INTRODUCTION

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MULTIPLE NEURONAL SUBTYPES have been identified within the mammalian intrinsic cardiac nervous system, both anatomically (4, 6, 8, 10) and functionally (1, 2). It has been proposed that the intrinsic cardiac nervous system is important for the maintenance of adequate cardiac output (11), particularly in disease states such as myocardial ischemia (2). Canine intrinsic cardiac neurons display complex behavioral patterns that rely, to a considerable degree, on their cardiovascular sensory inputs. These inputs, in turn, are dependent on cardiovascular status (1, 2). It is known that the human cardiac efferent nervous system displays functional characteristics similar to those found in animals (5, 9). It remains to be established whether neurons in the human heart behave in a manner similar to that identified in animal models. Furthermore, we do not know what effects cardiopulmonary bypass (CPB) and cardioplegia have on the human intrinsic cardiac nervous system.

The present experiments were designed to determine whether human intrinsic cardiac neurons generate spontaneous activity and, if so, how they respond to altered cardiovascular status. Additionally, we sought to determine whether cardiac afferent inputs to the human intrinsic cardiac nervous system alter its behavior. Finally, we sought to determine whether CPB and cardioplegia exert deleterious effects on human intrinsic cardiac neuronal function.

	MATERIALS AND METHODS

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General methods. Ethical approval for human experimentation at our institution is reached through a clinical and layperson peer-reviewed process at the Queen Elizabeth II Hospital, Halifax, Nova Scotia, Canada. Once full ethical approval of the study was achieved, patients were approached for entry into the study in accordance with guidelines set forth by the Queen Elizabeth II Hospital Ethics Committee. To qualify for entry into this study, a patient needed to have angiographic evidence of a lesion in at least one major coronary artery that required the coronary artery bypass grafting (CABG) procedure (Table 1). Patients were excluded from this study if they had depressed left ventricular function, required another procedure combined with the CABG procedure, or refused to consent to the recording procedure. Ten patients scheduled for CABG procedures entered this study. Basic demographic data and comorbid variables were collected from each patient upon entry into the study. Left ventricular ejection fractions were determined in each patient by means of transthoracic or transesophageal echocardiography, radionucleotide imaging, or left ventriculography performed at time of selective coronary catheterization. Operative variables relating to pump and aortic cross- clamp times, times to extubation postprocedure, as well as intensive care unit and total length of stay were assessed for each patient.

Operative procedures. After instigation of general anesthesia, a midline sternotomy was performed to expose the heart, and the appropriate bypass conduit was harvested in the usual fashion for CABG. Once the pericardotomy was completed, baseline systemic and pulmonary artery pressures were recorded along with a two-lead (leads II and V₅) electrocardiogram (ECG). Patients were heparinized and underwent aorto-right atriocaval cannulation in case CPB compromised hemodynamics, so that CPB could be instigated immediately.

Recording neuronal activity. Fatty tissue on the lateral surface of the right atrium that contains the right atrial ganglionated plexus (RAGP) (4, 6, 10) was exposed. A tungsten recording microelectrode was employed to record the extracellular activity generated by right atrial neurons (7). The microelectrode had a shank diameter of 100 µm, an exposed tip of 5 µm, and an impedance of 9-11 M at 1,000 Hz. The electrode was held in place by a micromanipulator attached to a variable extension arm (Octopus 1; Medtronic, Minneapolis, MN) to stabilize its motion.

Intraoperative procedures. Once an active site was identified, various loci on the exposed epicardial surfaces of the right and left ventricles were touched gently. The epicardial mechanical stimuli so applied were insufficient to distort the heart and thus alter the position of the recording electrode tip. This procedure was performed to determine whether identified RAGP neurons received mechanosensory inputs from such epicardial regions. Then systemic arterial pressure was reduced or increased by ~30% after administration of nitroglycerin (50-100 µg iv boluses) or phenylephrine (50-100 µg iv boluses), respectively.

Data analysis. Cardiac variables were analyzed over 30-s periods of time before and during peak responses elicited by each of the interventions described above. Action potentials with signal-to-noise ratios greater than 3:1 generated within a locus of the RAGP were counted for 30-s periods of time to establish average activity immediately before and during maximal responses elicited by each intervention. Fluctuations in the amplitude of action potentials generated by individual neurons varied by <25 µV over several minutes, retaining their same configurations over time. Action potentials recorded in a given locus with the same configuration and amplitude were considered to be generated by the somata and/or dendrites of a single neuron. The means (± SE) of data recorded during control states as well as during each intervention were calculated. ANOVA and paired t-test with Bonferroni correction for multiple tests were employed for statistical analysis where appropriate. A significance value of P < 0.01 was used for these determinations.

	RESULTS

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Patient and operative variables. Patient demographics, comorbid variables, and medications are listed in Table 1. The average age of patients was 62 yr, with 80% of the patients being male. All patients had preserved left ventricular function. All but one patient received at least two grafts that consisted of a left internal mammary artery to the left anterior descending artery and either a radial artery and/or a reversed saphenous vein graft to the remaining diseased territories. Two patients were classified as "in-house" urgent cases, having been in the coronary care unit awaiting the CABG procedure. There were no complications arising from the intraoperative recording procedure. No in-hospital deaths occurred in this cohort of patients. The majority of patients was extubated within 4-7 h postoperatively. Both in-house urgent patients had prolonged hospital stays (17 and 18 days). One of these urgent patients developed left lower lobe pneumonia postoperatively. The other patient had low cardiac output postoperatively requiring low doses of dopamine and epinephrine for 24 h. The rest of the patients had uncomplicated recoveries.

