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The Journal of Physiology logoLink to The Journal of Physiology
. 1999 Aug 1;518(Pt 3):857–866. doi: 10.1111/j.1469-7793.1999.0857p.x

Role of protons in activation of cardiac sympathetic C-fibre afferents during ischaemia in cats

Hui-Lin Pan *,†, John C Longhurst ‡, James C Eisenach *, Shao-Rui Chen *
PMCID: PMC2269450  PMID: 10420020

Abstract

  1. Chest pain caused by myocardial ischaemia is mediated by cardiac sympathetic afferents. The mechanisms of activation of cardiac afferents during ischaemia remain poorly understood. Increased lactic acid production is associated closely with myocardial ischaemia. The present study examined the role of protons generated during ischaemia in activation of cardiac sympathetic C-fibre afferents.

  2. Single-unit activity of cardiac afferents innervating both ventricles was recorded from the left sympathetic chain in anaesthetized cats. Epicardial tissue pH was measured within 1-1.5 mm of the surface by a pH-sensitive needle electrode. Responses of cardiac afferents to myocardial ischaemia, lactic acid, sodium lactate, acidic phosphate buffer and hypercapnia were determined.

  3. Occlusion of the coronary artery for 5 min decreased epicardial tissue pH from 7.35 ± 0.21 to 6.98 ± 0.22 (P < 0.05). Epicardial placement of isotonic neutral phosphate buffer, but not saline, prevented the ischaemia-induced decrease in epicardial pH. This manoeuvre significantly attenuated the response of 16 afferents to 5 min of ischaemia (1.56 ± 0.23 pre-treatment vs. 0.67 ± 0.18 impulses s−1). Topical application of 10-100 μg ml−1 of lactic acid, but not sodium lactate, concentration-dependently stimulated 18 cardiac afferents. Inhalation with high-CO2 gas failed to activate 12 separate cardiac afferents. Furthermore, lactic acid stimulated cardiac afferents to a greater extent than acidic phosphate buffer solution, applied at a similar pH to the same afferents.

  4. Collectively, this study provides important in vivo evidence that protons contribute to activation/sensitization of cardiac sympathetic C-fibre afferents during myocardial ischaemia.

Chest pain, or angina pectoris, is one of the hallmarks of myocardial ischaemia although ‘silent’ ischaemia (lack of pain perception) also occurs in some patients with coronary artery disease. Sympathetic and vagal nerves innervating the heart contain not only autonomic efferent axons but also afferent fibres that transmit signals generated by cardiac sensory receptors (White, 1957; Cervero, 1994). Cardiac primary afferents running in the sympathetic nerves, especially finely myelinated Aδ- and unmyelinated C-fibre afferents, generally are considered to be the essential pathways for transmission of cardiac nociception to the central nervous system during myocardial ischaemia (White, 1957; Baker et al. 1980; Cervero, 1994). In this regard, removal of both stellate ganglia and excision of the first to the fifth thoracic sympathetic ganglia relieves cardiac pain in patients with ischaemic heart disease (White, 1957). Occlusion of coronary arteries also produces severe pain and pseudaffective reactions in dogs and cats, which can be abolished by thoracic sympathectomy but not vagotomy (Moore & Singleton, 1935; Brown, 1967). There is ample evidence demonstrating that myocardial ischaemia excites a subgroup of cardiac sympathetic afferents, namely, ischaemically sensitive afferents, which transmit nociceptive information to the central nervous system to elicit cardiac pain perception (Brown, 1967; Nishi et al. 1977; Bosnjak et al. 1979; Chandler et al. 1989,1998; Pal et al. 1989). Furthermore, activation of cardiac sympathetic afferents during ischaemia is known to initiate neural reflexes, which lead to haemodynamic alterations and arrhythmias (Malliani et al. 1969). Although adenosine was initially considered as the metabolite responsible for activation of cardiac afferents during ischaemia (Thames et al. 1993; Gnecchi-Ruscone et al. 1995), several recent studies have failed to demonstrate that adenosine is capable of stimulating cardiac sympathetic afferents (Pan & Longhurst, 1995; Veelken et al. 1996; Abe et al. 1998). Thus, the mechanisms of activation of cardiac nociceptors during ischaemia remain unclear.

Under physiological conditions, extracellular hydrogen ion concentrations are regulated within a very narrow range, and buffering of protons in the extracellular space minimizes changes in pH around sensory nerve endings (Poole-Wilson, 1978). It has long been recognized that myocardial ischaemia is associated with local acidosis, and that both intracellular and extracellular pH fall markedly during ischaemia (Opie et al. 1973; Poole-Wilson, 1978). Protons have been known to play a dominant role in excitation/sensitization of cutaneous nociceptors, pulmonary vagal afferents and abdominal sympathetic afferents (Steen et al. 1992, 1995; Stahl & Longhurst, 1992; Hong et al. 1997). Furthermore, intracoronary injection of lactic acid elicits a pseudaffective response in lightly anaesthetized animals (Guzman et al. 1962). However, the contribution of endogenously accumulated protons to activation of cardiac sympathetic afferents during ischaemia has not been studied directly. In the present study, by directly recording single-unit activity of cardiac sympathetic C-fibre afferents, we tested the hypothesis that protons produced during myocardial ischaemia play a role in activation of ischaemia-sensitive cardiac afferents.

