and cog-wheel phase all decreased substantially (Table 1).
The data presented here demonstrate a synchronization There was also a decrease in the integrated cog-wheel ECG between RV contraction and cog-wheel valve contraction (Figs (Table 1), signifying a weakening of cog-wheel muscle 2–5), that the timing of these events can be altered (Table 1), contraction. From our limited data, it appeared that the lower that the timing accounts for the observed central pressure stimulation frequencies (10 and 20 Hz) were more effective profiles (Figs 2, 3) and that the delay between these two events than the high stimulation frequency (50 Hz) at eliciting these with subsequent valve contraction is likely to control P→S effects, although no clear pattern between vagal stimulation shunting (Fig. 3B). Further, we show that contraction of the frequency and cog-wheel ECG delay emerged.
cog-wheel valve follows RV depolarization by a defined delay All four animals which had ACh applied topically to the cog- (see also White, 1968), implicating a nodal conduction delay wheel valve muscle showed a decrease in heart rate and a linking the RV ECG with that in the cog-wheel muscle (Figs 4, considerable weakening of the integrated cog-wheel ECG (Table 5). From these results, it is apparent that active control over 1). The effect on the conduction delay was variable, ranging the timing of valve closure allows pulmonary input resistance from an increase of almost 60 % to a decrease of 40 % to and thus shunting to be controlled somewhat independently of complete blockade of cog-wheel muscle contraction. Together systemic blood pressure or chronic pulmonary resistance and with the reduction in the integrated cog-wheel ECG was a allows the shunt to be initiated or terminated in, perhaps, a reduction in the secondary rise of pressure in the RV (Figs 2B,D, 3C). All these effects were reversible as the ACh washed out.
Although we believe that synchronization of RV The effects of application of Xylocaine to the cog-wheel depolarization with valve contraction is the primary mechanism muscle were variable in the two instances it was attempted that initiates/controls shunting, a number of secondary Delayed depolarization of the cog-wheel valve 1849 mechanisms by which P→S shunting may be influenced have in close proximity to the cog-wheel valve may expand the also been proposed. These include: (i) alterations in RV subpulmonary conus to such a degree that the cog-wheel valve contractility, (ii) alterations in pulmonary resistance, including could not effectively occlude the pulmonary outflow tract. It those associated with ventilation, (iii) changes in systemic will be most interesting to learn what the effects of adrenergic vascular resistance, and (iv) changes in right ventricular end- stimulation on the pulmonary outflow tract are and what the diastolic volume (via a Starling effect) and, thus, changes in RV anatomy of the cog-wheel valve musculature is in relation to pressure development (Shelton and Jones, 1991; Axelsson and its behaviour under adrenergic stimulation.
More recently, Franklin and Axelsson (2000) report that Cog-wheel valve contraction and shunting shunting may be influenced through β-adrenergic control of In our anaesthetized alligators, there appeared to be a resistance in the subpulmonary conus, which contains the substantial resistance in the pulmonary outflow tract that was cog-wheel valve. Injection of the competitive β-adrenergic independent of cog-wheel valve contraction (but see Axelsson antagonist sotalol into the right side of the heart increased et al. (1996) for an example in crocodiles where there is very resistance in the pulmonary outflow tract and induced little resistance). This resistance caused a large pressure drop shunting in isolated, perfused hearts of the estuarine crocodile between the RV and PA during early systole, before the cog- Crocodylus porosus. The subsequent addition of a saturating wheel ECG (Figs 2A,C, 3A) (see also Shelton and Jones, 1991; concentration of adrenaline caused the shunt to be abolished.
Grigg and Johansen, 1987), and it did not disappear when the While such observations support the idea that β-adrenergic cog-wheel muscle was inactivated (Figs 2B,D, 3C). The stimulation can affect the shunt, the mechanism they propose catheter used to measured PA pressure was placed just distal (direct, adrenergic stimulation of the cog-wheel muscle to the cog-wheel valve, and the majority of the resistance we causing valve relaxation) is seemingly at odds with the normal measured would therefore have resided within or very close to response of cardiac muscle to adrenergic stimulation. If the the valve. However, despite the substantial pulmonary cog-wheel muscle mass is of the cardiac type, which it appears resistance when the cog-wheel muscle was not active, RV to be, adrenergic stimulation would be expected to cause valve pressures never attained levels required for shunting (Fig. 2) closure and thus promote P→S shunting, and removal of β- and, as far as we are aware, the shunt is always accompanied adrenergic stimulation by sotalol treatment would diminish by a biphasic RV pressure profile showing the dramatic the force of cog-wheel valve contraction and inhibit shunting; secondary rise in pressure associated with phasic, cog-wheel this is exactly opposite to what was observed. This leads us valve contraction. The resistance appears to lessen as to the conclusion that either the cog-wheel muscle’s response pulmonary and RV pressures rise (Jones and Shelton, 1993; D.
