INTRODUCTION

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CLINICAL STUDIES AND DATA obtained from animal experimentation indicate that insulin-dependent diabetes mellitus (IDDM) is associated with a reduced ability to regulate heart rate. Diabetic patients have an impaired positive chronotropic response to exercise (7, 17), and streptozotocin (STZ)-induced diabetes in animals alters both basal heart rate (4, 14) and chronotropic responses to adrenergic and cholinergic stimulation (2, 3, 14). Diabetic rats present a resting bradycardia (4, 14), and right atria isolated from this animal model show a depression in basal spontaneous pacemaker rate (14) and an increased sensitivity to the negative chronotropic actions of cholinergic agonists (2, 3). Data regarding the adrenergic responsiveness of isolated right atria are conflicting. Ramanadham and Tenner (14) reported a decreased maximum response to isoproterenol, whereas Foy and Lucas (5) found that the chronotropic responses to norepinephrine and isoproterenol were slightly increased.

The majority of available data from the STZ-treated rat model of IDDM has been obtained after treatment with 60-65 mg/kg STZ, a dose associated with severely elevated blood glucose and ketoacidosis. An elevation of ketone bodies has been observed as early as 1 wk after treatment with 60-65 mg/kg STZ (11, 12). Thus it is not possible to determine whether observed cardiac changes are the result of hyperglycemia or a ketoacidotic state. In contrast, lower doses of STZ have been shown to elevate blood sugar without producing a severe ketoacidosis; in one study, urinary ketone bodies were not detected 8 wk after administration of a 50 mg/kg dose (13). The present study was designed to determine whether STZ-induced IDDM-mediated changes in basal pacemaker rate and autonomic responsiveness of right atria isolated from rats are dependent on the presence of a ketoacidotic state. Diabetes was induced using 45, 50, and 65 mg/kg STZ to provide hyperglycemic models with and without ketoacidosis.

	METHODS

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All experiments were conducted in accordance with institutional guidelines and the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals [DHHS Publication No. (NIH) 85-23, Revised 1986, Office of Science and Health Reports, Bethesda, MD 20892].

Animal model. Male Sprague-Dawley rats weighing 300-350 g were maintained in the Division of Laboratory Medicine at the University of Arkansas for Medical Sciences with free access to food and water. Seven days after arrival, animals were randomly separated into either seven or three groups. Initial experiments were conducted in seven groups: 1) naive controls; 2) diluent-treated controls; 3) body weight controls; 4) 45 mg/kg STZ-induced diabetics; 5) 65 mg/kg STZ-induced diabetics; 6) insulin-treated, 45 mg/kg STZ-induced diabetics; and 7) insulin-treated, 65 mg/kg STZ-induced diabetics. Subsequent studies utilized three groups: 1) diluent-treated controls; 2) 50 mg/kg STZ-induced diabetics; and 3) insulin-treated, 50 mg/kg STZ-induced diabetics. Diabetes was induced by intravenous (via tail vein) administration of STZ (dissolved in 0.05 M citrate buffer, pH = 4.5) and confirmed by sustained blood glucose values of 300 mg/dl and above; all STZ-treated animals were diabetic within 1 wk after the injection. Diluent-control rats were given an equivalent volume of diluent (1 ml/kg iv). Body weight controls were food restricted in order to maintain body weight at a level similar to that of the diabetic animals. The food-restricted rats were provided 12-18 g of chow daily with body weight being monitored each week. Insulin treatment (5-7 U sc extended insulin-zinc suspension daily) was initiated 5 wk after the administration of STZ and continued for the remainder of the 12-wk study. Previous studies had shown that resting heart rate was significantly diminished in diabetic rats at this 5-wk time point and that this bradycardia was reversible with insulin treatment (9). Blood glucose values were monitored before injection of STZ (or diluent) and at weekly intervals thereafter. Blood was obtained from a tail vein, and glucose was measured using an Accu-Check II monitor (Boehringer Mannheim Diagnostics, Indianapolis, IN). Serum ketones were measured at the end of the 12-wk period using Acetest tablets (Miles, Elkhart, IN). This test identified serum ketones as being in the negative (<10 mg/dl), small (20 mg/dl), moderate (30-40 mg/dl), or large (80-100 mg/dl) range.

