Heart Of Streptozotocin-diabetic Rats Research Articles

Liver X Receptor Agonist TO901317 Prevents Diacylglycerols Accumulation in the Heart of Streptozotocin-Diabetic Rats

Background/Aims: Liver X receptors (LXRα and LXRβ) are ligand-activated transcription factors that regulate expression of genes involved in lipid and cholesterol metabolism. LXR expression has been identified in the heart, and enhanced LXR activity in the streptozotocin (STZ) diabetic myocardium was reported recently. The aim of this study was to investigate effect of in vivo LXR activation on myocardial lipid metabolism under conditions of STZ-induced diabetes. Methods: Wistar rats were randomly divided into three experimental groups: non-diabetic control, treated with STZ, and treated with STZ and LXR agonist - TO901317. Diabetes was induced by a single intraperitonal injection of STZ at a dose of 55 mg/kg. LXR agonist was administrated once daily in the morning by an oral gavage at a dose of 10 mg/kg/d during the last week of the experiment. After anesthesia samples of blood and the left ventricle were taken. Results: TO901317 administration increased expression of both LXR isoforms and its target genes: sterol response element binding protein 1c (SREBP-1c) and acetyl-coenzyme A carboxylase 1 (ACC1) in the heart of streptozotocin-diabetic rats. Treatment with LXR agonist had no effect on plasma lipids and glucose in the diabetic rats. Concomitantly, content of the examined lipid classes in the diabetic heart (nonesterified fatty acids, triacylglycerols, phospholipids, cholesterol esters, ceramide) was unchanged after treatment with TO901317. On the contrary, myocardial level of cholesterol and diacylglycerols (DAG) was decreased after LXR activation in diabetic rats, the change in DAG level was associated with downregulated expression of adipose triglyceride lipase (ATGL). Conclusion: Activation of LXRs by TO901317 protects cardiomyocytes against DAG accumulation and thus may reverse disturbances in lipid metabolism observed in streptozotocin-diabetic heart.

Early diabetes treatment does not prevent sympathetic dysinnervation in the streptozotocin diabetic rat heart

BackgroundPositron emission tomography (PET) studies have demonstrated reduced sympathetic neuronal integrity in high-fat diet fed streptozotocin insulin-resistant diabetic rats in parallel with abnormal early-to-atrial transmitral velocity. We hypothesized that administration of anti-glycemic drugs early after diabetes induction would prevent sympathetic neuronal dysfunction. Methods and ResultsMale Sprague-Dawley rats fed high-fat diet were administered streptozotocin (45 mg·kg−1, ip, n = 23) to induce diabetes or vehicle alone (n = 6). Diabetic rats were randomized to receive insulin (4 U·day−1), metformin (650 mg·kg−1·day−1), rosiglitazone (4 mg·kg−1·day−1), or no treatment 1 week after streptozotocin. Small animal PET imaging using the norepinephrine analog [11C]meta-hydroxyephedrine (HED) at baseline and 8 weeks of diabetes determined sympathetic neuronal integrity. Echocardiography assessed cardiac function. Plasma norepinephrine levels were determined in parallel. Ex vivo immunoblotting was performed at the end of the experiment to compare the relative expression of various proteins involved in metabolic and noradrenergic signaling. Insulin restored blood glucose and lipid levels to normal. Despite improved plasma lipid levels, neither metformin nor rosiglitazone reduced blood glucose. At 8 weeks, untreated and treated diabetics displayed a 39%-42% reduction in myocardial HED standardized uptake values (P < .05). In all diabetic groups, plasma norepinephrine was elevated (2.3- to 3.3-fold, P < .05) and norepinephrine reuptake transporter expression reduced (28%-35%, P < .05) compared to non-diabetics. Doppler echocardiography revealed delayed development of prolonged mitral valve deceleration and elevated early-to-atrial filling velocity ratio among treated diabetic rats. ConclusionEarly glycemic treatment of insulin-resistant diabetic rats did not prevent deterioration of sympathetic neuronal integrity though ventricular filling abnormalities were delayed.

