L-Carnitine improves mechanical responses of cardiomyocytes and restores Ca2+ homeostasis during aging.

L-Carnitine (ß-hydroxy-γ-trimethylaminobutyric acid, LC) is a crucial molecule for the mitochondrial oxidation of fatty acids. It facilitates the transport of long-chain fatty acids into the mitochondrial matrix. The reduction in LC levels during the aging process has been linked to numerous cardiovascular disorders, including contractility dysfunction, and disrupted intracellular Ca2+ homeostasis. The aim of this study was to examine the effects of long-term (7 months) LC administration on cardiomyocyte contraction and intracellular Ca2+ transients ([Ca2+]i) in aging rats. Male albino Wistar rats were randomly assigned to either the control or LC-treated groups. LC (50 mg/kg body weight/day) was dissolved in distilled water and orally administered for a period of 7 months. The control group received distilled water alone. Subsequently, ventricular single cardiomyocytes were isolated, and the contractility and Ca2+ transients were recorded in aging (18 months) rats. This study demonstrates, for the first time, a novel inotropic effect of long-term LC treatment on rat ventricular cardiomyocyte contraction. LC increased cardiomyocyte cell shortening and resting sarcomere length. Furthermore, LC supplementation led to a reduction in resting [Ca2+]i level and an increase in the amplitude of [Ca2+]i transients, indicative of enhanced contraction. Consistent with these results, decay time of Ca2+ transients also decreased significantly in the LC-treated group. The long-term administration of LC may help restore the Ca2+ homeostasis altered during aging and could be used as a cardioprotective medication in cases where myocyte contractility is diminished.

Biomechanical Effects of Angiotensin 1-7 in Diabetes Rats Femur / Efectos Biomecánicos de la Angiotensina 1-7 en el Fémur de Ratas Diabéticas

SUMMARY: It is known that diabetes mellitus has late complications, including microvascular and macrovascular diseases. Diabetes can affect bones through biochemical markers of bone structure, density, and turnover. This study aimed to biomechanically investigate the bone-protective effects of angiotensin 1-7 (Ang 1-7), one of the active peptides in the renin-angiotensin system, in rats with diabetes. Thirty male Wistar albino rats, three months old and weighing 250-300 g, were divided into four groups: diabetes, Ang 1- 7, diabetes plus Ang 1-7, and control. One month later, diabetes developed in rats; the rats were sacrificed, and their right femur was removed. Three-point bending biomechanical tests were performed on the femurs. The diabetic group had significantly higher bone fragility than the other groups (Pr >.05). Bone fragility was lower, and bone flexibility was higher in the Ang 1-7 groups (Pr>F value 0.05). As a result of our study, the effect of Ang 1-7 on the bones of rats with diabetes was investigated biomechanically. Ang 1-7 has a protective impact on the bones of rats with diabetes.

Se sabe que la diabetes mellitus tiene complicaciones tardías, incluyendo enfermedades microvasculares y macrovasculares. La diabetes puede afectar los huesos a través de los marcadores bioquímicos de la estructura, la densidad y el recambio óseo. Este estudio tuvo como objetivo investigar biomecánicamente los efectos protectores en los huesos de la angiotensina 1-7 (Ang 1-7), uno de los péptidos activos en el sistema renina-angiotensina, en ratas con diabetes. Treinta ratas albinas Wistar macho, de tres meses de edad y con un peso de 250-300 g, se dividieron en cuatro grupos: diabetes, Ang 1-7, diabetes más Ang 1-7 y control. Un mes después, se desarrolló diabetes en ratas; se sacrificaron los animales y se extrajo su fémur derecho. Se realizaron pruebas biomecánicas de flexión de tres puntos en los fémures. El grupo diabéticos tenía una fragilidad ósea significativamente mayor que los otros grupos (Pr > 0,05). La fragilidad ósea fue menor y la flexibilidad ósea fue mayor en los grupos Ang 1-7 (valor Pr>F 0,05). Como resultado de nuestro estudio, se determinó biomecánicamente el efecto de Ang 1-7 en los huesos de ratas con diabetes. Se concluye que Ang 1-7 tiene un impacto protector en los huesos de ratas diabéticas.