METHODS

Surgical preparations

The surgical preparations and experimental protocols were approved by the Animal Care and Use Committee at Wake Forest University School of Medicine and the University of California at Davis. Adult cats of either sex were anaesthetized with ketamine (30 mg kg−1, i.m.) and anaesthesia was maintained with α-chloralose (60-80 mg kg−1, i.v.). Supplemental doses of α-chloralose (5-10 mg kg−1) were given as necessary to maintain an adequate depth of anaesthesia, assessed by lack of nociceptive reflexes and fluctuation of blood pressure and heart rate. A femoral artery and vein were cannulated for measurement of pressure and administration of fluids and drugs, respectively. The trachea was intubated and respiration maintained artificially with an animal ventilator (model CIV-101, Columbus Instruments, Columbus, OH, USA). The left carotid artery was cannulated with a Millar catheter (catheter-tip pressure transducer), which was passed retrogradely into the left ventricle for monitoring the left ventricular pressure. A PE 60 catheter was also introduced into the left atrium through the left atrial appendage for intracardiac injection of drugs. Arterial blood pressure was measured with a pressure transducer (model PT300, Grass Instruments). Arterial blood gases were analysed with a blood gas analyser and maintained within physiological limits (PO2 > 100 mmHg, PCO2 35-40 mmHg, pH 7.35-7.45). When necessary, arterial PO2 was increased by enriching the inspired O2 supply; pH was corrected by administering NaHCO3 (1 M, i.v.) and/or adjusting ventilation. The haemodynamic parameters were monitored and maintained stable (mean arterial pressure: 70-99 mmHg; heart rate: 110-130 beats min−1; left ventricular end-diastolic pressure: 3-4 mmHg) throughout the experiment. Blood from a donor cat or 6 % dextran was infused when necessary to keep the haemodynamics in the above range. Body temperature was maintained in the range 37-38°C with a circulating water heating pad and heat lamps. Animals were killed at the end of experiments by an intravenous injection of an overdose of sodium pentobarbital.

Recording of cardiac sympathetic afferents

A midline sternotomy was performed and the first to seventh left ribs and the upper lobe of the left lung were removed. The fascia overlying the left paravertebral sympathetic chain from T2 to T6 was removed. The chain and rami communicantes were then draped over a plastic platform and covered with warm mineral oil. Small nerve filaments were teased gently from the chain or rami communicantes between T2 and T5 under an operating microscope (model M900, D.F. Vasconcellos S.A., São Paulo, Brazil). The rostral end was placed across a recording electrode, which was connected to a high impedance probe. The nerve filaments were dissected gradually until single-unit activity of a cardiac afferent nerve was isolated (Pan & Longhurst, 1995; Huang et al. 1995; Tjen-A-Looi et al. 1998). The action potential of the afferent was amplified and processed through an audioamplifier (model AM8, Grass Instruments) and displayed on an oscilloscope (model 450, Gould). The neurogram, blood pressure and left ventricular pressure were simultaneously monitored on a recorder (model K2G, Astro-Med, W. Warwick, RI, USA). In addition, afferent nerve activity was fed into a Pentium computer through an analog-to-digital interface card for subsequent off-line quantitative analysis. Discharge frequency was quantified by using a data acquisition and analysis software (DataWave Technology, Inc., Longmont, CO, USA) and a histogram was created for each afferent nerve. Accurate counting of the afferent nerve discharge frequency was verified for each afferent by comparing the constructed histogram with the original neurogram. The precise location of afferent nerve endings was confirmed using a stimulating electrode placed directly on the receptive field of the afferent to electrically evoke the action potential of the afferent fibre. We have found that this method is the most accurate means of locating afferent nerve endings on the beating heart (Pan & Longhurst, 1995; Tjen-A-Looi et al. 1998). Conduction time was determined by measuring the time interval from the signal of electrical stimulation to the recording of the action potential from the evoked afferent. Conduction distance was estimated from the receptive field along the course of the inferior cardiac nerve through the left stellate ganglion, and to the recording electrode following the course of the sympathetic chain (Kuo et al. 1984; Pan & Longhurst, 1995). C- and Aδ-fibre afferent nerves were classified as those with a conduction velocity < 2.5 and 2.5-30 m s−1, respectively (Pan & Longhurst, 1995; Huang et al. 1995; Tjen-A-Looi et al. 1998).