to adrenergic stimulation is very unusual (relaxation) or A. S., K. G. and D. R. J., unpublished observations), which perhaps that the fibre orientation in the valve is such that would be consistent with the cartilaginous nodules of the cog- contraction leads to valve opening (M. Axelsson, personal wheel valve being ‘blown open’ at higher pressures.
Inhibition of cog-wheel valve contraction, whether by Interestingly, adrenergic stimulation of the aortic outflow ethanol, ACh, vagal stimulation or Xylocaine, caused RV tract in crocodiles leads to vasoconstriction (Axelsson and pressure to track the lower PA pressure throughout systole, and Franklin, 2001), presumably through an α-adrenergic there was no possibility of a shunt. Similar pressure profiles in response. If the pulmonary outflow tract behaves similarly the RV and pulmonary outflow tract, reflecting activity and (which we do not know at this point), then adrenergic inactivity of the cog-wheel valve, have also been observed stimulation would promote shunting. This would not be during chronic recordings from the RV and PA of consistent with the effects of adrenergic stimulation on unanaesthetized, estuarine crocodiles (Axelsson and Franklin, shunting noted by Franklin and Axelsson (2000) nor with its 1997). Thus, while we agree with Franklin and Axelsson (2000) effects in animals; adrenergic stimulation in alligators that shunting in crocodilians is influenced by the relative increases systemic blood pressure (Shelton and Jones, 1991) to resistances in the lung versus systemic circulations and that the levels that may exceed the pressure that can be developed by major control site of this resistance is the subpulmonary conus, the RV, and disturbing instrumented alligators increases it does not appear that maintained tonus in the subpulmonary pulmonary blood flow and always terminates shunting (D. A.
conus induced by adrenergic withdrawal is adequate in itself to S., K. G. and D. R. J., unpublished observations). However, a elicit shunting; active contraction of the valve following muscle β-adrenergic mechanism that promoted vasodilation in the depolarization is required. In support of this, we provide direct pulmonary outflow tract would be in accord with a loss of the evidence correlating valve contraction and closure (the cog- shunt under adrenergic tone. Sustained, β-adrenergic dilation wheel valve ECG) with the secondary rise in RV pressure that of the pulmonary outflow tract could decrease the ability of the always precedes shunting (Figs 2, 3).
animal to shunt by two mechanisms. First, dilation of thepulmonary outflow tract may significantly increase blood flow into the PA during early systole, leaving only a small volume In our study, the wave of depolarization spread across the of blood in the RV. This may prevent the RV from developing RV from the apex towards the base with a conduction velocity the pressure required to initiate a P→S shunt. Second, dilation of 91 cm s–1. This conduction pattern is similar to that observed 1850 D. A. Syme, K. Gamperl and D. R. Jones by Christian and Grigg (1999) in crocodiles; however, the the lungs, and a shunt would not occur. Alternatively, if the conduction velocity they report (65 cm s–1) is much slower.
timing of cog-wheel valve contraction were shifted earlier in Differences in temperature or species may contribute to this systole, more of the cardiac output could be shunted back to discrepancy; Christian and Grigg (1999) do not report the temperature used in their experiments. The cog-wheel valve Vagal stimulation in alligators resulted in a marked decrease muscle, located at the base of the RV, appeared to be activated in the absolute delay and phase of the cog-wheel ECG by a depolarization that originated in the RV, and in each (Table 1). Further, Malvin et al. (1995) found that efferent vagal animal the two events were synchronized by a relatively stimulation in alligators promoted pulmonary vasoconstriction.