Isolated right atrial preparations. Animals of each group were killed for in vitro experimentation 12 wk after the administration of STZ or diluent. This 12-wk interval was chosen because cardiac changes are frequently monitored in this animal model at this time. Rats were anesthetized with halothane (Halocarbon Laboratories, Hackensack, NJ), and hearts were removed and immediately perfused through the aorta with a Krebs-Henseleit (KH) solution of the following composition (in mM): 118.0 NaCl, 25.0 NaHCO₃, 4.8 KCl, 1.2 MgSO₄, 1.4 CaCl₂, and 11.1 glucose. The solution was buffered to pH 7.4 by saturation with 95% O₂-5% CO₂ gas, and temperature was maintained at 37°C.

After all detectable blood was washed from the hearts, entire right atria were isolated and allowed to beat spontaneously in a temperature-controlled (37°C) tissue bath containing the KH solution described above. Pacemaker frequency was monitored via tachographs (using signals from bipolar contact electrodes) and recorded continuously on a polygraph (Grass model 7, Grass Instruments, Quincy, MA). Atria were equilibrated for 90 min in the buffer solution before experiments were begun; during this period, the medium was replaced every 15 min. After equilibration, pacemaker rate was stable and, under control conditions, it did not change by more than 5 beats/min during time periods comparable to those used for experimental procedures.

Experiments were designed to evaluate the effect of diabetes with and without ketoacidosis on chronotropic responsiveness to adrenergic and cholinergic stimulation. Cumulative dose-response curves for the adrenergic agonists norepinephrine and isoproterenol and the cholinergic agonists acetylcholine and carbachol were obtained by sequential addition of drug to the bathing medium. Preparations used for adrenergic and cholinergic studies were isolated from separate animals. Adrenergic responsiveness was examined in the presence of 10⁶ M atropine; cholinergic responsiveness was monitored in the presence of 10⁶ M nadolol. These antagonists, which were added to prevent effects of possible local neurotransmitter release, had no effect on basal pacemaker rate (data not shown) but were found in preliminary studies to produce significant (at least 10-fold) rightward shifts in the dose-response curves for respective agonists.

When cumulative dose-response curves were obtained, the next higher concentration of agonist was added to the KH solution only after the tissue reached a steady-state response at the previous level (60-90 s). This time allowed evaluation of the maximum response with each dose and prevented possible shifts in the dose-response curve due to hydrolysis. The absence of such a shift or of significant agonist-induced acute desensitization was verified by comparing responses to given single concentrations of agonist with corresponding values on the cumulative dose-response curve and by comparing cumulative dose-response curves before and after exposure to a maximally effective concentration of agonist (data not shown).

To determine whether diabetes alters the rate of neurotransmitter removal, dose-response curves for norepinephrine and acetylcholine were also examined after the primary physiological mechanisms involved in termination of their action were inhibited. Effects of norepinephrine were monitored after pretreatment with cocaine, an inhibitor of neuronal reuptake. Cocaine (5 × 10⁶ M) was added to the bathing solution after the equilibration period; norepinephrine dose-response curves were obtained as described above 15 min after the addition of cocaine. Preliminary studies showed that the chosen concentration of cocaine increased norepinephrine sensitivity without altering basal pacemaker rate or the maximum response to -adrenoceptor stimulation. Similarly, chronotropic responsiveness to acetylcholine was determined after pretreatment with diisopropylfluorophosphate (DFP), an irreversible inhibitor of acetylcholinesterase (16). DFP (5 × 10⁵ M) was added to the bathing medium after the equilibration period; after a 15-min exposure, unbound DFP was removed by three washes with drug-free buffer. Pacemaker frequency was reduced significantly by DFP in all preparations (see below); higher concentrations or longer exposure periods eliminated spontaneous activity in some preparations. Acetylcholine dose-response curves were obtained as described above after pacemaker rate became stable in these DFP-treated tissues.

Materials. Acetylcholine chloride, atropine sulfate, carbachol chloride, cocaine, DFP, dl-isoproterenol hydrochloride, nadolol, dl-norepinephrine bitartrate, and STZ were purchased from Sigma Chemical (St. Louis, MO). Extended insulin-zinc suspension (bovine-porcine; 100 U/ml) was purchased from Eli Lilly (Indianapolis, IN). All other chemicals were reagent grade.

Stock solutions of the adrenergic and cholinergic agonists were prepared in 0.01 N HCl immediately before each experiment. Control experiments demonstrated that the amount of HCl added to the bathing medium did not affect buffer pH or pacemaker frequency. Cocaine solution was prepared in double-distilled H₂O. DFP was diluted in dimethyl sulfoxide; the final concentration of dimethyl sulfoxide in the organ bath (<0.15%) did not affect pacemaker frequency or the response to agonists.