Measurement of epicardial pH

Epicardial tissue pH was measured with a pH-sensitive needle electrode (20 gauge, 0.9 mm o.d., model 401, Micro Probe, Hamden, CT, USA), which has a response time of < 3 s. The needle electrode was connected to an ATI Orion digital pH meter (model 330, Boston, MA, USA) and was calibrated in vitro using standard pH solutions (7.40, 6.85 and 6.40), as described previously (Stahl & Longhurst, 1992). The electrode was then inserted into the myocardium within 1-1.5 mm of the surface. The pH electrode was inserted into the region of epicardium perfused by the left anterior descending coronary artery or the left circumflex coronary artery since most ischaemically sensitive afferent nerve endings are located in these two areas when recording of afferent nerve activity is performed with fibres in the left sympathetic chain from T2 to T5 (Pan & Longhurst, 1995; Tjen-A-Looi et al. 1998). The output voltage signal from the pH meter was recorded continuously on the chart recorder.

Experimental protocols

Epicardial pH during myocardial ischaemia

This protocol was utilized to determine the epicardial pH changes during ischaemia since most cardiac sympathetic afferent nerve endings are distributed near the epicardial surface (Baker et al. 1980; Barber et al. 1984; Pan & Longhurst, 1995). This protocol was also used to evaluate the effect of topical placement of neutral phosphate buffer on ischaemia-induced epicardial pH changes. The pH electrode was allowed to stabilize for at least 45 min to minimize pH changes due to tissue damage caused by insertion of the electrode (Stahl & Longhurst, 1995). Regional myocardial ischaemia was induced by constricting the corresponding coronary vessel with a thread placed around the coronary artery. In six animals, epicardial pH in the ischaemic and non-ischaemic zones was measured continuously during 5 min of control, 5 min of ischaemia and 2 min of reperfusion. Repeat myocardial ischaemia was induced 30-45 min after the first period of ischaemia in the presence of a topical application of saline to ensure that the ischaemia-induced pH changes were reproducible. In another seven animals, after the first period of myocardial ischaemia, a 2 cm2 filter paper patch pre-immersed in a solution of isotonic neutral phosphate buffer was placed directly on the epicardium where the pH electrode was inserted. Isotonic neutral phosphate buffer was prepared by combining 140 mM Na2HPO4 and 135 mM NaH2PO4. The pH (7.4) and osmolarity (290 mosmol (l H2O)−1) of the phosphate buffer were measured and adjusted with use of an ATI Orion digital pH meter and a μOSMETER (Precision Systems, Inc., Natick, MA, USA), respectively. Five minutes of myocardial ischaemia was reinduced 30 min later to determine the changes in epicardial pH during ischaemia in the presence of buffer solution.

Effects of exogenous protons on cardiac sympathetic afferents

This protocol examined the effect of protons derived from various sources on ischaemically sensitive cardiac sympathetic afferents. The effect of the acidic phosphate buffer on cardiac afferents was compared with the response of afferents to lactic acid, sodium lactate and hypercapnia. The differential effect of various sources of protons on ischaemically sensitive and insensitive cardiac afferents was also investigated. After the location of the receptive field of the afferent nerves was confirmed, the ischaemically sensitive or insensitive afferent fibres were identified following 5 min of regional myocardial ischaemia. Myocardial ischaemia was induced by constricting the coronary artery supplying the receptive field of cardiac ventricular afferent nerves with a thread placed around the vessel. Under an operating microscope, ligatures were placed around the proximal left anterior descending or left circumflex coronary artery, with care being taken not to disrupt nerve fibres that course along the vessel. Lactic acid (10-100 μg ml−1, Sigma), acidic phosphate buffer (pH = 5.42), or vehicle (normal saline) was applied, in random order, to the receptive field of afferent nerves using a cotton-tipped applicator soaked with these solutions or injected (2 ml) into the heart through the left atrial catheter (Pan & Longhurst, 1995). Allowing for dilution during application of lactic acid from the epicardial surface into the interstitial space where the nerve endings are located, the concentrations of lactic acid are approximately within the range occurring during tissue ischaemia (Stahl & Longhurst, 1992). Sodium lactate (100-300 μg ml−1, Sigma) was applied or injected into the heart to determine if afferents respond to lactic acid rather than to the dissociated lactate ions. Receptive fields were washed with saline and blood pH was corrected after each chemical had been administered. Furthermore, since high tissue CO2 during myocardial ischaemia also constitutes a source of protons (Opie et al. 1973), we studied the effect of hypercapnia on cardiac afferents. After ischaemically sensitive cardiac afferents were identified, the animal was ventilated with gas containing a high percentage of CO2 (12 % CO2, 21 % O2, and N2 balance) for 5 min while the impulse activity of the afferent was recorded continuously (Stahl & Longhurst, 1992). In addition, to investigate further the possible differential effect of protons on ischaemically sensitive versus ischaemically insensitive cardiac afferents, the latter group of cardiac afferents was subjected to topical application or intracardiac injection of lactic acid. The epicardial pH was measured during hypercapnia and during topical application of the test solutions.

