Severe diabetes mellitus is associated with a low thyroid state in patients and animals that is characterized by low serum thyroid hormone (T4) values (1,2). Hypothyroidism and diabetes mellitus have several similar noxious effects on metabolic, hemodynamic, and pharmacological cardiovascular parameters. Both pathological conditions are characterized by decreased myosin ATPase activity (3,4), impaired uptake of Ca2+ ions by the sarcoplasmic reticulum (SR) (3,5-7), impaired myocardial contractility and relaxation rate (8,9), and altered responsiveness to adrenoceptor stimulation (10,11). Therefore, the diabetes-induced hemodynamic and pharmacological cardiovascular changes may be caused partly by the low thyroid state accompanying diabetes. Indeed, treatment of diabetic rats with T3 (triiodothyronine) or T4 (thyroxine) effectively reversed the decrease in myosin ATPase activity (12-14), whereas the enhanced positive inotropic response to α-adrenergic stimulation in isolated atria of diabetic rats was prevented by T3 treatment .
In a previous study, we observed increased responses to α1-adrenoceptor stimulation in isolated hearts from diabetic rats that also had low serum T4 values (15). The possibility that these functional alterations could be due to the low thyroid hormone values resulting from the disease should at least be taken into consideration. For these reasons, we wished to investigate the efficacy of different dosages of streptozotocin (STZ), administered in the fasted and nonfasted state, to induce diabetes mellitus and a hypothyroid state. Furthermore, we investigated the influence of diabetes mellitus and hypothyroidism on cardiac function and on the inotropic responsiveness to the α1-adrenoceptor agonist cirazoline in isolated perfused hearts.
Male Wistar rats aged 6-8 weeks were obtained from IFFA CREDO, Les Oncins, France. At age 10-12 weeks, both fasted (24 h) and nonfasted rats received 20, 40, or 60 mg/kg STZ by a single intravenous (i.v.) injection into a lateral tail vein. We assessed the diabetic state by measuring blood glucose levels at 3 days and every 2 weeks thereafter.
At age 14-16 weeks, male Wistar rats (IFFA CREDO) received 0.5 g/L 6-n-propyl-2-thiouracil (PTU) in their drinking water for 4 weeks.
All rats had free access to standard rat chow and drinking water and were kept until age 18-20 weeks. After the animals had been anesthetized with pentobarbital (75 mg/kg intraperitoneally, i.p.), they were connected to an artificial respirator by a polyethylene tube inserted in the trachea (respiration frequency 40/min, respiration volume 160-180 ml/min). Heparin (250 IU) was administered through a polyethylene cannula inserted in a carotid artery. Carotid blood pressure (BP) was measured intraarterially by a Statham P23 Db pressure transducer and recorded with a Maclab data acquisition system (ADInstruments Pty Ltd, Castle Hill, New South Wales, Australia). Blood samples were obtained for measurement of serum T4.
Isolated perfused hearts
The heart and lungs were rapidly removed and immersed in a physiologic salt solution (PSS) at 4°C. The aorta was mounted on a cannula (2.0 mm ID) attached to a perfusion device. The hearts were perfused according to the Langendorff technique at a constant perfusion pressure of 55 mm Hg, with gassed (95% O2/5% CO2) nonrecirculated Tyrode's solution (PSS) of the following composition (in mM): NaCl 124.0, KCl 4.0, MgCl2 1.1, CaCl2 1.8, NaHCO3 24.9, NaH2PO4 0.3, and glucose 11.1. The temperature was maintained at 37°C and the pH was maintained at 7.4. The hearts were paced throughout at a frequency of 5 Hz (pulse width 5 ms, voltage 10% above threshold, 2-4 V) by platinum electrodes placed at the level of the valves. The left ventricular pressure (LVP) was measured by a water-filled balloon inserted in the left ventricle, connected to a Statham P23 Db pressure transducer, and recorded by a Maclab data acquisition system. The maximal contraction velocity was expressed as +dP/dtmax, and the maximal relaxation velocity was expressed as -dP/dtmax. The balloon filling pressure, corresponding to the diastolic BP (DBP), was maintained at 10 mm Hg.