Statistical analysis. Data were analyzed by analysis of variance with Duncan's multiple range test or by Student's t-test where appropriate. Half-maximal effective concentration (EC₅₀) values were obtained by graphical evaluation of individual dose-response curves. All data are presented as means ± SE. Criterion for significance was a P value <0.05.

	RESULTS

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Body weight, blood glucose, and ketones. Table 1 shows mean body weights and blood glucose values for the seven groups included in the initial study. These data were obtained 12 wk after injection of STZ or diluent. As shown in Table 1, body weight in the 65 mg/kg STZ-induced diabetic group was lower than that in rats treated with 45 mg/kg STZ. The body weight control group had a weight similar to that in the 45 mg/kg STZ-induced diabetic group but greater than that in 65 mg/kg STZ-induced diabetic animals. Insulin treatment resulted in body weights greater than those in corresponding diabetic rats; however, weight was not totally restored to control values by 7 wk of insulin treatment.

Blood glucose was elevated 12 wk after administration of STZ, with the level observed in the 65 mg/kg STZ-induced diabetic group being greater than that in the 45 mg/kg STZ-treated group. Insulin treatment returned blood glucose to values that were not significantly different from those in the three control groups. As shown in Table 2, 45 mg/kg STZ-induced diabetic rats had serum ketone levels in the negative to small range, whereas the 65 mg/kg STZ-induced diabetic group showed significantly higher values, with ketone levels ranging from small to large. The assay for ketones was always negative in control animals and during insulin treatment.

Because initial results showed no difference in adrenergic and cholinergic responsiveness when comparing tissues isolated from the two groups of diabetic rats (45 and 65 mg/kg STZ), continued experiments used a single dose of STZ (50 mg/kg). This dose decreased body weight (301 ± 23 vs. 484 ± 28 g in controls) and increased blood glucose (397 ± 22 vs. 114 ± 10 mg/dl in controls) when measured 12 wk after administration of STZ or diluent. Blood glucose in the 50 mg/kg STZ-treated group was less than that in the 65 mg/kg group (493 ± 7 mg/dl) but was not significantly different from that in the 45 mg/kg group (418 ± 11 mg/dl). Body weight in the 50 mg/kg STZ-treated group was not significantly different from that in either the 45 mg/kg group (331 ± 12 g) or the 65 mg/kg group (242 ± 18 g). Insulin treatment of the 50 mg/kg STZ-treated rats (from weeks 5 to 12) returned both body weight (455 ± 17 g) and blood glucose (120 ± 14 mg/dl) to values that were not significantly different from control. Serum ketone levels were only slightly elevated in the 50 mg/kg STZ-induced diabetic animals after the 12-wk period. In a group of eight rats, three showed ketone levels to be negative (<10 mg/dl), four had values in the small range (20 mg/dl), and one showed a moderate level (30-40 mg/dl). The assay for ketones was always negative in control animals and after insulin treatment.

Basal spontaneous pacemaker rate. Initial experiments showed reductions in basal pacemaker rate in right atria isolated from animals treated with either 45 or 65 mg/kg STZ (Table 3). Spontaneous rate tended to be lower in preparations isolated from 65 mg/kg STZ-induced diabetic animals; however, values in the two groups were not significantly different. Basal rate did not differ significantly in the three control groups (naive, diluent treated, and body weight adjusted). The diabetes-associated depression in rate was reversible with insulin treatment in both the 45 and 65 mg/kg STZ-treated groups; values observed in preparations isolated from control and insulin-treated diabetic rats were not significantly different. Spontaneous rate was not affected by treatment with nadolol (1 × 10⁶ M) or atropine (1 × 10⁶ M) in any group.

Results of continued studies demonstrated a similar insulin-reversible reduction in rate in atria isolated from 50 mg/kg STZ-induced diabetic rats. Basal pacemaker rate was 339 ± 7, 280 ± 6, and 327 ± 6 beats/min in right atria isolated from control, 50 mg/kg STZ-induced diabetic and insulin-treated diabetic rats (n = 31-37/group), respectively.

Adrenergic responsiveness. Initial experiments examined the response to norepinephrine in right atria from control, 45 mg/kg STZ-induced diabetic, 65 mg/kg STZ-induced diabetic, and insulin-treated diabetic animals. As shown in Table 3, the dose-dependent positive chronotropic response was not different when compared among the seven groups. Neither the EC₅₀ value for norepinephrine nor the maximum response to this agonist was statistically different when compared among the various control, diabetic, and insulin-treated diabetic groups. As with basal rate, the maximum spontaneous rate observed during exposure to the adrenergic agonist was significantly reduced in atria from diabetic animals (574 ± 10, 476 ± 21, and 462 ± 6 beats/min in diluent/controls, 45 mg/kg STZ-induced diabetics and 65 mg/kg STZ-induced diabetics, respectively); however, the increase in rate above basal values was not different.