Role of endogenously produced protons in activation of cardiac sympathetic afferents during myocardial ischaemia

In 18 animals, after the receptive fields of the afferents were located precisely, ischaemically sensitive cardiac afferents were identified following 5 min of myocardial ischaemia. A 2 cm2 filter paper patch was immersed in solution of isotonic neutral phosphate buffer and then placed directly on the epicardium where the afferent nerve ending was located. We have found in our previous studies that ischaemically sensitive afferents usually have one receptive field, and the size of the receptive field of cardiac afferent nerves is generally < 0.5-1 cm (Pan & Longhurst, 1995; Huang et al. 1995; Tjen-A-Looi et al. 1998). Five minutes of myocardial ischaemia was repeated 30-45 min later to assess the response of the afferent nerve fibre to ischaemia in the presence of saline on the receptive field of the afferent. In a separate group of animals (n = 14 animals), the response of cardiac sympathetic afferents to 5 min of ischaemia was determined before and after application of a 2 cm2 filter paper patch saturated with normal saline on the receptive field of the afferent. This procedure was used to establish a proper vehicle control to show that alteration of the afferent response to ischaemia following application of phosphate buffer was not caused simply by dilution of other metabolites. We have documented that two 5 min periods of myocardial ischaemia, separated by 30-45 min, induce reproducible responses from cardiac afferents without damaging or sensitizing the nerve endings (Pan & Longhurst, 1995; Tjen-A-Looi et al. 1998). We did not measure epicardial pH and afferent nerve activity at the same time due to the interfering electrical noise from the pH meter. In addition, dichloroacetate has been reported to decrease myocardial lactic acid accumulation by stimulating pyruvate dehydrogenase during partial occlusion of the coronary artery in dogs (Sakai et al. 1990). Thus, we evaluated the effect of systemic administration of dichloroacetate (200-600 mg, i.v.) on myocardial lactic acid concentrations and epicardial pH in our feline model of myocardial ischaemia in nine cats.

Data analysis

Results are given as means ±s.e.m. The discharge activity of afferents was averaged during a 5 min pre-ischaemic control period, a 5 min period of myocardial ischaemia and a 2 min period after reperfusion (Pan & Longhurst, 1995; Huang et al. 1995; Tjen-A-Looi et al. 1998). Afferents were considered to be ischaemically sensitive if their discharge frequency during 5 min of myocardial ischaemia was increased and was sustained at least twofold above baseline activity (Pan & Longhurst, 1995; Tjen-A-Looi et al. 1998). The response of afferents to lactic acid, hypercapnia, acidic phosphate buffer and sodium lactate was measured by averaging discharge rates during the entire period of the response of afferents. Comparisons between control and experimental interventions were made either by Student's paired t test or a repeated measures analysis of variance followed by Duncan's post hoc test. Differences were considered to be statistically significant when P < 0.05.

RESULTS

Acid-base status and haemodynamic profiles

The acid-base balance (arterial blood gas) was kept in the following range during the experiments: PCO2, 35-40; HCO3−, 22-28 mmol l−1; and pH, 7.36-7.42. The haemodynamic parameters throughout the experiments were as follows: mean arterial pressure, 78 ± 15 mmHg; heart rate, 117 ± 11 beats min−1; left ventricular end-diastolic pressure, 3 ± 1 mmHg. Afferent recordings from three animals were eliminated due to ventricular fibrillation during the 5 min period of ischaemia. Inhalation of hypercapnic gases significantly increased mean arterial blood pressure from 78 ± 15 to 158 ± 22 mmHg.

Epicardial pH during myocardial ischaemia

In seven animals, 5 min of myocardial ischaemia significantly decreased the epicardial pH (Fig. 1A). Topical application of phosphate buffer effectively prevented ischaemia-induced decreases in epicardial pH (Fig. 1B). In six other animals, topical placement of saline solution did not attenuate the decrease in epicardial pH caused by ischaemia, compared with that during the initial ischaemic period (Fig. 2). There was no significant difference between changes in epicardial pH induced by occlusion of the left anterior coronary artery (6.98 ± 0.21, n = 7 animals) or the left circumflex coronary artery (7.03 ± 0.23, n = 5 animals). In five additional animals, we measured epicardial interstitial pH in the right ventricle during 5 min of ischaemia. Epicardial pH decreased from 7.32 ± 0.14 during control to 6.98 ± 0.08 during 5 min of ischaemia, similar to that observed in the left ventricle (see Fig. 2).

Figure 1. Effect of isotonic neutral buffer on changes in epicardial pH induced by myocardial ischaemia.

Figure 1

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Original record showing the epicardial pH changes during 5 min of myocardial ischaemia in the presence of saline (A) and isotonic neutral phosphate buffer (B) in one animal.

