Although adriamycin (doxorubicin) is an effective antineoplasmic drug against a variety of cancers, its use is limited by the risk of developing congestive heart failure in a dose-dependent manner (1). Adriamycin-induced cardiomyopathy is characterized by the loss of myofibrils, distention of the sarcoplasmic reticulum, and vacuolization of the cytoplasm. Animal models of adriamycin-induced heart failure are characterized by decreased blood pressure and cardiac output, elevated plasma norepinephrine (NE) concentration, and decreased myocardial NE content (2). Although there is much evidence of increased cardiac NE spillover in heart failure (3,4), how chronic adriamycin treatment affects the local neuronal release of catecholamines at the cardiac sympathetic nerve terminals remains to be determined. The aim of the present study is to examine whether local neuronal release of catecholamines at the cardiac sympathetic nerve terminals is altered in rabbits with chronic adriamycin treatment. Because not only plasma NE, but also plasma epinephrine (Epi) concentration is elevated in congestive heart failure (5,6), we measured myocardial interstitial NE and Epi levels by means of a cardiac microdialysis technique (7-13).
Animal care proceeded in strict accordance with the guiding principles of the Physiological Society of Japan. Fourteen male rabbits (Japanese white; weight, 2.4-3.2 kg) were divided into control (CNT) (n = 6) and adriamycin-treated (ADR) (n = 8) groups. We injected 4 mg/kg adriamycin weekly from the marginal ear vein to achieve a cumulative dose of 24 mg/kg. This dose of adriamycin was reported to be sufficient to induce chronic heart failure in rabbits (2). A 2-week recovery period was allowed before performing cardiac microdialysis. None of the animals were lost during the course of adriamycin treatment.
The experimental animals were anesthetized with a mixture of urethane (500 mg/kg) and α-chloralose (80 mg/kg). Additional doses of these drugs were applied as necessary to maintain an appropriate level of anesthesia. The animals were intubated and ventilated with room air mixed with oxygen. Body temperature was maintained at around 37°C with a heating pad and lamp. Arterial blood pressure and heart rate were monitored through a catheter inserted via the right femoral artery. After midline thoracotomy and incision of the pericardium, a dialysis probe was implanted into the left ventricular free wall using a fine guiding needle. Heparin sodium (100 U/kg) was intravenously administered to prevent blood coagulation. Experimental animals were killed at the end of the experiments with an overdose of pentobarbital sodium. We then measured total ventricular mass and left ventricular mass, as well as verifying the position of the implanted dialysis probe in the middle layer of the left ventricular myocardium.
We measured dialysate NE and Epi concentrations as indices of myocardial interstitial NE and Epi levels, respectively. The materials and properties of the dialysis probe are described elsewhere (9). Briefly, a dialysis fiber (8 mm × 0.31 mm O.D. and 0.2 mm I.D.; PAN-1200, 50 000 molecular mass cut-off; Asahi Chemical, Tokyo, Japan) was glued contiguously at both ends to polyethylene tubing (25 cm × 0.5 mm O.D. and 0.2 mm I.D.). The dialysis probe was perfused with Ringer's solution at a rate of 10 μl/min. To allow the NE and Epi levels to reach a steady state, the dialysate sampling was started from 120 min after implantation of the dialysis probe (6). Taking into account the dead space volume between the dialysis membrane and sample tube, actual dialysate sampling lagged behind a given collection period by 1 min. After removing interfering compounds in the dialysate by an alumina procedure, we measured NE and Epi concentrations using high-performance liquid chromatography with electrochemical detection (Eicom, Kyoto, Japan).
We induced exocytotic release of catecholamines from the cardiac sympathetic nerve terminals by local administration of KCl (100 mM) through the dialysis probe (10,12,14). The dialysate was sampled for 6 min under control conditions and after the beginning of KCl administration. According to our previous studies, the dialysate NE and Epi concentrations reach maximum responses within the first 6 min of KCl administration (10,12). Arterial blood samples (0.5 ml) were collected before the KCl administration. The blood samples were centrifuged, and then plasma NE and Epi concentrations were measured using the alumina procedure.
All data were presented as means ± SD values. When the myocardial interstitial Epi level was undetectable, we treated it as a value of the detection limit of Epi (4 pg/ml or 200 fg/50 μl injection) in our liquid chromatographic system (9). We used the Mann-Whitney rank-sum test to examine differences in myocardial catecholamine levels, systemic circulatory parameters, and ventricular mass between the CNT and ADR groups (15). Differences were considered statistically significant when p < 0.05.
Myocardial interstitial NE levels are shown in the left panel of Fig. 1. Baseline NE levels did not differ between the CNT and ADR groups. The NE levels during the local KCl administration were significantly lower in the ADR group than in the CNT group. Shown in the right panel of Fig. 1 are myocardial interstitial Epi levels. Baseline Epi levels did not differ between the CNT and ADR groups. The Epi levels during the local KCl administration were significantly lower in the ADR group than in the CNT group. The Epi response to the local KCl administration was totally abolished in the ADR group.
Table 1 summarizes systemic circulatory parameters and ventricular masses obtained from the CNT and ADR groups. Changes in blood pressure, heart rate, and plasma NE and Epi concentrations were not statistically significant between the two groups. Neither total nor left ventricular mass differed between the CNT and ADR groups.