consistent delay. Approximately 90 % of the delay between the Both these responses would favour P→S shunting. However, RV ECG and the cog-wheel valve ECG appeared to reside at the integrated cog-wheel ECG was decreased under vagal the junction between the two muscle masses (Fig. 5). These stimulation (Table 1), which presumably reflects an inhibition data imply that a mechanism akin to AV nodal delay exists in of valve function and a decreased capacity to shunt. In turtle hearts, peripheral stimulation of the cut vagus or application of The existence of such a node and the physiological ACh causes the ventricular depolarization pattern to shift mechanism responsible for the delay have not previously been transiently from that seen during apnoea to that observed during described in the alligator heart. Burggren (1978) noted a breathing, and the absolute magnitude of the conduction delay similar phenomenon in turtles, where a delay of 200–300 ms is longer during breathing than during apnoea (Burggren, 1978).
existed between depolarization of the ventricle and the bulbus Both these changes would favour blood flow to the lungs cordis surrounding the PA. He attributed the delay to a slow (inhibit shunting), the latter by the mechanism we propose. In conduction velocity (2 cm s–1 or one-fifth to one-tenth of that our experiments, the effect of ACh on the conduction delay was of the ventricle) in the transition zone between the cavum variable (Table 1) and at present does not support a role for a venosum of the ventricle and the bulbus cordis. When direct effect of ACh on the timing of valve closure and shunting.
watching the contraction of the cog-wheel muscle in alligators, However ACh, like vagal stimulation, did appear to weaken the it appeared that the entire muscle mass contracted cog-wheel ECG, and this may inhibit shunting. Shelton and synchronously and, hence, it is unlikely that the delay we Jones (1991) did not see any effect of ACh administration on report was due to very slow propagation across the cog-wheel the relative timing of events in the left and right ventricles. In muscle mass itself. However, we did not measure conduction contrast, White (1970) noted that atropine injection reversed velocities across the small cog-wheel muscle to confirm this.
both the diving-induced bradycardia and the large RV–PA White (1968) alludes to an unpublished observation of a pressure gradient in alligators, suggesting that the shunt may 350 ms delay in alligators at 25 °C. In our alligators, the conduction delay averaged 248 ms at 30 °C. Pressurerecordings indicate that this is long enough to allow some PA flow during early systole (a rise in PA pressure), but short The cog-wheel valve in alligators is surrounded by a muscle enough to obstruct the PA before the full stroke volume is mass that is somewhat isolated from the RV. This muscle delivered to the lungs. Sufficient blood then remains in the RV produces a distinct ECG signal that is temporally separated by to cause a substantial secondary rise in RV pressure during the approximately 250 ms from the RV ECG measured at the base latter half of systole and, thus, P→S shunting (Figs 2, 3).
of the heart. The cog-wheel ECG signals the onset of valve The absolute conduction delay was not dependent on heart closure and obstruction of the pulmonary outflow tract, rate, but the phase was (Fig. 4A). Thus, changes in phase resulting in a phasic, secondary rise in RV pressure and a fall appear to be more indicative of changes in heart rate (diastolic in PA pressure, and sets up the haemodynamic conditions interval) than of changes in ECG delay. It may be that the required for P→S shunting. The extent of the delay could have absolute delay is maintained within a relatively constant range a major influence on RV pressure and the degree of shunting.
because the timing of cog-wheel valve closure relative to the Vagal and cholinergic stimulation had significant but varied onset of RV systole would be critical in controlling shunting, effects on the cog-wheel ECG. Both appeared to weaken cog- rather than the phase per se. The significant relationship wheel valve contraction, which may inhibit shunting. Vagal between phase and ECG delay (Fig. 4B) may simply reflect the stimulation decreased the delay between the RV and cog-wheel constancy of heart rates in these animals, such that any change ECGs, which could promote shunting. Although these latter in delay would translate directly into a change in phase.
results do not provide a clear picture of how autonomic Changing the delay of the cog-wheel ECG or the ECG nervous tone would control the extent of P→S shunting, they magnitude (strength of cog-wheel contraction) may be do, in combination with the results of Franklin and Axelsson mechanisms by which the degree of shunting can be controlled.
(2000) and Axelsson and Franklin (2001), provide strong The existence of specialized fibres whose conduction velocity evidence that nervous and/or humoral mechanisms acting on is under autonomic control is well established in reptilian the subpulmonary conus and valve can markedly influence the hearts (Burggren, 1978; Christian and Grigg, 1999; and magnitude of P→S shunting in crocodilians.
references therein). If the cog-wheel ECG occurred very latein ventricular systole, most of the RV output would be sent to Supported by NSERC grants to D.R.J. and D.A.S.
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