Figure 2. Bar graph summarizing changes in epicardial pH during control and 5 min of ischaemia in the presence of saline and phosphate buffer.

Figure 2

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Columns and error bars represent means ±s.e.m.*P < 0.05 compared with pre-ischaemia control.

Exposure to hypercapnic gases decreased the arterial pH to 6.97 ± 0.23 and the epicardial pH to 6.95 ± 0.26 in six animals. Intracardiac injection of 2 ml of lactic acid decreased epicardial pH to 7.01 ± 0.27 (n = 9 animals), which was comparable to that measured during topical applications of lactic acid and during 5 min of ischaemia.

Effects of exogenous protons on cardiac sympathetic afferents

Five minutes of myocardial ischaemia increased the discharge frequency of 18 cardiac C-fibre afferent nerves from 0.22 ± 0.08 to 1.63 ± 0.24 impulses s−1. Topical application or intracardiac injection of lactic acid stimulated these afferent nerve fibres in a concentration-dependent fashion (Fig. 3 and Fig. 4). However, topical application or intracardiac injection of sodium lactate failed in all cases to stimulate the same afferents. The impulse discharge activity of these afferent fibres was 0.23 ± 0.08 and 0.22 ± 0.07 impulses s−1 before and after administration of sodium lactate, respectively.

Figure 3. Original representative tracings showing responses of an ischaemically sensitive cardiac afferent to topical application (↑) of lactic acid or sodium lactate.

Figure 3

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The afferent ending was located in the anterior left ventricle and had a conduction velocity of 0.46 m s−1. During topical applications of lactic acid at 20, 50 and 100 μg ml−1 and of sodium lactate at 100 μg ml−1, the epicardial pH values measured by the tissue electrode were 7.18, 7.03, 6.83 and 7.34, respectively.

Figure 4. Concentration-dependent responses of 18 ischaemically sensitive cardiac afferents to topical (epicardial) application of lactic acid.

Figure 4

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Data are presented as means ±s.e.m.*P < 0.05 compared with the discharge activity during control. During topical applications of lactic acid at 20, 50 and 100 μg ml−1, the epicardial pH values were 7.20 ± 0.04, 7.00 ± 0.04 and 6.80 ± 0.06, respectively.

We observed that inhalation of hypercapnic gases for 5 min in four animals effectively decreased the epicardial pH to 6.96 ± 0.21, which was similar to the values observed during 5 min of ischaemia. In 12 separate ischaemically sensitive afferents, inhalation of hypercapnic gases for 5 min did not activate any of these afferent nerve endings (Fig. 5) although the arterial blood pressure was increased significantly. The response of eight other ischaemically sensitive afferent nerves to acidic phosphate buffer was significantly less than their response to 50 μg ml−1 of lactic acid (Fig. 6), although the pH of these two solutions was identical (pH = 5.42). The epicardial interstitial pH was measured when lactic acid and acidic phosphate buffer at a similar pH (5.42) were topically applied. These two solutions caused an identical decrease in epicardial interstitial pH (7.0 ± 0.08 for lactic acid vs. 6.9 ± 0.08 for acidic phosphate buffer, n = 6 afferents). The locations of these 38 C-fibre afferent nerve endings are shown in Table 1.

Figure 5. Bar graph showing responses of 12 ischaemically sensitive cardiac afferents to 5 min of ischaemia and inhalation of high-CO2 gas.

Figure 5

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Columns and error bars represent means ±s.e.m.*P < 0.05 compared with pre-ischaemia control. Tissue pH values during ischaemia and hypercapnia were 6.98 ± 0.21 and 6.96 ± 0.21, respectively.

Figure 6. Bar graph showing responses of 8 ischaemically sensitive cardiac afferents to topical application of lactic acid or isotonic phosphate buffer.

Figure 6

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With both lactic acid (50 μg ml−1), and isotonic phosphate buffer, pH = 5.42. Columns and error bars represent means ±s.e.m.*P < 0.05 compared with the afferent activity during control. **P < 0.05 compared with afferent response to lactic acid. The epicardial interstitial pH was 7.0 ± 0.08 during topical application of lactic acid and 6.9 ± 0.08 during application of acidic phosphate buffer.

Table 1. Location of nerve endings of ischaemically sensitive cardiac sympathetic afferents.

Lactic acid (n = 18) Hypercapnia(n = 12) Acidic phosphate(n = 8) Phosphate buffer(n = 16) Saline (n = 14)
Left ventricle
 Anterior 7 3 2 6 5
 Posterior 4 6 5 2 2
Right ventricle
 Anterior 3 1 — 5 3
 Posterior 3 2 1 1 2
Apex 1 — — 2 2
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Topical application of 100 μg ml−1 of lactic acid only weakly activated 3 of 16 ischaemically insensitive cardiac afferent nerve fibres (increase in afferent nerve activity from 0.32 ± 0.11 to 0.56 ± 0.14 impulses s−1). The remaining 13 afferent nerves were unresponsive to topical application or intracardiac injection of lactic acid.