Coronary flow was determined volumetrically. After 20 min of equilibrium cumulative concentration-response curves (CRC) were constructed for the inotropic effect of the α1-adrenoceptor agonist cirazoline (concentration range 10-9-10-6M).
Serum T4 was measured by conventional radioimmunoassay methods (16). Blood glucose was measured by means of sensor electrodes for MediSense Companion 2 (MediSense, U.S.A.).
Data evaluation and statistics
CRC, based on the relation E = Emax × AP × (AP + EC50P)-1, were fitted to concentration-response data of six individual experiments by a computer programme (GraphPad, Institute for Scientific Information, San Diego, CA, U.S.A.). In the equation, E is the response obtained at a given concentration A, Emax is the maximally attainable response, EC50 (M) is the concentration required to obtain the half-maximal effect, and the exponent P describes the slope of the relationship (Hill coefficient). All data are mean ± SE. Statistical significance comparing all experimental groups with controls was evaluated by Dunnett's test. Student's t test was used to compare fasted and nonfasted groups. The level of significance was considered p < 0.05.
Pentobarbital was obtained from Onderlinge Pharmaceutische Groothandel (Utrecht, The Netherlands), heparin sodium was obtained from Novo Nordisk A/S (Denmark), and STZ, and PTU were obtained from Sigma Chemical (St. Louis, MO, U.S.A.). Cirazoline HCl was donated by Synthélabo, Paris, France. Other chemicals were of analytic grade and were obtained from E. Merck (Darmstadt, Germany). STZ was dissolved in PSS.
Blood glucose, body weight, mean arterial pressure (MAP), heart rate (HR), and serum T4 values of the rats before the experiment are shown in Table 1. None of the rats receiving 20 mg STZ in the nonfasted state became diabetic. In the fasted state, 3 days after receiving 20 mg STZ, 2 of 7 rats had increased glucose values (>18 mM). However, 8 weeks after the STZ injection, only 1 of these rats had a glucose level > 18 mM (data not shown). All rats receiving 40 or 60 mg STZ became diabetic. Seven rats received 60 mg in the fasted state. Six of the 7 animals died ≤4 days. None of the 60 fasted rats survived >4 weeks.
Both the hyperglycemic and hypothyroid rats had body weights lower than those of control animals. After STZ treatment, nonfasted rats receiving 40 mg STZ had slower weight gain, whereas the fasted rats receiving 40 mg STZ lost some body weight. Both groups receiving 60 mg STZ exhibited severe weight loss. After receiving PTU in the drinking water, the hypothyroid group ceased gaining weight.
MAP was reduced only in the hypothyroid group, whereas STZ treatment had no effect on MAP. The HR was significantly reduced in the hypothyroid group, whereas the diabetic groups tended to have a decreased HR. T4 values were decreased in the 40-mg STZ nonfasted group and the hypothyroid group as compared with control values, whereas fasted rats receiving 40 mg STZ and nonfasted rats receiving 60 mg STZ had excessively low T4 levels. Fasted rats receiving 40 mg STZ had lower T4 values as compared with the nonfasted rats receiving 40 mg STZ.
The basal parameters of the isolated perfused hearts obtained from the various groups of animals are shown in Table 2. LVP, +dP/dtmax, -dP/dTmax, and coronary flow were not influenced by the diabetic state of the donor animals. Hearts obtained from hypothyroid animals showed no difference in LVP, -dP/dtmax, or coronary flow as compared with hearts from control rats. Maximal contractility, however, was reduced in these hearts.