Vitamin D3 Promotes Structural and Functional Recovery After Vincristine-Induced Peripheral Neuropathy in Rats: An Experimental Study.

Background Vincristine-induced peripheral neuropathy (VIPN) is a distal axonopathy characterized by the loss of distal myelinated axons. This study aimed to assess the potential neuroregenerative roles of vitamin D3 using functional and electron microscopic analyses in a rat model of VIPN. Methodology A total of 40 female Wistar rats were randomly divided into four main groups: Group 1 (control, n = 10), Group 2 (vincristine, n = 10), Group 3 (vincristine + vitamin D3, n = 10), and Group 4 (vincristine + vehicle, n = 10). Vincristine was administered intraperitoneally at a dose of 0.15 mg/kg, for two weeks, to induce peripheral neuropathy. Following successful induction, vitamin D3 (500 IU/kg/day) and vehicle treatments were applied weekly over four weeks. Structural (electron microscopic analysis) and functional analysis (von Frey test, pinch test, and electrophysiological analysis) were performed to assess functional recovery after peripheral nerve impairment. Results Withdrawal responses to mechanical allodynia and pinch tests were significantly higher in the vitamin D3-treated group (P < 0.05). The electrophysiological analysis also supported these results. Electron microscopic evaluation revealed that the remyelinated nerve fibers in the vitamin D3-treated group (Group 3) had thick myelin sheaths and normal axonal morphology. Conclusions Our study demonstrated that vitamin D3 could promote functional and structural recovery in a rat model of VIPN. Further studies should be conducted to elucidate the underlying mechanisms by which vitamin D3 exerts its regenerative effects in VIPN, using alternative administration protocols.

Pharmacological blockade of angiotensin II receptor restores diabetes-associated reduction of store operated Ca2+ entry in adult cardiomyocytes.

The store-operated Ca2+ entry (SOCE) represents an important route for generating cellular Ca2+ signals that are implicated in physiological and various pathological scenarios that include diabetic cardiomyopathy (DM-CMP) which is well known to have Ca2+ dysregulation among other salient features. In this study, we investigated the role of SOCE in Ca2+ handling of cardiomyocytes obtained from adult male Wistar rats that were made diabetic by intraperitoneal administration of streptozotocin (STZ 50 mg/kg). We also included another group of rats with diabetes induced by STZ administration but received an angiotensin II receptor blocker - losartan. In whole cell recordings with isolated cardiomyocytes, the SOCE-representative whole-cell current ICRAC was found to be significantly reduced for the diabetic group compared to the control group and chronic losartan treatment could restore ICRAC to a level comparable to the control group. However, in contrast to the observed reduction in ICRAC, Orai1 and Orai3 proteins were found to be significantly upregulated in diabetic condition whereas no significant change in the expression levels of Stim1, Stim2 and Orai2 was observed. Also, losartan treatment did not affect the expression pattern of these key proteins for SOCE in diabetic group. The observed imbalance between the functional read out of SOCE (peak ICRAC size) and expression levels of the underlying proteins was puzzling but could be, among other possibilities, due to impairment of interaction between Stim and Orai proteins. We argue that the observed changes in SOCE with diabetes could be a contributing factor for the Ca2+ dyshomeostasis associated with diabetic cardiomyopathies and blockade of angiotensin II receptor may potentially restore normal SOCE in diabetic cardiomyocytes.

Comparison of nerve conduction velocity distribution methods by cold exposure and ischemia.