Role of endogenously produced protons in activation of cardiac sympathetic afferents during myocardial ischaemia

Figure 7 shows the response of an ischaemically sensitive cardiac afferent nerve to 5 min of ischaemia in the absence and presence of isotonic neutral phosphate buffer solution. The response of the afferent to ischaemia was reduced after placement of the buffer solution. For 16 cardiac sympathetic afferent fibres recorded in 15 animals, the initial 5 min of myocardial ischaemia led to a significant increase in discharge activity. Buffering the pH changes in the receptive field of these afferents with isotonic neutral phosphate buffer significantly attenuated the response of these afferents to repeated 5 min periods of ischaemia (Fig. 8A). In 14 other animals, the response of 14 cardiac afferent nerves to repeated 5 min periods of ischaemia was not significantly altered in the presence of saline solution, compared with that during the initial period of ischaemia (Fig. 8B). The epicardial pH was 7.02 ± 0.23 during 5 min of ischaemia in the absence of saline, and topical application of saline did not significantly change the epicardial tissue pH during ischaemia (7.02 ± 0.22, n = 6 afferents). The locations of these 30 C-fibre afferent nerve endings are shown in Table 1.

Figure 7. Representative histograms showing the discharge activity of a cardiac afferent during control, ischaemia and reperfusion before (A) and after (B) treatment with isotonic neutral phosphate buffer.

Figure 7

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The afferent ending was located in the anterior left ventricle and had a conduction velocity of 0.64 m s−1. Traces 1 and 2: original tracings of this afferent recorded at the times indicated by bars above histograms.

Figure 8. Bar graphs showing the response of cardiac sympathetic afferents to repeated 5 min periods of ischaemia in the absence and presence of isotonic neutral phosphate buffer (A) or saline (B).

Figure 8

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Columns and error bars represent means ±s.e.m.*P < 0.05 compared with respective pre-ischaemic control. **P < 0.05 compared with the initial afferent response to ischaemia.

In an attempt to determine more specifically the role of endogenous lactic acid in cardiac afferent activation during myocardial ischaemia, we measured both epicardial pH and lactic acid sampled from the coronary vein before and after treatment with dichloroacetate (600 mg, i.v., n = 9 animals). Before treatment with dichloroacetate, the epicardial pH decreased from 7.34 ± 0.11 during pre-ischaemic control to 6.96 ± 0.08 during 5 min of coronary artery occlusion. The lactic acid in coronary venous samples increased from 3.3 ± 0.6 during control to 7.5 ± 1.2 mM during 5 min of myocardial ischaemia. Treatment with dicholoracetate did not significantly attenuate ischaemia-induced changes in epicardial pH (from 7.32 ± 0.11 to 6.94 ± 0.07) and coronary venous lactic acid (from 3.2 ± 0.6 to 7.8 ± 1.3 mM). Thus, we were unable to use this approach to study further the role of endogenous lactic acid in ischaemia-induced cardiac afferent activation.

DISCUSSION

We focused our current study on C-fibre afferents because the heart is innervated predominantly by sympathetic C-fibre afferents (Pan & Longhurst, 1995; Huang et al. 1995; Tjen-A-Looi et al. 1998). In the present study, we only recorded three Aδ-fibre afferents whose nerve endings were in the heart. None of these fibres was responsive to 5 min of myocardial ischaemia or administration of lactic acid.

There are two important findings in the present study. First, we found that protons, probably derived from lactic acid, stimulated ischaemically sensitive cardiac afferent fibres in a concentration-dependent fashion. In this regard, hypercapnia, sodium lactate and acidic phosphate buffer either had no effect on ischaemically sensitive cardiac afferent endings or only slightly increased the discharge activity of these fibres. Furthermore, our data demonstrate that buffering the receptive field of afferents, which stabilizes epicardial pH, significantly attenuated the increase in discharge activity of cardiac sympathetic afferents induced by ischaemia. Therefore, the present electrophysiological study provides in vivo evidence for the first time that endogenously produced protons, most probably those derived from lactic acid, play an important role in activation of cardiac sympathetic C-fibre afferents during ischaemia.