The ratio of wet heart weight to body weight was increased in the fasted rats receiving 40 mg STZ and in nonfasted rats receiving 60 mg STZ. Fasted rats receiving 40 mg STZ had a higher heart weight than the nonfasted 40-mg STZ control group. Wet heart weight/body weight ratio in the hypothyroid group was not different from the control values.
CRC's constructed for the positive inotropic effect of the α1-adrenoceptor agonist cirazoline showed that hearts from both groups receiving 40 mg STZ (both fasted and nonfasted) developed a more pronounced (p < 0.05) increase in LVP (Emax) than did hearts obtained from control rats. Inotropic responses obtained in hearts from the 20-mg STZ (fasted and nonfasted), 60-mg STZ (nonfasted), and hypothyroid groups were not significantly different from effects obtained in hearts from control rats. The sensitivity to cirazoline (expressed as log EC50) was not different in the seven experimental groups (Table 3).
CRC's for cirazoline in hearts from nonfasted rats made diabetic with 60 mg STZ could be constructed in only 3 rats. The small number of rats and (consequently) the considerable SE made it difficult to compare results obtained in this group with the other data.
Maximal increase in coronary flow, in the concentration range used, is shown in Table 3. Hearts from fasted rats receiving 40 mg STZ showed a more pronounced increase in coronary flow than did hearts from control animals, whereas the coronary flow in hearts from hypothyroid rats decreased.
A dose of 40 mg STZ administered in the nonfasted state proved sufficient to induce a clear diabetic state in male Wistar rats; 60 mg STZ in fasted and nonfasted rats led to increased mortality rates and severe weight loss. The 3 nonfasted rats that received 60 mg STZ and survived 8 weeks were in poor physical condition.
A decrease in T4 production, probably caused by intracellular energy shortage, has been reported in diabetes mellitus and also during modified fasting (17). The extremely low T4 values observed in the 40-mg STZ fasted and the 60-mg STZ nonfasted groups may therefore be explained by simultaneously occurring diabetes and, consequently, a severe reduction in body weight.
As demonstrated by our results, STZ caused a more severe diabetic state when administered to rats that had been fasted for several hours. STZ has been demonstrated to cause DNA damage in isolated pancreatic islet cells. This process initiates a chain of events leading to cellular dysfunction and eventual cell death (18,19). The similar structure of STZ and glucose causes them to compete for the same uptake mechanisms in pancreatic β-cells. Each measure that affects the blood glucose levels, such as fasting, may therefore influence the efficacy of STZ treatment.
Reports of the action of STZ on BP in rats are conflicting (20,21). In the present study, STZ treatment had no effect on BP, whereas HR tended to decrease in the diabetic rats. Heart rate and MAP in hypothyroid rats were decreased as compared with those in control rats. Bradycardia in diabetic rats has been described previously and was attributed to the accompanying hypothyroid state (10,14).
Both in conditions of hypothyroidism and diabetes mellitus, impaired myocardial contractility and a lower relaxation rate have been demonstrated (5,8,9,22). These phenomena appear to be due to decreased myosin ATPase activity, a change in myosin isoenzyme predominance (3,4), and decreased SR Ca2+ uptake (3,5-7), respectively. Treatment of diabetic rats with pharmacological doses of T3 and T4 reversed the decrease in Ca2+-activated myosin ATPase, although myosin isoenzyme distribution, SR calcium transport, and the decreased cardiac function were not restored (7,12-14).
When hearts were perfused according to the Langendorff procedure, no difference in LVP, +dP/dtmax, -dP/dtmax, or coronary flow was evident in hearts from the diabetic rats, although there was a tendency to decreased contractile and relaxation parameters in these hearts. In previous studies, no difference in basal contractile parameters was shown in hearts from diabetic animals (23), but decreased cardiac performance was demonstrated at higher filling pressures in isolated working hearts obtained from diabetic Wistar rats (9).