PURPOSE: Non-invasive estimation of the conduction velocity distribution (CVD) of a peripheral nerve has the potential to both improve clinical diagnoses of pathology and to observe the progress of the disease or the efficacy of treatments. Comparisons were made of the performance of three non-invasive CVD estimation methods proposed by independent research groups on peripheral nerve bundles under different conditions. METHODS: The first method (Cummins) uses a nerve compound action potential (CAP) with temporal dispersion and a mathematical single fiber action potential (SFAP). The second method (Barker) uses two CAPs and a non-mathematical SFAP waveform. The third method (Hirose) uses two CAPs recorded from distal and proximal sites. The Cummins and Barker methods have iterative solutions in the time domain while the Hirose method is a deconvolution estimator in the frequency domain. In order to compare these methods, we used cold exposure to affect primarily motor fibers and ischemia to affect primarily sensory fibers on rat caudal nerve bundles. RESULTS: The Cummins method is sensitive to changes in motor and sensory fiber percentages in CVD if it is used with the volume conductor model. The Barker and Hirose methods are sensitive to motor fiber percentages in CVD but they cannot detect changes in sensory fiber percentages accurately. CONCLUSIONS: Estimation of the CVD using a priori SFAP created with a volume conductor model can non-invasively supply accurate and precise information about fiber groups in a peripheral nerve bundle.

Sulfur Dioxide Derivative Prevents the Prolongation of Action Potential During the Isoproterenol-Induced Hypertrophy of Rat Cardiomyocytes.

Exogenous SO2 is toxic especially to the pulmonary and cardiovascular system, similar to nitric-oxide, carbon-monoxide, and hydrogen-sulfide. Endogenous SO2 is produced in many cell types. The SO2 content of the rat heart has been observed to substantially decrease during isoproterenol-induced hypertrophy. This study sought to determine whether an SO2 derivative could inhibit the prolongation of action potentials during the isoproterenol-induced hypertrophy of rat cardiomyocytes and explore the ionic currents. Alongside electrocardiogram recordings, the voltage and current-clamped measurements were conducted in the enzymatically isolated left ventricular cardiomyocytes of Wistar rats. The consistency of the results was evaluated by the novel mathematical electrophysiology model. Our results show that SO2 significantly blocked the prolongation of QT-interval and action potential duration. Furthermore, SO2 did not substantially affect the Na+ currents and did not improve the decreased steady-state and transient outward K+ currents, but it reverted the reduced L-type Ca2+ currents (I CaL) to the physiological levels. Altered inactivation of I CaL was remarkably recovered by SO2. Interestingly, SO2 significantly increased the Ca2+ transients in hypertrophic rat hearts. Our mathematical model also confirmed the mechanism of the SO2 effect. Our findings suggest that the shortening mechanism of SO2 is related to the Ca2+ dependent inactivation kinetics of the Ca2+ current.

Concealed role of red blood cells in pathogenesis of pulmonary arterial hypertension: Decreased red blood cell nitric oxide generation and effect of Rho-Kinase inhibitor fasudil.

BACKGROUND: Pulmonary arterial hypertension (PAH) is a devastating disease characterized with alterations in pulmonary vasculature yielding increased pulmonary arterial resistance. Emerging evidences suggest important regulatory roles of red blood cells (RBCs) on nitric oxide (NO) bioavailability, mainly by modulating their endothelial nitric oxide synthase (eNOS) enzyme activity. OBJECTIVE: The aim of this pilot study was to evaluate the alterations in RBC eNOS activity and intracellular NO generation in PAH patients and the modulatory effects of Rho-Kinase (ROCK) inhibitors. METHODS: RBCs were isolated from patients with PAH and age-matched healthy subjects and were analyzed for their eNOS activity and NO generation capacity under the conditions of the presence or absence of ROCK inhibitor, fasudil. Phosphotidylserine (PS) exposure was also defined. RESULTS: eNOS activity and intracellular NO generation were lower in RBC from PAH patients. ROCK inhibitor increased basal eNOS activity and improved NO generation capacity of RBC of PAH patients to healthy control levels. PS exposure levels were also higher in RBC of PAH patients. CONCLUSIONS: This study provides first evidences for decreased RBC eNOS activity due to its ROCK mediated negative regulation in PAH patients. Considering increased ROCK activity contribution to progression of PAH, ROCK inhibition influences NO bioavailability through RBC eNOS, in addition to endothelial eNOS.