Increased production of some metabolites during myocardial ischaemia has been proposed to contribute to excitation of nerve endings of primary cardiac sympathetic afferents (Nishi et al. 1977; Baker et al. 1980). For example, we have shown that bradykinin and free radicals, but not adenosine, contribute to activation of ischaemically sensitive cardiac sympathetic afferents (Pan & Longhurst, 1995; Huang et al. 1995; Tjen-A-Looi et al. 1998). However, in vivo myocardial ischaemia is a complex entity and many metabolites probably act in concert to activate cardiac sympathetic afferents. In this regard, elimination of the action of free radicals or blockade of kinin B2 receptors generally does not eliminate completely ischaemia-induced activation of cardiac sympathetic afferents (Huang et al. 1995; Tjen-A- Looi et al. 1998). Thus, we believe that other mechanisms are still present which account for activation/sensitization of cardiac afferents during ischaemia. Since lactic acid is produced in large quantities during myocardial ischaemia and because exogenous lactic acid elicits nociceptive responses in conscious animals (Guzman et al. 1962; Opie et al. 1973; Nishi et al. 1977), this metabolite has been long suspected of playing a role in myocardial ischaemia-induced chest pain. However, there is little in vivo evidence supporting the hypothesis that endogenously produced protons contribute to activation of cardiac nociceptors during ischaemia. Evidence that outward proton flux from myocytes occurs rapidly during ischaemia has been presented by Yan & Kleber (1992), who found that proton efflux during ischaemia, coupled with the poor buffering capacity of the extracellular versus the intracellular milieu, is sufficient to lower extracellular pH more than intracellular pH. Although it has been established previously that myocardial pH decreases during ischaemia (Poole-Wilson, 1978), epicardial pH changes during myocardial ischaemia have not been examined in particular. In the present study, we found that epicardial pH decreased progressively during 5 min of ischaemia. Because the nerve endings of cardiac sympathetic afferents are distributed near the epicardial surface (Baker et al. 1980), we measured the epicardial tissue pH changes, which are most relevant to the environment of cardiac sympathetic afferents during ischaemia. In preliminary studies, we performed extensive experiments using several approaches, including treatments with dichloroacetate (200-600 mg kg−1, i.v.) and acetazolamide (60-100 mg kg−1, i.v.) (data not shown) in an attempt to define further the role of lactic acid in activation of cardiac afferents during ischaemia. However, we could not demonstrate significant attenuation of ischaemia-induced alterations in epicardial pH and lactic acid by either dichloroacetate or acetazolamide. A previous study has shown that dichloroacetate effectively attenuated myocardial acidosis in a canine model of partial occlusion of the coronary artery (Sakai et al. 1990). We were unable to demonstrate such an effect in our feline model of complete occlusion of the coronary artery, and therefore the present study is limited by the lack of a suitable protocol to restrict the accumulation of lactic acid in our feline model of myocardial ischaemia.

Taking advantage of the anatomical distribution of the nerve endings of cardiac sympathetic afferents, we observed that epicardial application of isotonic phosphate buffer prevented the decrease in pH during ischaemia. Thus, buffering the epicardium pH provided a useful means of evaluating the role of endogenously produced protons in the activation of cardiac afferents during ischaemia. The role of protons from lactate ions and other sources in the activation of ischaemically sensitive cardiac afferents was determined in our study. The cause of acidosis during myocardial ischaemia is mainly due to the retention of acid metabolites. The major sources of protons during myocardial ischaemia are glycolysis associated with the production of lactic acid, generation of CO2, and abnormal lipid metabolism (Opie et al. 1973; Poole-Wilson, 1978). Steen et al. (1995) found that, at pathophysiologically relevant concentrations, protons played a dominant role in activating cutaneous afferents. Uchida & Murao (1975) reported that application of low concentrations (7.5-75 μg ml−1) of lactic acid preferentially stimulated cardiac unmyelinated C-fibre afferents but not myelinated afferents (the afferent response to ischaemia was not tested in this study). We have shown previously that exogenous lactic acid is capable of activating ischaemically sensitive afferent nerve endings innervating abdominal viscera (Stahl & Longhurst, 1992). The present study indicates that protons are primarily important in the activation of ischaemically sensitive afferents since sodium lactate (lactate ions) alone had no direct effect on this group of afferents. Our results also suggest that protons derived from lactic acid, but not from hypercapnia, stimulate cardiac sympathetic afferents in a concentration-dependent fashion. Unlike results from in vitro studies on cutaneous afferents (Steen et al. 1992, 1995), we found that, although hypercapnia caused a similar reduction in epicardial pH to ischaemia, it did not stimulate cardiac afferents. This finding is consistent with our previous studies showing that hypercapnia has no effect on abdominal visceral afferents (Stahl & Longhurst, 1992). It is unclear why the response of visceral afferents to protons derived from hypercapnia differs from that of cutaneous afferents. This discrepancy may be due to the difference in the biochemical transduction mechanism of the afferent nerve endings (i.e. the generator potential of receptors) and/or the tissue type in which the nerve endings reside. Furthermore, it remains uncertain why protons from different sources have a different effect on the cardiac nociceptors. This disparity has been demonstrated in other visceral afferents, including abdominal sympathetic afferents and pulmonary afferents (Stahl & Longhurst, 1992; Hong et al. 1997). The lactate and proton exchange in the nerve endings may be important in activation of cardiac afferents during ischaemia (Schneider et al. 1993). Thus, the combination of lactic ions and protons is probably a stronger stimulus for cardiac afferent endings, as has been demonstrated for pulmonary afferents (Hong et al. 1997). This notion is supported by our finding that lactic acid stimulated cardiac sympathetic afferents to a much greater extent than acidic phosphate buffer solution, although both solutions caused a similar decrease in epicardial pH when applied to the surface of the heart.