Hearts isolated from hypothyroid Wistar rats showed a decrease in maximal contraction velocity, a finding in agreement with the reduced LV function observed in hypothyroid rats (5) and patients (8). An increase in wet heart weight/body weight ratio, a measure of relative cardiac hypertrophy, was observed in hearts from the diabetic rats, with the exception of the nonfasted animals that received 40 mg STZ. No increase in wet heart weight/body weight ratio was observed in the hypothyroid animals.
Both hypothyroidism and diabetes mellitus have been associated with enhanced responses of isolated left atria to α-adrenergic stimulation (11). Goyal and colleagues demonstrated that the enhanced positive inotropic responses to methoxamine in left and right atria of diabetic rats were prevented by T3 treatment (10). Sato and co-workers, however, demonstrated an impaired response to norepinephrine and to transmural nerve stimulation in isolated left atria of STZ-diabetic and STZ-hypothyroid rats. T4 treatment did not improve the decreased response in atria from diabetic rats, whereas the response in atria from hypothyroid rats was normalized by T4 treatment (24).
Our present data and previously published data (15) demonstrate an increased inotropic response to cirazoline in hearts from diabetic rats as compared with hearts of control rats. Despite differences in T4 values, no difference was observed in the maximal inotropic response to cirazoline in hearts from either fasted or nonfasted rats receiving 40 mg STZ. Therefore, we conclude that the level of T4 does not influence the maximal response to the α1-adrenoceptor agonist cirazoline. This conclusion is confirmed by the maximal inotropic responses obtained in hearts from hypothyroid rats, which were not significantly weaker than those in control hearts. Therefore the increased response to α1-adrenoceptor stimulation in isolated Langendorff hearts in this study cannot be explained by the accompanying low thyroid state of the animals. Because cardiac α-adrenoceptor density is decreased in diabetic rats (25,26), alterations in post-receptor mechanisms may be involved in the enhanced inotropic responsiveness to α1-adrenoceptor stimulation. A more pronounced interaction of inositol 1,4,5 triphosphate (IP3) with an altered cardiac intracellular calcium metabolism as described for the diabetic state (27) would be in agreement with our findings of an increased contractile response in hearts from diabetic animals. In cardiac tissue from hypothyroid rats, a decrease in α-adrenoceptor number (28) and an impaired activity of IP3(5) have been reported, which may explain the slightly weaker (although not significantly different) inotropic response to α-adrenoceptor stimulation in the hypothyroid rats. One confounding factor, however, may be that the inotropic effect of cirazoline in the hypothyroid state may be reversed by a direct vasoconstrictor effect of cirazoline on the coronary arteries. In most cases, in mesenteric arteries and aortas from diabetic animals, an enhanced responsiveness to α1-adrenoceptor stimulation is evident as compared with responses obtained in arteries from control rats (29,30). In hypothyroid rat aorta, decreased vasoconstrictor responses to α1-adrenoceptor stimulation were demonstrated (31), whereas the responses in mesenteric arteries were not significantly reduced (32). No data are available on responsiveness to α1-adrenoceptor stimulation in isolated coronary arteries of such animals. Therefore, the vasodilation in coronary arteries from diabetic rats and the vasoconstriction in arteries obtained from hypothyroid rats observed in our experiments require further clarification.
Our results indicate that 40 mg STZ administered to nonfasted rats induces a diabetic state accompaned by a moderate decrease in serum T4 levels. However, 40 mg STZ in fasted rats and 60 mg STZ in both nonfasted and fasted rats causes a severe diabetic state and extremely low T4 values. The sharp decrease in serum T4 concentrations may be considered the expression of a severe, probably nonspecific, state of disease. Although diabetes mellitus and hypothyroidism are associated with various similar metabolic and hemodynamic parameters, the increased inotropic response to α1-adrenoceptor stimulation observed in isolated perfused hearts of diabetic rats cannot be explained by the decrease in serum T4 levels.
Acknowledgment: We thank Anton B. vd Wardt for excellent technical assistance.
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