We have shown that the neuronal release of NE and Epi at the cardiac sympathetic nerve terminals in response to local KCl administration was suppressed in the ADR group when compared with the CNT group. The local KCl administration depolarizes the sympathetic nerve terminals and evokes exocytotic release of neurotransmitters independent of cardiac sympathetic nerve activity (10,12,14). Therefore, the present results indicate that the chronic adriamycin treatment impaired the ability of exocytotic release of catecholamines at the cardiac sympathetic nerve terminals. According to a cytofluorescence localization of adriamycin in the peripheral nervous system, intravenously injected adriamycin reaches the dorsal root ganglia, trigeminal ganglia, and cervical sympathetic ganglia (16). As the superior cardiac sympathetic nerve arises from the cervical sympathetic ganglia, a neurotoxic action of adriamycin on the cervical sympathetic ganglia would result in loss of small synaptic vesicles that contain catecholamines at the cardiac sympathetic nerve terminals. Therefore, the neurotoxic action of adriamycin may be involved in the development of heart failure, although adriamycin directly affects the myocardium and also induces cardiomyopathy (1,17).
The fact that the neuronal exocytotic release of catecholamines was suppressed more in the ADR group than in the CNT group was inconsistent with the increased cardiac NE spillover in heart failure (3,4). Methodological differences may account for the discrepancy. The myocardial interstitial catecholamine levels measured by cardiac microdialysis reflect local nerve terminal function, whereas the NE spillover may be affected not only by local nerve terminal function, but also by sympathetic nerve activity directed to the heart. Minatoguchi and Majewski (18) demonstrated that the NE release rate in response to spinal stimulation in pithed rabbits did not differ between control and adriamycin-treated groups, although the plasma NE level was elevated in the adriamycin-treated group. Tong et al. (19) demonstrated that a turnover rate of injected labeled NE in the heart was accelerated despite increased NE uptake in 6-week adriamycin-treated rats, suggesting that the NE synthesis was decreased in the late stage of heart failure. Takano et al. (20) showed that nerve fibers were atrophic in regions of reduced [123I-]meta-iodobenzylguanidine uptake in adriamycin-induced cardiomyopathy. These studies indicated that the increased sympathetic nerve activity to maintain systemic hemodynamics in adriamycin-induced heart failure would override the impaired local nerve terminal function of catecholamine release.
Arterial blood pressure, heart rate, plasma NE and Epi concentrations, and total and left ventricular mass did not differ between the CNT and ADR groups in the present study. However, Yoshikawa et al. (2) reported that plasma NE concentration and left and right ventricular mass were increased in the 16-week adriamycin-treated rabbits with the same cumulative dose of adriamycin (24 mg/kg). The present results indicated that nerve terminal function of exocytotic catecholamine release was impaired at the cardiac sympathetic nerve terminals before systemic circulatory parameters or ventricular mass showed significant changes by adriamycin treatment.
Although cardiac microdialysis was useful for analyzing the local NE and Epi release at the cardiac sympathetic nerve terminals, there are several limitations in the present study. First, as local KCl administration evokes the sustained depolarization of sympathetic nerve terminals, the results of exocytotic release might be different if other methods were applied such as electrical sympathetic nerve stimulation. Second, we investigated local Epi and NE release at cardiac sympathetic nerve terminals in anesthetized rabbits. Therefore, the possibility cannot be ruled out that the anesthesia altered systemic sympathetic nerve activity, and thereby masked differences in baseline myocardial interstitial NE and Epi levels between the ADR and CNT groups. Third, adriamycin-induced myocardial fibrosis may have affected the in vivo recovery rate of catecholamines. However, chronic adriamycin treatment partially suppressed the exocytotic NE release, whereas it totally abolished the exocytotic Epi release. Changes in the in vivo recovery rate might not account for the differential suppression between the NE and Epi responses in the ADR group.
In conclusion, chronic adriamycin treatment impaired the local exocytotic release of NE and Epi at the cardiac sympathetic nerve terminals. The impairment of neuronal catecholamine release became significant during a compensated stage of heart failure when systemic hemodynamics were relatively maintained. The neurotoxicity may be one of the mechanisms responsible for adriamycin-induced heart failure.
Acknowledgement: This study was supported by Research Grants for Cardiovascular Diseases (9C-1, 11C-3, 11C-7) from the Ministry of Health and Welfare of Japan, by a Health Sciences Research Grant for Advanced Medical Technology from the Ministry of Health and Welfare of Japan, by Special Funds for Encourage System of COE from the Science and Technology Agency of Japan, by a Ground-Based Research Grant for the Space Utilization promoted by NASDA and Japan Space Forum, by a Bilateral International Joint Research Grant from the Science and Technology Agency of Japan, by Grant-in-Aid for Scientific Research (B: 11694337, C: 11680862, 11670730), by Grant-in-Aid for Encouragement of Young Scientists (11770390, 11770391), and by a Grant provided by the Ichiro Kanehara Foundation.
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The symposium and the publication of this supplement were supported by an educational grant from Novartis Pharma K.K. Tokyo, Japan.