Because results indicated that the chronotropic response to -adrenoceptor stimulation was not different when compared in preparations isolated from 45 and 65 mg/kg STZ-treated rats, continued studies used a single dose of STZ. Experiments examined the responses to norepinephrine and the selective -adrenoceptor agonist isoproterenol in right atria isolated from control, 50 mg/kg STZ-induced diabetic, and insulin-treated, 50 mg/kg STZ-induced diabetic animals. As observed with norepinephrine in earlier experiments, the dose-dependent positive chronotropic responses to norepinephrine and isoproterenol were not affected by diabetes (Figs. 1 and 2). The EC₅₀ values for these two agonists were not altered by diabetes or insulin-treatment (Table 4). The maximum spontaneous rate observed during exposure to the adrenergic agonists was significantly reduced in atria from 50 mg/kg STZ-induced diabetic animals; however, the actual increase in rate above basal values was not different in the three groups. The reduction in maximum rate was reversible with insulin treatment.

View larger version (23K): [in this window] [in a new window]   Fig. 1.   Effect of norepinephrine on spontaneous rate in right atria isolated from control (filled circles), 50 mg/kg streptozotocin (STZ)-induced diabetic (squares), and insulin-treated diabetic (open circles) rats. Preparations (n = 7-8/group) were isolated 12 wk after administration of STZ or diluent and bathed in Krebs-Henseleit (KH) solution containing 106 M atropine. Dose-dependent effects of norepinephrine were examined by cumulative addition; vertical bars represent ±SE.

View larger version (19K): [in this window] [in a new window]   Fig. 2.   Effect of isoproterenol on spontaneous rate in right atria isolated from control (filled circles), 50 mg/kg STZ-induced diabetic (squares), and insulin-treated diabetic (open circles) rats. Preparations (n = 11-14/group) were isolated 12 wk after administration of STZ or diluent and bathed in KH solution containing 106 M atropine. Dose-dependent effects of isoproterenol were examined by cumulative addition; vertical bars represent ±SE.

The chronotropic response to norepinephrine was also examined after pretreatment with 5 × 10⁶ M cocaine. The cocaine-induced shift in the norepinephrine dose-response curve was not significantly different among treatment groups; norepinephrine EC₅₀ values in the presence of cocaine were 71 ± 7, 80 ± 6, and 76 ± 7% of precocaine values in controls, 50 mg/kg STZ-induced diabetics, and insulin-treated diabetics, respectively.

Cholinergic responsiveness. Preparations isolated from rats 12 wk after 50 mg/kg STZ treatment displayed an enhanced sensitivity to acetylcholine (the cholinergic neurotransmitter) as reflected by a leftward shift in the dose-response curve (Fig. 3) and a reduction in EC₅₀ values (Table 5). The increased sensitivity to acetylcholine was restored toward control levels by insulin treatment; EC₅₀ values in this group fell between those obtained in right atria isolated from control and diabetic rats. EC₅₀ values were not significantly different when compared in preparations isolated from 45, 50, and 65 mg/kg STZ-induced diabetic animals (2.08 ± 0.50, 1.60 ± 0.45, and 1.05 ± 0.56 × 10⁵ M in the 45, 50, and 65 mg/kg STZ-treated groups, respectively). In contrast to results with acetylcholine, the responsiveness to carbachol (a cholinergic agonist not readily metabolized by acetylcholinesterase) was not affected by 50 mg/kg STZ-induced diabetes or the corresponding insulin treatment (Fig. 4 and Table 5). Furthermore, the enhanced sensitivity to acetylcholine was masked by 15 min of exposure of right atria to 5 × 10⁵ M DFP. As shown in Table 5, DFP treatment decreased basal spontaneous pacemaker rate in all three groups and resulted in EC₅₀ values for acetylcholine that were not significantly different when compared among preparations isolated from control, diabetic, and insulin-treated diabetic rats.

View larger version (23K): [in this window] [in a new window]   Fig. 3.   Effect of acetylcholine on spontaneous rate in right atria isolated from control (filled circles), 50 mg/kg STZ-induced diabetic (squares), and insulin-treated diabetic (open circles) rats. Preparations (n = 12-14/group) were isolated 12 wk after administration of STZ or diluent and bathed in KH solution containing 106 M nadolol. Dose-dependent effects of acetylcholine were examined by cumulative addition; vertical bars represent ±SE.