Neuronal activity. Using the criteria mentioned above, we identified spontaneous activity generated by two to three intrinsic cardiac neurons in each patient before any intervention. Multiple neuronal activity was evidenced by the different amplitudes of their recorded action potentials (Fig. 1; Table 2). The configuration and amplitude of each identified neuronal unit did not vary over time, allowing for comparison of changes in neuronal activity before and after an intervention. The activity so identified was sporadic in nature and, for the most part, not related to a specific phase of the cardiac cycle (Fig. 1). Immediately before the CPB, the activity generated by right atrial neurons in anesthetized patients averaged 59 ± 11 impulses per minute. At that time, these patients' systemic arterial pressure was, on average, 90/56 mmHg (Table 2).

View larger version (61K): [in this window] [in a new window]   Fig. 1.   Continuous recording of the activity generated by right atrial neurons and cardiovascular indexes obtained before and after administering a bolus dose of phenylephrine into the circulation (arrow, bottom). Neuronal activity increased soon after this occurred and before any change in recorded cardiovascular variables was detected (note that a paper fold prevented recording partway through the record). ECG, electrocardiogram; AP, aortic pressure; PAP, pulmonary pressure (the same abbreviations are used in the other figures).

View larger version (86K): [in this window] [in a new window]   Fig. 2.   Neuronal responses elicited by a gentle touch of a locus on the left ventricular ventral epicardium (horizontal line below). A burst of activity was generated by right atrial neurons (neural, bottom) during application of this stimulus. Monitored cardiovascular variables remained unchanged.

View larger version (93K): [in this window] [in a new window]   Fig. 3.   A and B represent a continuous record of the ECG, AP, and right atrial neuronal activity obtained from the beginning of infusing cardioplegia solution until the point of ECG quiescence. Note that neuronal activity persisted in the presence of suppressed cardiac electrical activity (B). C: data obtained after cardioplegia infusion had been instituted for some time, just before the first distal anastomosis was performed.

View larger version (101K): [in this window] [in a new window]   Fig. 4.   A burst of activity was generated by right atrial neurons soon after the administration of protamine into the circulation commenced (arrow, bottom). Recorded cardiovascular indexes remained unchanged.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The results obtained in these experiments demonstrate that populations of human intrinsic cardiac neurons generate spontaneous activity in patients undergoing cardiac surgery after anesthesia is induced and a midline sternotomy is performed. These data support those derived from the canine model (7) in as much as, first, the activity generated by right atrial neurons was for the most part sporadic in nature and thus unrelated to the cardiac cycle (Fig. 1). Second, as has been found in experimental animals, populations of human intrinsic cardiac neurons receive inputs from ventricular mechanosensory neurites. Third, cardioplegia or CPB procedures do not appear to reduce the capacity of human intrinsic cardiac neurons to generate spontaneous activity after their discontinuance (Table 2). Fourth, the activity generated by human intrinsic cardiac neurons is dependent, in part, on cardiodynamic status.

Right atrial neurons were activated in most patients when limited loci on the ventral epicardial surfaces of either ventricle were touched briefly (Fig. 2). Presumably, not all investigated neurons responded to this intervention (Table 2) because not all of them received mechanosensory inputs from the epicardial areas investigated with local mechanical stimuli, as occurs in animal models (2, 7). The fact that human intrinsic cardiac neurons receive cardiac mechanosensory inputs may explain, in part, the fact that the activity generated by many identified right atrial neurons changed in concordance with alterations in cardiovascular status (Fig. 1). For instance, when systemic arterial pressure increased as a consequence of the administration of phenylephrine (representing an increase in afterload), neuronal activity increased. Likewise, when systemic arterial pressure decreased after systemic administration of nitroglycerin (representing a reduction in afterload and preload), neuronal activity decreased. These data support those derived from the canine model, in as much as animal intrinsic cardiac neurons are known to receive direct inputs from ventricular mechanosensory neurites and thus are sensitive to changing cardiodynamics (2, 7). In accord with that, most identified neurons became inactive when systemic arterial pressure fell below 60 mmHg. Presumably, that was primarily due to a relative reduction of cardiac mechanosensory inputs to the intrinsic cardiac nervous system. This agrees with the fact that neuronal activity was lowest during cardiac standstill when the ECG was quiescent.

Administering the -adrenergic agonist phenylephrine increased right atrial neuronal activity concomitant with increases in systemic arterial pressure (Table 2). These neuronal responses accompanied changes in monitored cardiovascular indexes, presumably reflecting increased sensory inputs as well as increasing inputs arising from central reflexes as a result of global changes in cardiovascular status. In some patients, activation of right atrial neurons occurred even before any detectable changes in monitored cardiovascular indexes became evident (Fig. 1). Populations of canine intrinsic cardiac neurons are known to be sensitive to exogenous applied -adrenoceptor agonists (3). Thus -adrenoceptor agonists may directly affect some human intrinsic cardiac neurons. Human intrinsic cardiac neurons also proved to be sensitive to protamine (Fig. 4). The fact that some atrial neurons were modified by this peptide is in accord with the fact that canine intrinsic cardiac neurons are sensitive to multiple chemicals, including peptides and amino acids (2).

Because intrinsic cardiac neuronal activity was restored to baseline values by the end of bypass (Table 2), cardioplegia and CPB do not appear to adversely affect the capacity of human intrinsic cardiac neurons to generate spontaneous activity. We have proposed that proper functioning of the final common regulator of cardiac behavior, the intrinsic cardiac nervous system, may be important in the maintenance of adequate cardiac output (11). This aspect of the human intrinsic cardiac nervous system may be relevant with respect to modifying cardiac function in the perioperative period.

Perspectives