Cardioprotective effect of propranolol on diabetes-induced altered intracellular Ca2+ signaling in rat

We have previously shown that chronic treatment with propranolol had beneficial effects on heart function in rats during increasing-age in a gender-dependent manner. Herein, we hypothesize that propranolol would improve cardiac function in diabetic cardiomyopathy and investigated the benefits of chronic oral administration of propranolol on the parameters of Ca(2+) signaling in the heart of streptozotocin-diabetic rats. Male diabetic rats received propranolol (25 mg/kg, daily) for 12 weeks, 1 week after diabetes induction. Treatment of the diabetic rats with propranolol did not produce a hypoglycaemic effect whereas it attenuated the increased cell size. Basal and β-agonist response levels of left ventricular developed pressure were significantly higher in propranolol-treated diabetic rats relative to untreated diabetics while left ventricular end diastolic pressure of the treated diabetics was comparable to the controls. Propranolol treatment normalized also the prolongation of the action potential in papillary muscles from the diabetic rat hearts. This treatment attenuated the parameters of Ca(2+) transients, depressed Ca(2+) loading of the sarcoplasmic reticulum, and of the basal intracellular Ca(2+) level of diabetic cardiomyocytes. Furthermore, Western blot data indicated that the diabetes-induced alterations in the cardiac ryanodine receptor Ca(2+) release channel's hyperphosphorylation decreased the FKBP12.6 protein level. Also, the high phosphorylated levels of PKA and CaMKII were prevented with propranolol treatment. Chronic treatment with propranolol seems to prevent diabetes-related changes in heart function by controlling intracellular Ca(2+) signaling and preventing the development of left ventricular remodeling in diabetic cardiomyopathy.

Insulin induced translocation of Na+/K+-ATPase is decreased in the heart of streptozotocin diabetic rats

To investigate the effect of acute insulin administration on the subcellular localization of Na(+)/K(+)-ATPase isoforms in cardiac muscle of healthy and streptozotocin-induced diabetic rats. Membrane fractions were isolated with subcellular fractionation and with cell surface biotinylation technique. Na(+)/K(+)-ATPase subunit isoforms were analysed with ouabain binding assay and Western blotting. Enzyme activity was measured using 3-O-methylfluorescein-phosphatase activity. In control rat heart muscle alpha1 isoform of Na(+)/K(+) ATPase resides mainly in the plasma membrane fraction, while alpha2 isoform in the intracellular membrane pool. Diabetes decreased the abundance of alpha1 isoform (25 %, P<0.05) in plasma membrane and alpha2 isoform (50%, P<0.01) in the intracellular membrane fraction. When plasma membrane fractions were isolated by discontinuous sucrose gradients, insulin-stimulated translocation of alpha2- but not alpha1-subunits was detected. Alpha1-subunit translocation was only detectable by cell surface biotinylation technique. After insulin administration protein level of alpha2 increased by 3.3-fold, alpha1 by 1.37-fold and beta1 by 1.51-fold (P<0.02) in the plasma membrane of control, and less than 1.92-fold (P<0.02), 1.19-fold (not significant) and 1.34-fold (P<0.02) in diabetes. The insulin-induced translocation was wortmannin sensitive. This study demonstrates that insulin influences the plasma membrane localization of Na(+)/K(+)-ATPase isoforms in the heart. alpha2 isoform translocation is the most vulnerable to the reduced insulin response in diabetes. alpha1 isoform also translocates in response to insulin treatment in healthy rat. Insulin mediates Na(+)/K(+)-ATPase alpha1- and alpha2-subunit translocation to the cardiac muscle plasma membrane via a PI3-kinase-dependent mechanism.

Cardiac expression of adiponectin and its receptors in streptozotocin-induced diabetic rats