Mathematical model of the ventricular action potential and effects of isoproterenol-induced cardiac hypertrophy in rats.

Mathematical action potential (AP) modeling is a well-established but still-developing area of research to better understand physiological and pathological processes. In particular, changes in AP mechanisms in the isoproterenol (ISO) -induced hypertrophic heart model are incompletely understood. Here we present a mathematical model of the rat AP based on recordings from rat ventricular myocytes. In our model, for the first time, all channel kinetics are defined with a single type of function that is simple and easy to apply. The model AP and channels dynamics are consistent with the APs recorded from rats for both Control (absence of ISO) and ISO-treated cases. Our mathematical model helps us to understand the reason for the prolongation in AP duration after ISO application while ISO treatment helps us to validate our mathematical model. We reveal that the smaller density and the slower gating kinetics of the transient K+ current help explain the prolonged AP duration after ISO treatment and the increasing amplitude of the rapid and the slow inward rectifier currents also contribute to this prolongation alongside the flux in Ca2+ currents. ISO induced an increase in the density of the Na+ current that can explain the faster upstroke. We believe that AP dynamics from rat ventricular myocytes can be reproduced very well with this mathematical model and that it provides a powerful tool for improved insights into the underlying dynamics of clinically important AP properties such as ISO application.

Rho-kinase is a negative regulator of red blood cell eNOS under basal conditions.

BACKGROUND: Rho-kinase, an effector of the small GTPase RhoA, is known to be a novel inhibitory regulator of eNOS in endothelial cells under basal conditions and disease states. However, although RBC possesses active RhoA/Rho-kinase pathway, Rho-kinase mediated eNOS regulation has not been investigated in RBC, so far. OBJECTIVE: The aim of the present study is to investigate whether eNOS activity is regulated by Rho-kinase under basal conditions and to evaluate whether inhibition of this enzyme causes eNOS activation and intracellular NO production in RBC. METHODS: RBC packeds were isolated from healthy volunteers and resuspended in Hepes solution at a hematocrit of 0.01 l/l. Intracellular NO and Ca+2 levels and eNOS activation measured by flow cytometry in response to Rho-kinase inhibitors, fasudil and Y-27632, in the absence and presence of NOS, and PI3K inhibitors. RESULTS: Rho-kinase inhibitors fasudil and Y-27632 found to increase intracellular NO concentrations. These inhibitors also cause enhancement of intracellular Ca+2 and serine 1177 phosphorylated eNOS levels. Besides, although these responses have shown to be suppressed by NOS enzyme, PI3K inhibition had no effect on this mechanism. CONCLUSIONS: The results of the present study demonstrated that RBC eNOS enzyme activity is regulated by inhibitory Rho-kinase pathway under basal conditions and inhibition of this pathway enhances the activity of eNOS in RBC. This activation is mediated by both intracellular Ca+2 and Serine 1177 phosphorylated eNOS increment, with no contribution of AKT activation, in RBC. The mechanism we described here gives first evidences about Rho-kinase mediated eNOS regulation in RBC under basal conditions. This pathway could also be more important under disease states.

Extracellular ATP activates eNOS and increases intracellular NO generation in Red Blood Cells.