We observed that lactic acid stimulates ischaemically sensitive C-fibre afferent nerves but has a rare mild effect on ischaemically insensitive cardiac afferent nerves. The reason behind this unique action of lactic acid is not clear. A proton-induced current has been discovered in a subpopulation of rat dorsal root ganglion neurons, which is specifically gated by downward steps in extracellular pH (Bevan & Yeats, 1989; Peterson & LaMotte, 1993). Indeed, the acid-sensing ionic channel expressed in the dorsal root ganglion and central neurons has been cloned recently (Waldmann et al. 1997). This channel type may exist mainly in ischaemically sensitive sensory nerve endings (Rang et al. 1991; Peterson & LaMotte, 1993), and could provide a molecular basis for activation of this group of cardiac afferents.

In contrast to previous electrophysiological studies on cardiac afferents, our study assessed directly the role of endogenous protons in excitation of cardiac sympathetic afferents during ischaemia. Since the nerve endings of cardiac sympathetic afferents are mainly located near the epicardial surface and the isotonic neutral phosphate buffer was demonstrated to effectively prevent the epicardial pH change caused by ischaemia, this intervention was used to explore the role of protons in activation of cardiac afferents during ischaemia. Because topical saline application did not alter the response of cardiac afferents to ischaemia, it is unlikely that dilution of other metabolites contributed to the attenuating effect of the buffer on cardiac afferents. Although no histological measurements were made, we were confident that both the pH electrode and the receptive field of the afferents were located in the ischaemic zone for the following reasons. First, regional ischaemia was clearly indicated by visible cyanosis of the ischaemic zone during occlusion of the coronary artery. Furthermore, occlusion of the corresponding coronary artery always decreased the epicardial pH and increased the discharge frequency of afferents, confirming that the pH electrode and the afferent endings were indeed located in the ischaemic zone. In addition, in our experiments, all afferent nerve endings were precisely located on the surface of the heart using a stimulating electrode. Based on our previous experience (Pan & Longhurst, 1995; Huang et al. 1995; Tjen-A-Looi et al. 1998), the pH electrode was placed in the area in which most of the afferent endings are located (i.e. the anterior left ventricle). Thus, we believe that epicardial pH changes were closely related to afferent responses to ischaemia. This notion is also strongly supported by the data showing that buffering the epicardium (where the afferent ending was located) significantly attenuated the afferent responses to ischaemia.

We recognize that, in addition to its direct stimulating effect on cardiac afferents, accumulation of protons in the ischaemic myocardium also may sensitize cardiac afferents in response to other stimuli, as demonstrated for cutaneous afferents (Steen et al. 1992). Results from our study, however, cannot distinguish a stimulating vs. a sensitizing action of protons on cardiac afferents. It is likely that accumulating protons in ischaemic myocardium could contribute to activation of cardiac afferents through both direct and indirect actions. Furthermore, tissue ischaemia is a complex process and many ischaemic metabolites are produced during myocardial ischaemia. It is clear from the present study that endogenously produced protons are not entirely responsible for activation of cardiac sympathetic afferents during ischaemia since buffering the receptive field of afferents did not completely abolish the response of afferent endings to ischaemia. Considering the results from previous studies (Huang et al. 1995; Tjen-A-Looi et al. 1998), it is possible that protons interact synergistically with many other metabolites, such as oxygen-derived free radicals, bradykinin and prostaglandins, to fully stimulate cardiac sympathetic afferents during ischaemia. These ischaemic metabolites also may play a distinct role in afferent activation during different phases of ischaemia. For example, previous studies have shown that bradykinin is generated rapidly during brief ischaemia (Kimura et al. 1973), which may account for a short latency in the activation of afferent endings during ischaemia. Data shown in Figs 1 and 7 appear to suggest that protons play a more important role in activation of cardiac afferents when ischaemia is prolonged to more than 1-2 min. Further studies are warranted to determine precisely the role of their interactions in ischaemia-induced activation of cardiac afferents.

In conclusion, this study has provided important in vivo evidence that protons, probably derived from lactic acid but not from tissue hypercapnia, contribute to activation/sensitization of cardiac sympathetic C-fibre afferents during myocardial ischaemia. Our data suggest that increased production of protons/lactic acid during myocardial ischaemia is responsible for stimulation of the nerve endings of cardiac sympathetic afferents, which triggers perception of chest pain and, through a reflex mechanism, evokes excitatory cardiovascular responses and tachyarrythmias.

Acknowledgments

This study was supported by grant GS-30 (H.-L.P.) from the American Heart Association, Mid-Atlantic Affiliate, and by grants HL-60026 (H.-L.P.) and HL-52165 (J.C.L.) from the National Institutes of Health.

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