Adiponectin can improve both glucose metabolism and insulin resistance via the adenosine monophosphate–activated protein kinase (AMPK) signaling pathway. Activated AMPK phosphorylates a variety of intracellular proteins, including acetyl coenzyme A carboxylase (ACC) that is involved in fatty acid oxidation. Adenosine monophosphate–activated protein kinase increases glucose transport by stimulating the translocation of glucose transporter 4 (GLUT4) to the sarcolemma in the heart. Adiponectin exerts its effect through adiponectin receptors, which are predominantly expressed in the liver and skeletal muscle. It is unknown whether the cardiac expression of adiponectin and its receptors is changed in diabetic rats. In the present study, we investigated the protein expression of adiponectin and its receptors in streptozotocin (STZ)–induced diabetic rat hearts. We also explored whether the levels of AMPK, ACC, and GLUT4 will be altered with the changed adiponectin and its receptors in STZ diabetic rat hearts. Plasma and cardiac adiponectin levels were measured by radioimmunoassay. Plasma and cardiac interleukin 6 and plasma tumor necrosis factor α (TNF- α) were assayed by enzyme-linked immunosorbent assay. Cardiac adiponectin receptors, AMPK- α, ACC, GLUT4, and TNF- α were analyzed by Western blot in control and STZ diabetic rats. The plasma adiponectin level was decreased, but the cardiac protein expression of adiponectin receptor 1 was increased in diabetic rats. There was no difference in the cardiac adiponectin level and the cardiac adiponectin receptor 2 protein expression between control and diabetic rats. The phosphorylation of AMPK- α and protein expression of GLUT4 were decreased, but the phosphorylation of ACC was unchanged in diabetic rat hearts. Plasma and cardiac levels of interleukin 6 and TNF- α were increased in diabetic rats. In conclusion, STZ-induced diabetes up-regulates adiponectin receptors in the heart. Despite an increase in cardiac adiponectin receptor 1 expression, there is an increased cardiac inflammatory response and a decreased GLUT4 protein expression associated with a reduction in circulating adiponectin.

Inhibition of nitric oxide synthase enhances contractile response of ventricular myocytes from streptozotocin-diabetic rats

The contractile hyporesponsiveness of the streptozotocin diabetic rat heart in vitro to beta-adrenergic agonists is eliminated when the heart is perfused with N(G)-nitro-L-arginine methyl ester (L-NAME), a non-selective inhibitor of nitric oxide synthase (NOS). The following study evaluated the hypothesis that an increased production of NO/cGMP within the diabetic myocyte inhibits the beta-adrenergic-stimulated increase in calcium current and contractile response. Male Sprague-Dawley rats were given an intravenous injection of streptozotocin (60 mg/kg). After 8 weeks, L-type calcium currents were recorded in ventricular myocytes using the whole cell voltage-clamp method. Shortening of isolated myocytes was determined using a video edge detection system. cAMP and cGMP were measured using radioimmunoassay. Nitric oxide production was determined using the Griess assay kit. Basal cGMP levels and nitric oxide production were elevated in diabetic myocytes. Shortening of the diabetic myocytes in response to isoproterenol (1 microM) was markedly diminished. However, there was no detectable difference in the isoproterenol-stimulated L-type calcium current or cAMP levels between control and diabetic myocytes. Acute superfusion of the diabetic myocyte with L-NAME (1 mM) decreased basal cGMP and markedly enhanced the shortening response to isoproterenol but did not alter isoproterenol-stimulated calcium current. These data suggest that increased production of NO/cGMP within the diabetic myocyte suppressed beta-adrenergic stimulated shortening of the myocyte. However, NO/cGMP apparently does not suppress shortening of the myocyte by inhibition of the beta-stimulated calcium current.

Protection against myocardial ischemia/reperfusion injury by short-term diabetes: enhancement of VEGF formation, capillary density, and activation of cell survival signaling

The aims of this study were to determine effects of diabetes duration on myocardial ischemia/reperfusion (I/R) injury and test whether time-dependent differences in sensitivity of the streptozotocin diabetic rat heart to I/R are related to differences in vascular density, levels of vascular endothelial growth factor (VEGF) or endothelial nitric oxide synthase (eNOS) expression, NO formation, activation of Akt, and/or oxidative stress. After 2 or 6 weeks of streptozotocin-induced diabetes, I/R injury was induced by occlusion (30 min) and reperfusion of the left descending coronary artery. After 2 weeks of diabetes, infarct size and cleavage of caspase-3, a proapoptosis signal, were decreased as compared with normoglycemic controls or rats that had been diabetic for 6 weeks, whereas capillary density and levels of VEGF and eNOS protein and cardiac NO(x) levels were all increased. Phosphorylation of Akt, a prosurvival signal, was also significantly increased after 2 weeks of diabetes. Cardiac lipid peroxidation was comparable to controls after 2 weeks of diabetes, whereas levels of nitrotyrosine, a peroxynitrite biomarker, were reduced. After 6 weeks of diabetes, lipid peroxidation was increased and levels of VEGF and plasma NO were reduced as compared with controls or rats diabetic for 2 weeks. Our results indicate endogenous cardioprotective mechanisms become transiently activated in this early stage of diabetes and that this may protect the heart from I/R injury through enhancement of VEGF and eNOS expression, NO formation, activation of cell survival signals, and decreased oxidative stress.