BACKGROUND: It has been well documented that ATP activates NOS enzymes and causes increased NO production in several cell types. Although RBC known to possesses eNOS enzyme activity, it has not been investigated whether RBC eNOS could be induced by extracellular ATP. OBJECTIVE: The aim of the present study is to evaluate extracellular ATP mediated eNOS activation and NO production in RBC. METHODS: RBC packed were isolated from healthy volunteers and re-suspended in Hepes solution at a hematocrit of 0.01 l/l. Intracellular NO and Ca+2 levels and eNOS activation measured by flow cytometry in response to P2X receptor agonist, Bz-ATP, in the absence and presence of NOS, P2 receptors and PI3K inhibitors. RESULTS: P2X receptor agonist Bz-ATP found to increase intracellular NO, Ca+2 and serine 1177 phosphorylated eNOS levels and these responses have shown to be suppressed by NOS enzyme, P2 receptors and PI3K inhibitors. CONCLUSIONS: The results of the study clearly demonstrated extracellular ATP induced NO generation in RBC through intracellular Ca+2 and PI3K/Akt pathways. The mechanism we described here might be important at basal conditions and also in conditions with increased ATP release.

Effect of magnesium supplementation on blood rheology in NOS inhibition-induced hypertension model.

This study investigated the effects of magnesium on blood rheological properties and blood pressure in nitric oxide synthase (NOS) inhibition-induced hypertension model. Hypertension was induced by oral administration of the nonselective NOS inhibitor N-nitro-L-arginine methyl ester (L-NAME, 25 mg/kg/day) for 6 weeks and systolic blood pressure was measured by the tail-cuff method. The groups receiving magnesium supplementation were fed with rat chow containing 0.8% magnesium oxide during the experiment. At the end of experiment, blood samples were obtained from abdominal aorta, using ether anesthesia. Plasma and erythrocyte magnesium levels were determined by the atomic absorption spectrometer. RBC deformability and aggregation were determined by rotational ektacytometry. Plasma fibrinogen concentration was evaluated by ELISA. Whole blood and plasma viscosities were determined by viscometer and intracellular free Ca++ level was measured by using spectroflurometric method. Blood pressure was elevated in hypertensive groups and suppressed by magnesium therapy. Plasma viscosity and RBC aggregation were found to be higher in hypertensive rats than control animals and these parameters significantly decreased in magnesium supplemented hypertensive animals. Other measurements were not different between experimental groups. These results confirm that blood pressure, plasma viscosity and RBC aggregation increased in NOS inhibition-induced hypertension model and oral magnesium supplementation improved these parameters.

Melatonin leads to axonal regeneration, reduction in oxidative stress, and improved functional recovery following sciatic nerve injury.

Despite recent advances in microsurgical techniques and equipments, recovery of function following repair of transected nerves often remains suboptimal. Contrary to traumatic injuries vascular damage that causes peripheral nerve injury has not been well-documented in the literature. In the present study a total of 40 female rats were randomly divided into four groups: Group 1: intact controls (n: 10), Group 2: sham-operated (n: 10), Group 3: vehicle-treated (n: 10), Group 4: melatonin-treated (n: 10). Sciatic nerve damage was created by stripping of the epineurial vessels around the nerve. 50 mg/kg Melatonin was injected intraperitoneally immediately after epineurial stripping in Group 4 in the course of 4 postoperative weeks. We found that melatonin administration after stripping of the epineurial vessels exerted a beneficial effect on axonal regeneration and functional recovery was confirmed by functional (sensory-motor, biochemical, and electrophysiological analyses) and morphological (light microscopic and ultrastructural analyses) data. In the light of these results we concluded that melatonin in a model of sciatic nerve injury leads to axonal regeneration, reducing in oxidative stress, and improved functional recovery.

Serum lipid peroxidation markers are correlated with those in brain samples in different stress models.