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Effect of carbachol on spontaneous rate in right atria isolated from control (filled circles), 50 mg/kg STZ-induced diabetic (squares), and insulin-treated diabetic (open circles) rats. Preparations (n = 12-14/group) were isolated 12 wk after administration of STZ or diluent and bathed in KH solution containing 106 M nadolol. Dose-dependent effects of carbachol were examined by cumulative addition; vertical bars represent ±SE.

	DISCUSSION

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Results of this study indicate that STZ-induced IDDM in rats is associated with a decline in basal pacemaker rate and an enhanced sensitivity to the negative chronotropic action of acetylcholine. These observed alterations were not significantly different when compared in preparations isolated from animals treated with 45, 50, and 65 mg/kg STZ. Because higher doses of STZ produced greater increases in both blood glucose and serum ketones, data suggest that observed changes were associated with moderate hyperglycemia (400 mg/dl); ketosis and additional elevations in blood glucose seemed to have little further influence.

Present data provide little information regarding the mechanism of the decline in basal spontaneous pacemaker rate. Results suggest that it is caused by diabetes rather than by a direct effect of STZ on the heart and that it is not mediated by differences in endogenous neurotransmitter release. Thus it would seem that STZ-induced diabetes alters the intrinsic pacemaking activity of the sinoatrial node. Alterations in ionic currents could reduce spontaneous rate by decreasing the slope of diastolic depolarization or by hyperpolarizing maximum diastolic potential. Ion channel function may be influenced by numerous diabetes-associated alterations, including glycosylation, changes in membrane lipid composition, or changes in protein expression.

Current data suggest that the enhanced sensitivity to the negative chronotropic action of acetylcholine is the result of a diminished acetylcholinesterase activity. Tissues isolated from diabetic animals showed a significant reduction in EC₅₀ values for this cholinergic neurotransmitter, whereas the sensitivity to carbachol, a cholinergic agonist not readily metabolized by acetylcholinesterase, was not significantly affected. Furthermore, treatment with DFP, an irreversible cholinesterase inhibitor (8), abolished the enhanced sensitivity to acetylcholine.

Carrier and co-workers (2, 3) previously reported a supersensitivity to the chronotropic effects of acetylcholine that was mediated, at least in part, by a reduced acetylcholinesterase activity. In fact, they found decreases in both right atrial muscarinic receptor density and cholinesterase enzyme activity. Current results thus support their data with acetylcholine. However, in contrast to our data, experiments by Carrier and co-workers (2, 3) and by Li et al. (10) demonstrated an enhanced sensitivity to cholinergic agonists, which are not susceptible to acetylcholinesterase-mediated hydrolysis. The cause of this apparent disparity is unknown. Current data suggest that it is not explained by the use of different doses of STZ, since the doses administered by both Li et al. (10) and Carrier and co-workers (2, 3) were within the range used in our study. In fact, the only apparent difference among the studies is the age of the animals at the time of STZ treatment.

Results of present studies indicate that STZ-induced diabetes does not affect the responsiveness to adrenergic agonists. The maximum observed spontaneous rate in the presence of norepinephrine or isoproterenol was reduced in atria isolated from diabetic rats; however, the actual increase in rate elicited by these agents was not altered. In addition, no alterations in EC₅₀ values for either agonist were observed. These results were similar to those of Ramanadham and Tenner (14) but conflicted with the data of Foy and Lucas (5), who reported increased positive chronotropic responses to isoproterenol and norepinephrine. As discussed above with respect to cholinergic responsiveness, the cause of this disparity among studies is unknown. Current experiments ruled out the possibility that it is the result of differing doses of STZ. However, a comparison of the studies suggests that it may be related to the longer duration of the diabetic state used in current experiments and in the work by Ramanadham and Tenner (14).

The absence of alterations in norepinephrine sensitivity did not eliminate the possibility that diabetes elicits a change in neuronal catecholamine reuptake. In fact, reported elevations in plasma and cardiac norepinephrine levels (1, 6) suggest that neurotransmitter synthesis, storage, release, and/or reuptake are altered in STZ-treated rats. However, current data indicate that the magnitude of the cocaine-induced leftward shift in the norepinephrine dose-response curve is not significantly affected by diabetes. Although not direct proof, this finding suggests that adrenergic nerve terminals and neuronal reuptake are intact in right atria isolated from diabetic rats. A similar conclusion can be drawn when one considers that this animal model of diabetes did not affect responses to either norepinephrine or isoproterenol, a -adrenergic agonist not readily subject to neuronal reuptake.