Green tea impedes dyslipidemia, lipid peroxidation, protein glycation and ameliorates Ca 2+-ATPase and Na +/K +-ATPase activity in the heart of streptozotocin-diabetic rats

Diabetes-induced hyperlipidemia, oxidative stress and protein glycation impair cellular calcium and sodium homeostasis associated with abnormal membrane-bound enzyme activities resulting in cardiac dysfunction in diabetes. To explore the cardioprotective mechanism of green tea in diabetes, we measured the changes in the levels of calcium, sodium, potassium and the activities of Na +/K +-ATPase and Ca 2+-ATPase in green tea treated diabetic rat hearts. The effect of green tea on triglycerides, lipid peroxidation and protein glycation in diabetic heart were also measured to elucidate the underlying mechanisms. Diabetes was induced by streptozotocin (STZ, 60 mg/kg i.p.). Six weeks after the induction of diabetes, some of the diabetic rats were treated orally with green tea extract (GTE) (300 mg/kg/day) for 4 weeks. GTE produced reduction in blood glucose and lowered the levels of lipid peroxides, triglycerides and extent of protein glycation in the heart of diabetic rats. GTE blunted the rise in cardiac [Ca 2+] and [Na +] whereas increased the activities of Ca 2+-ATPase and Na +/K +-ATPase in diabetic rats. In conclusion, the data provide support to the therapeutic effect of GTE and suggest that a possible mechanism of action may be associated with the attenuation of the rise in [Ca 2+] and [Na +] by ameliorating Ca 2+-ATPase and Na +/K +-ATPase activities.

Therapeutic effect of green tea extract on oxidative stress in aorta and heart of streptozotocin diabetic rats

Hyperglycemia induced oxidative stress has been proposed as a cause of many complications of diabetes including cardiac dysfunction. The present study depicts the therapeutic effect of green tea extract on oxidative stress in aorta as well as heart of streptozotocin diabetic rats. Six weeks after diabetes induction, green tea was administered orally for 4 weeks [300 mg (kg body weight) −1 day −1]. In aorta and heart of diabetic rats there was a significant increase in the activity of superoxide dismutase, catalase and glutathione peroxidase with an increase in lipid peroxides. Diabetic rats showed a significant decrease in the levels of serum and cardiac glutathione. Green tea administration to diabetic rats reduced lipid peroxides and activity of antioxidant enzymes whereas increased glutathione content. The results demonstrate that the induction of antioxidant enzymes in diabetic rats is not efficient and sufficient to reduce the oxidative stress. But green tea by providing a competent antioxidative mechanism ameliorates the oxidative stress in the aorta and heart of diabetic rats. The study suggests that green tea may provide a useful therapeutic option in the reversal of oxidative stress induced cardiac dysfunction in diabetes mellitus.

The Effect of NCX4016 [2-Acetoxy-benzoate 2-(2-nitroxymethyl)-phenyl Ester] on the Consequences of Ischemia and Reperfusion in the Streptozotocin Diabetic Rat

