Diabetic Rat Cardiomyocytes Research Articles

Early molecular events in the development of the diabetic cardiomyopathy.

Oxidative damage to DNA has been well documented in cardiac cells isolated from diabetic patients and rats with streptozotocin-induced diabetes mellitus (DM). This study evaluates possible molecular mechanisms for early events in the development of DM-induced cardiomyopathy. To analyze the mechanism of overexpression of p21(WAF1/CIP1) and inhibition of cyclin D(1) expression in cardiomyocytes of diabetic rats we examined the methylation status of these genes by MS-PCR and assessed the possibility of epigenetic control of their expression. We found that the steady-state expression of both genes is influenced by their methylation status, as an epigenetic event, of their 5'-flanking regions upon development of diabetes. Oxidative damage contributes to the development of cardiomyopathy via p53-dependent activation of cardiac cell death. This pathway includes de novomethylation of the P53-inducible p21(WAF1/CIP1)-gene encoding a protein which binds to and inhibits a broad range of cyclin-cyclin-dependent kinase complexes.

PLC/IP3 signaling defects underly abnormal contractile activity induced by phosphatidic acid in diabetic rat cardiomyocytes

PLC/IP3 signaling defects underly abnormal contractile activity induced by phosphatidic acid in diabetic rat cardiomyocytes

Human Tissue Kallikrein Gene Delivery Attenuates Cardiac Glycogen Accumulation in Streptozotocin-Induced Diabetic Rats

P58 The tissue kallikrein-kinin system is locally present in the heart and is reduced under diabetic conditions. In the present study, we explored the effects of kallikrein gene delivery in the heart under diabetic conditions. Intravenous injection of streptozotocin (STZ) (60mg/kg) into 8-week-old male Sprague-Dawley rats produced hyperglycemia (460 ± 10 mg/dl, n=30). Three weeks after STZ treatment, adenovirus containing the human tissue kallikrein gene (Ad.CMV-cHK) or green fluorescence protein gene (Ad.CMV-GFP) under the control of CMV promoter/enhancer were injected via the tail vein into STZ-diabetic rats. Adenovirus-mediated kallikrein gene delivery caused a significant reduction of blood pressure as compared to control rats receiving control virus (170.8 ± 3.5 vs. 187.4 ± 4.2 mmHg, n=8, P<0.01). Rats were sacrificed and tissues were collected for analysis at 16 days after gene delivery. Histological examination of heart sections stained with periodic acid-Schiff (PAS) showed marked accumulation of glycogen in cardiomyocytes of diabetic rats. Kallikrein gene delivery significantly reduced cardiac glycogen accumulation as compared to rats receiving control adenovirus. Quantitative analysis confirmed the morphological observation that kallikrein gene delivery significantly reduced cardiac glycogen levels as compared to control diabetic rats injected withAd.CMV-GFP (0.5 ± 0.07 vs. 2.15 ± 0.83 mg glycogen/mg protein, n=6, P<0.05). Cardiac cAMP levels were significantly increased after kallikrein gene delivery. This is the first study to demonstrate that kallikrein gene delivery exerts a protective effect in the reduction of glycogen accumulation in cardiomyocytes of STZ-induced diabetic rats.

Stimulus interval-dependent differences in Ca2+ transients and contractile responses of diabetic rat cardiomyocytes.

The aim of this study was to gain further insights into the consequences of insulin-dependent diabetes mellitus on cardiomyocyte calcium handling. The effects of steady state and transient changes in stimulus frequency on the intracellular Ca2+ transient and cell shortening were examined in left ventricular cardiomyocytes isolated from the hearts of control and streptozotocin-induced diabetic rats. During steady state stimulation diabetic rat cardiomyocytes displayed a slower decay of the Ca2+ transient and longer times for maximum cell shortening and re-lengthening. At 1.5 mM extracellular [Ca2+], increasing stimulus frequency over the range 0.2-1.0 Hz led to an increase in resting and peak [Ca2+]i as well as the amplitude of the transient in both the control and diabetic groups. At frequencies greater than 0.4 Hz the amplitude of the transient was significantly depressed in diabetic rat cells and this was not normalized by increasing extracellular [Ca2+] to 2.5 mM. Recovery of sarcoplasmic reticulum (SR) Ca2+ release was measured from the time course of restitution of the intracellular Ca2+ transient. In both control and diabetic rat cardiomyocytes recovery of the transient occurred in two phases. In diabetic rat myocytes, the initial rapid phase of restitution at intervals <1 s was markedly slowed. The fraction of Ca2+ recirculating between the SR and the cytosol was estimated from the decline in amplitude of transients following post-rest potentiation. There was no difference in this fraction between control and diabetic rat cells either at 1.5 or 2.5 mM extracellular [Ca2+]. The blunted frequency response of diabetic rat cardiomyocytes at frequencies greater than 0.4 Hz is consistent with reduced SR Ca2+ uptake leading to reduced SR Ca2+ content and subsequent release. At stimulus intervals greater than 1 Hz this is likely to be exacerbated by slower recovery of SR Ca2+ release. Despite the evidence for depressed SR Ca2+ uptake, the relative amount of Ca2+ recirculating within diabetic rat cardiomyocytes remains unaltered. This is most likely due to an accompanying reduction in Ca2+ efflux from the cell due either to depressed Na+/Ca2+ exchanger activity, or an elevation in intracellular Na+ levels.