The aim of this study was to assess the effect of chronic administration of NCX4016 [2 acetoxy-benzoate 2-(2-nitroxymethyl)-phenyl ester], a nitric oxide-releasing aspirin derivative on the consequences of coronary artery occlusion in streptozotocin-diabetic rats. Rats were made diabetic by injection of streptozotocin (60 mg kg(-1)) and received insulin (2.5 U kg(-1) s.c.) daily for 4 weeks. Animals received vehicle (1 ml kg(-1) polyethylene glycol), aspirin (65.2 mg kg(-1)), NCX4016 (60 mg kg(-1)), or (iv) NCX4016 (120 mg kg(-1)) orally, once daily for the last 5 days before coronary artery occlusion (CAO). One hour after the last dose, pentobarbital-anesthetized rats were subjected to CAO for 30 min followed by 120-min reperfusion. Neither drug significantly modified initial hemodynamics or plasma glucose levels compared with vehicle treatment in either nondiabetic or diabetic rats. Neither drug modified the total ventricular premature beat (VPB) count in normal animals, although NCX4016, but not aspirin, reduced the total VPB count and the incidence of ventricular tachycardia in diabetic rats. In nondiabetic animals, both aspirin and NCX4016 reduced infarct size. However, in diabetic rats, infarct size was reduced only by the larger dose of NCX4016 (120 mg kg(-1)) but not by aspirin or the lower dose of NCX4016. These results demonstrate that the cardioprotective effects of NCX4016 are reduced in the presence of diabetes compared with the effects seen in nondiabetic animals. In summary, the present study confirms the protective effect of NCX4016 against ischemia-reperfusion injury in the normal rat heart and demonstrates for the first time its protective effect in the heart of streptozotocin-diabetic rats.

Effect of cobalt on the oxidative status in heart and aorta of streptozotocin-induced diabetic rats.

Effects of cobalt on the antioxidant status of control and streptozotocin diabetic rat heart and aorta were examined at the second, fourth and sixth week of treatment. Rats were divided into four groups: control, diabetic, control treated with cobalt chloride and diabetic treated with cobalt chloride. Diabetes was induced by tail vein injection of streptozotocin (STZ). Cobalt treatment groups were given 0.5 mM of CoCl(2) in drinking water. The rats in both groups were further subdivided into three groups of six rats each. Rats in these subgroups were studied at 2-week intervals up to 6 weeks. At the end of the experiment, all animals were sacrificed by decapitation, heart and aorta samples were removed for determination of thiobarbituric acid reactive substance (TBARS) level and superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) activities. It was found that lipid peroxidation levels and antioxidant enzyme activities were increased in the streptozotocin-induced diabetic rats at all times studied. Cobalt treatment of diabetic rats (0.5 mM in drinking water) resulted in attenuation of the increased levels of TBARS and antioxidant enzyme activities in heart and aorta. Thus, the effect of oral administration of cobalt at this dose during the early stage of experimental diabetes can be considered as a consequence of altered endogenous defence mechanisms in heart and aorta.

Changes in catecholamines and CGRP levels in heart of streptozotocin-diabetic rats

Changes in catecholamines and CGRP levels in heart of streptozotocin-diabetic rats

Influence of diabetes on cardiac nitric oxide synthase expression and activity

There is some evidence that the endothelium dependent vasodilatation of coronary arteries is impaired in both types of diabetes. The underlying mechanisms are not yet clear, in particular whether this defect is caused by a direct effect of diabetes on the activity and the expression of nitric oxide synthases (NOS) or indirectly by an enhanced inactivation of nitric oxide. Methods: To study this question we determined the activity (conversion of L-arginine to citrulline) and the mRNAs encoding the isoforms of NOS (using polymerase chain reaction after reverse transcription of the mRNAs into cDNAs by reverse transcriptase) in hearts of streptozotocin diabetic rats and in rat heart endothelial cells (RHEC). The formation of reactive oxygen intermediates (ROI) was measured by the dichloro-dihydro-fluorescein method. Results: The activity of total NOS and the amounts of mRNAs encoding ecNOS and iNOS were dependent on the duration of diabetes. After a diabetes duration of 4 to 6 weeks both the total activity as well as the mRNAs encoding ecNOS and iNOS were elevated. A reduction of NOS activity and the amounts of mRNAs of ecNOS and iNOS was only seen after a diabetes duration longer than 20 weeks, a time at which a loss of endothelium has been described. In RHEC, high glucose (22 mM) and H 2O 2 (100 μM) were able to increase the mRNA encoding ecNOS, but not iNOS. This increase in ecNOS mRNA was inhibited by lipoic acid (1 μM). In addition, high glucose (22 and 30 mM) led to an enhanced formation of ROI and to activation of the transcription NFκB. Conclusion: These observations suggest that diabetes causes a temporary increase in NOS activity and ecNOS mRNA in the rat heart which is presumably the consequence of an enhanced oxidative stress exerted by hyperglycaemia. Together with previously published observations, our data suggest that the impairment of endothelium dependent vasodilatation in rat heart is not the consequence of a reduced activity and expression of NOS, but is caused by an enhanced inactivation of nitric oxide by ROI.

Diabetes mellitus induces long lasting changes in the glucose transporter of rat heart endothelial cells.

The accumulation of glucose exerts various cytotoxic effects on endothelial and other vascular cells, and thereby contributes to the development of microvascular complications in diabetes. Since tissues, in which vascular complications typically occur, do not take up glucose in an insulin regulated manner, it is an important question to know whether other mechanisms exist in these cells to restrict the uptake and the accumulation of glucose. To study this question, we used microvascular endothelial cells isolated from rat heart endothelial cells (RHEC). In RHEC, the non-insulin regulated glucose transporter (Glut-1) was detected as a broad protein band of 50-65 kD. In contrast, the Glut-1 from rat brain, which was taken as reference, had a molecular weight of 45 kD. After treatment with endoglycosidase F, both proteins formed a band of approximately 40 kD on SDS-PAGE, demonstrating a more extensive glycosylation of Glut-1 in RHEC as compared to brain. Incubation of the cells in high glucose (22 mM, up to 10 days) did not down-regulate either Glut-1 protein or mRNA. In contrast to high glucose, deprivation of the cells from glucose led to an increase in Glut-1 mRNA and protein which is partly non-glycosylated. In cells from hearts of streptozotocin-diabetic rats (DRHEC), Glut-1 protein, but not Glut-1 mRNA, was reduced by about 40%. Additionally, a significant amount of glycosyl residues was resistant to the enzymatic treatment with N-endoglycosidase F. Both changes in Glut-1 were also observed when the cells were cultivated in low glucose (5.5 mM) for several passages indicating a long lasting, hardly reversible modification of Glut-1 by diabetes. These data indicate that Glut-1 is not down-regulated in RHEC by high glucose, and that this important mechanism to protect the endothelium against an intracellular accumulation of glucose is missing in RHEC. As a consequence, increases in blood glucose may lead to a glucose overload with the described deleterious effects on the structure and function of endothelium.

Increased expression of Galphaq protein in the heart of streptozotocin-induced diabetic rats.

Heart disease is one of the major cause of death in diabetic patients, but the pathogenesis of diabetic cardio-myopathy remains unclear. In this experiment, to assess the significance of G protein signaling pathways in the pathogenesis of diabetic cardiomyopathy, we analyzed the expression of G proteins and the activities of second messenger dependent protein kinases: cAMP-dependent protein kinase (PKA), DAG-mediated protein kinase C (PKC), and calmodulin dependent protein kinase II (CaM kinase II) in the streptozotocin induced diabetic rat heart. The expression of Galphaq was increased by slightly over 10% (P<0.05) in diabetic rat heart, while Galphas, Galphai, and Gbeta remained unchanged. The PKA activity in the heart did not change significantly but increased by 27% (P<0.01) in the liver. Insulin treatment did not restore the increased activity in the liver. Total PKC activity in the heart was increased by 56% (P<0.01), and insulin treatment did not restore such increase. The CaM kinase II activity in the heart remained at the same level but was slightly increased in the liver (14% increase, P<0.05). These findings of increased expression of Galphaq in the streptozotocin-diabetic rat heart that are reflected by the increased level of PKC activity and insensitivity to insulin demonstrate that alteration of Galphaq may underlie, at least partly, the cardiac dysfunction that is associated with diabetes.

Acceleration of stiffness in underperfused diabetic rat hearts by glyburide, a KATP channel blocker, and its prevention by levcromakalim and insulin.