Effects of beta-adrenoceptor stimulation on contractility, [Ca2+]i, and Ca2+ current in diabetic rat cardiomyocytes.

The mechanism of the diminished inotropic response to beta-adrenoceptor stimulation in diabetic hearts was studied in enzymatically isolated diabetic rat ventricular myocytes in comparison with age-matched controls. The increases in contractions and intracellular Ca2+ concentration ([Ca2+]i) transients produced by isoproterenol were markedly diminished in diabetic myocytes. The inotropic and [Ca2+]i responses to forskolin and dibutyryl cAMP (DBcAMP) were also reduced. No significant difference was found in the stimulating effects of isoproterenol, forskolin, and DBcAMP on the L-type Ca2+ current (ICa) between control and diabetic myocytes. The rise of [Ca2+]i in response to rapid caffeine application, an index of sarcoplasmic reticulum (SR) Ca2+ content, was significantly decreased in diabetic myocytes. Isoproterenol, forskolin, and DBcAMP enhanced this [Ca2+]i response to caffeine in control myocytes more markedly than in diabetic myocytes. The changes in the isoproterenol responses observed in diabetic myocytes were prevented by insulin therapy. We conclude that 1) diabetes causes an impairment of the contractile and [Ca2+]i responses of cardiac myocytes when stimulated at both beta-adrenoceptors and the postreceptor level without affecting the ICa response and 2) altered SR functions of uptake and/or release of Ca2+ may primarily contribute to the diminished beta-adrenergic response.

Changes in microsomal membrane phospholipids and fatty acids and in activities of membrane-bound enzyme in diabetic rat heart.

Diabetes mellitus is associated with alterations in lipid metabolism and cardiac dysfunction despite an absence of coronary arteriosclerotic changes. To investigate mechanisms of cardiac dysfunction in diabetic cardiomyopathy, we studied the relation between activities of membrane-bound enzymes and surrounding phospholipids in rats with diabetes induced with a single intravenous injection of streptozotocin (65 mg/kg). We found that total phospholipid content of sarcoplasmic reticulum membrane increased significantly 8 weeks after treatment with streptozotocin owing to increases in phosphatidylcholine and phosphatidylethanolamine, a decrease in arachidonic acid, and an increase in docosahexaenoic acid in the early stage of diabetes. Sarcolemmal Na+/K(+)-ATPase activity and the number of receptors decreased in isolated cardiomyocytes of diabetic rats 8 weeks after streptozotocin administration. The Ca2+ uptake of both sarcoplasmic reticulum and mitochondria decreased simultaneously in permeabilized, isolated cardiomyocytes from diabetic rats. The depression of membrane-bound enzyme activities was correlated with alterations in phospholipids, which are closely related to the microenvironment of membrane-bound enzymes and influence intracellular Ca2+ metabolism. Because these changes in phospholipids and fatty acids were reversible with insulin therapy, they are diabetes-specific and might be a cause of cardiac dysfunction in diabetes.

Impaired function of mitochondria observed in situ in cardiomyocytes in diabetic rats

Impaired function of mitochondria observed in situ in cardiomyocytes in diabetic rats

G-protein-mediated regulation of the insulin-responsive glucose transporter in isolated cardiac myocytes

Isolated muscle cells from adult rat heart were used to study the involvement of G-proteins in the regulation of the glucose transporter by insulin and isoprenaline. Efficient modification of G-protein functions was established by measuring isoprenaline-stimulated cyclic AMP production, viability and ATP content after treating the cells with cholera toxin and pertussis toxin for 2 h. Under these conditions cholera toxin decreased the stimulatory action of insulin on 3-O-methylglucose transport by 56%, but pertussis toxin had no effect. Basal transport was not affected by toxin treatment. Isoprenaline increased 3-O-methylglucose transport by 63%. This effect was not mimicked by dibutyryl cyclic AMP, but was completely blocked by cholera toxin. Streptozotocin-diabetes abolished isoprenaline action and decreased stimulation of transport by 64%. Concomitantly, cholera-toxin sensitivity of glucose transport was lost in cells from diabetic animals. This was paralleled by a large decrease (87 +/- 4%) in mRNA expression of the insulin-regulatable glucose transporter, as shown by Northern-blot analysis of RNA isolated from cardiomyocytes of diabetic rats. These data suggest a functional association between the insulin-responsive glucose transporter and a cholera-toxin-sensitive G-protein mediating stimulation by insulin and isoprenaline.