To clarify the role of the KATP channels in myocardial dysfunction during underperfusion with norepinephrine (NE) in the diabetic heart, particularly the heart treated with sulphonylurea derivatives. Isolated 6-week streptozotocin-diabetic rat hearts with a balloon in the left ventricle (LV) were paced and perfused with normoxic Krebs-Henseleit solution. Agents were infused for 15-25 min before as well as during 60-min underperfusion (2 ml/min/g heart weight) with 10(-6) M NE. Regional myocardial flow distribution was measured using dye microspheres. The effects of ex vivo glyburide (10(-6) M, a sulphonylurea anti-diabetic drug and a specific KATP channel inhibitor) on contractile dysfunction and abnormal regional myocardial energy metabolism were examined during underperfusion with NE in the absence of presence of levcromakalim (10(-4) M, a selective K+ channel opener) and insulin (2 mU/min/g heart weight). The flow rate was greater in the LV subendocardium than the subepicardium during normal perfusion, and smaller at 60-min underperfusion with NE. The LV diastolic tension and pressure during underperfusion with NE increased more rapidly in the presence of glyburide. At 60-min underperfusion with NE, the diastolic pressure elevation was still higher in the glyburide-treated heart, and decreases in tissue ATP, creatine phosphate (CP), energy charge, phosphorylation potential and CP/inorganic phosphate (P(i)) ratio, and increases in AMP, P(i) and lactate were more marked in the glyburide-treated heart, particularly in the LV subendocardium. Thus, ex vivo glyburide enhanced the increase in LV stiffness and abnormal myocardial energy metabolism during underperfusion with NE in diabetic hearts. These changes were reduced by levcromakalim to the level during underperfusion with NE without glyburide. Insulin did not prevent the glyburide-induced earlier exacerbation of the increase in LV stiffness during underperfusion with NE, but reduced the detrimental effects 20 min after the onset of underperfusion. KATP channels in the diabetic myocardium probably open during underperfusion with NE, and it helps delay the initiation of the increase in cardiac stiffness. Glyburide may have harmful effects in the ischemic diabetic heart; the myocardial KATP channel blockade during underperfusion with NE enhanced the increase in LV stiffness and abnormal myocardial energy metabolism. The glyburide-induced detrimental effects in the ischemic diabetic heart are prevented by levcromakalim and partly by insulin.

Impairment of endothelium dependent relaxation in the diabetic rat heart: mechanisms and implications

An increased oxidative stress has been suggested to contribute to disturbances in the regulation of coronary flow and the increased cardiac risk in diabetes. Using the isolated perfused heart of streptozotocin-diabetic rats we could recently show [21] that the basal and the maximal coronary flow (tested by infusion of sodium nitroprusside) are not altered in diabetes, but that the 5-HT stimulated endothelial dependent increase in coronary flow becomes progressively impaired. This defect of the endothelium dependent vasodilatation was prevented by perfusion of the hearts with superoxide dismutase and pre-treatment of the diabetic rats with tocopherol-acetate. Perfusion of the hearts with indomethacin to inhibit the synthesis of vasoconstricting prostaglandin endoperoxides did not improve the disturbed 5-HT induced, endothelium dependent increase in coronary flow. Furthermore, acute variations of the glucose concentration in the perfusion medium did not affect the coronary flow significantly. In myocardium, the constitutive endothelial NO synthetase was nearly exclusively expressed with the highest activity in endothelium, as determined from experiments using isolated cardiac endothelial cells. The activity of NO synthetase determined by conversion of arginine to citrulline was significantly increased in diabetes. In line with this observation, the concentration of arginine in plasma was reduced, but that of citrulline elevated. Additionally, the renal elimination of nitrite was enhanced in diabetes. These data suggest that the impaired endothelium dependent relaxation observed in the diabetic myocardium is presumably not caused by a diminished synthesis and release of NO, but by an accelerated inactivation of NO by superoxide anions. We suggest that the increased NO generation reflects a compensatory mechanism to balance the enhanced inactivation of NO.

Endothelial relaxation is disturbed by oxidative stress in the diabetic rat heart: influence of tocopherol as antioxidant

Increased oxidative stress has been suggested to contribute to disturbances in the regulation of coronary flow and the increased cardiac risk in diabetes mellitus. Using the isolated perfused heart of streptozotocin-diabetic rats our study shows that basal and maximal coronary flow (tested by infusion of sodium nitroprusside) are not altered in diabetes, but that 5-hydroxytryptamine (5-HT) stimulated endothelium-dependent increase in coronary flow becomes progressively impaired. This defect of the endothelium-dependent vasodilatation was prevented by perfusion of the hearts with superoxide dismutase and pretreatment of the diabetic rats with tocopherol-acetate. Morphological studies also revealed that pretreatment with tocopherol-acetate was cardioprotective, and largely prevented severe alterations of myocardial structure typically observed after a diabetes duration of 3 months; deterioration and fragmentation of myofilament bundles were seen less, and the numbers of areas of focal necrosis and of contraction bands were clearly reduced. In contrast to untreated diabetic hearts the autonomic nerve fibers detected by catecholamine fluorescence were running in parallel in hearts of tocopherol-treated diabetic rats, and the amount of catecholamines was not different from that of healthy control rats. Trichrome staining and immunohistochemical staining of collagen I and III showed a dramatic increase in number and the size of deposits of collagen fibers at precapillary locations in the diabetic hearts which were significantly reduced by anti-oxidative treatment. These findings demonstrate that oxidative stress may not only play a major role in the impairment of endothelium-dependent regulation of coronary flow, but also in the development of perivascular fibrosis and severe changes of the autonomic nerves and contractile system in myocardium.

Endothelial relaxation is disturbed by oxidative stress in the diabetic rat heart: influence of tocopherol as antioxidant

Increased oxidative stress has been suggested to contribute to disturbances in the regulation of coronary flow and the increased cardiac risk in diabetes mellitus. Using the isolated perfused heart of streptozotocin-diabetic rats our study shows that basal and maximal coronary flow (tested by infusion of sodium nitroprusside) are not altered in diabetes, but that 5-hydroxytryptamine (5-HT) stimulated endothelium-dependent increase in coronary flow becomes progressively impaired. This defect of the endothelium-dependent vasodilatation was prevented by perfusion of the hearts with Superoxide dismutase and pretreatment of the diabetic rats with tocopherol-acetate. Morphological studies also revealed that pretreatment with tocopherol-acetate was cardioprotective, and largely prevented severe alterations of myocardial structure typically observed after a diabetes duration of 3 months; deterioration and fragmentation of myofilament bundles were seen less, and the numbers of areas of focal necrosis and of contraction bands were clearly reduced. In contrast to untreated diabetic hearts the autonomic nerve fibers detected by catecholamine fluorescence were running in parallel in hearts of tocopherol-treated diabetic rats, and the amount of catecholamines was not different from that of healthy control rats. Trichrome staining and immunohistochemical staining of collagen I and III showed a dramatic increase in the number and the size of deposits of collagen fibers at precapillary locations in the diabetic hearts which were significantly reduced by anti-oxidative treatment. These findings demonstrate that oxidative stress may not only play a major role in the impairment of endothelium-dependent regulation of coronary flow, but also in the development of perivascular fibrosis and severe changes of the autonomic nerves and contractile system in myocardium.

Alpha 1-adrenoceptor-mediated phosphoinositide breakdown and inotropic responses in diabetic hearts

The mechanism(s) involved in diabetes-induced changes in the heart is still unclear, but one defect appears to occur in the alpha 1-adrenoceptor system. We evaluated the possibility that the changes in the inotropic responsiveness to alpha 1-adrenoceptor stimulation in streptozotocin-diabetic rat hearts may be linked to altered phosphoinositide turnover. Stimulation of alpha 1-adrenoceptor by norepinephrine (in the presence of propranolol) in right ventricles resulted in the formation of D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] [measured with an Ins(1,4,5)P3 protein binding assay kit] in a time- and concentration-dependent manner in both control and diabetic rats. The increase in Ins(1,4,5)P3 preceded the increase in the norepinephrine-mediated positive inotropic effect. Diabetic hearts showed a greater maximum inotropic response to norepinephrine stimulation and also had higher Ins(1,4,5)P3 levels. These observations suggest that the changes in Ins(1,4,5)P3 levels may be implicated in the increased inotropic responsiveness to alpha 1-adrenoceptor stimulation in diabetic hearts. Ca2+ overload, induced by Ins(1,4,5)P3, could further be involved in the development of diabetic cardiomyopathy.