OBJECTIVE: Stress can stimulate increased production of oxygen radicals. We investigated the correlations between serum levels of lipid peroxidation markers and those in brain samples in different stress models. METHODS: Animals (n = 96) were divided equally into eight groups: a control group and groups treated with vitamin E (Vit E); exposed to immobilisation stress; exposed to immobilisation stress and treated with Vit E; exposed to cold stress; exposed to cold stress and treated with Vit E; exposed to both immobilisation and cold stress; and a final group exposed to both immobilisation and cold stress and treated with Vit E. Thiobarbituric acid-reactive substance (TBARS) in brain samples and levels of TBARS, corticosterone, conjugated dienes (CD), lipids, and paraoxonase-1 (PON1) activity in serum were analysed. RESULTS: Serum corticosterone (p < 0.001), CD (p < 0.05), lipid (p < 0.05) levels, and brain TBARS (p < 0.05) levels were significantly higher in all stress groups than in controls, and the elevated levels were reversed in the Vit E-treated stress groups (p < 0.05). Serum PON1 activity was not different among the groups (p > 0.05). Serum TBARS levels increased significantly in all stress groups (p < 0.05), but this elevation was only reversed in the group exposed to both immobilisation and cold stress and treated with Vit E (p < 0.001). CONCLUSION: These results suggest that serum levels of lipid peroxidation markers can be determined readily and may be useful as indicators to evaluate the effects of oxidative stress in the brain.

Long-term administration of rosuvastatin prevents contractile and electrical remodelling of diabetic rat heart.

In recent years, many findings have been presented about the potential benefit of statin therapy on diabetes-induced cardiovascular complications. Cardioprotective effects of statins were suggested to be mediated at least in part through inhibition of small GTPases, particularly those of the Rho family. The present study was designed to examine whether rosuvastatin can improve electrical remodeling and contractile dysfunction in type 1 diabetic rat heart via modulation of RhoA pathway. Type 1 diabetes was induced by single dose injection of STZ (50 mg/kg). One week after injection rosuvastatin (10 mg/kg/day) and sham treatment was given for 5 weeks in the diabetic rats, as well as in control groups. Shortening and Ca²âº transients were recorded in myocytes loaded with Fura2-AM. Membrane currents and Ca²âº transients were measured synchronously via whole-cell patch clamping. In untreated diabetic rats, relaxation of shortening and decay of the matched Ca²âº transients were prolonged. Fractional shortening and Ca²âº transients were also decreased. Rosuvastatin treatment reversed those changes. I(CaL) density did not change in either group but rosuvastatin recovered the loss of sarcoplasmic reticulum Ca²âº and Na⁺/Ca²âº exchange as evidenced from amplitude and decay of caffeine-induced Ca²âº transients, peak INCX and calculated sarcoplasmic reticulum Ca²âº content. Diabetes-induced attenuation of I(to) and I(sus) was also reversed, whilst I(K1) was unchanged in diabetes and unaffected by treatment. Rosuvastatin prevented the diabetes-induced increase in RhoA expression. Plasma cholesterol and triglyceride levels were higher in diabetic rats, but rosuvastatin reduced only the latter. In conclusion, HMG-CoA reductase inhibitor rosuvastatin can prevent diabetes-induced electrical and functional remodeling of heart due to inhibition of RhoA signalling rather than reduction of cholesterol level.

Effect of angiotensin II type 1 receptor blocker on osteoporotic rat femurs.

BACKGROUND: Osteoblasts and osteoclasts are known to express Ang II type I (AT1) receptor in cell cultures, suggesting the existence of local renin-angiotensin system (RAS) in bone. This study was designed to investigate the effects of losartan as AT1 receptor blocker on ovariectomized rats' femur. METHODS: Losartan (5 mg/kg/day) was administered via oral gavage for 8 weeks. Bone mineral density (BMD) was measured using dual energy X-ray absorptiometry, while tensile and three-point bending tests were performed for evaluation of biomechanical properties of bone. The trabecular porosity was analyzed by scanning electron microscopy. RESULTS: There was a significant decrease in BMD values of ovariectomized rats' femurs which were reversed by losartan treatment. According to tensile test results, ultimate tensile strength and strain values of losartan treated ovariectomized rats' femurs increased and decreased, respectively, when compared to that of ovariectomized animals. Losartan treatment also caused a significant recovery in flexural strength and modulus parameters regarding respective control values, which mean losartan treated ovariectomized rats' femur had more force tolerance until break than ovariectomized rats' femur. Quantitative microscopic analysis showed larger trabecular porosity in ovariectomized rats than control rat femurs and it was significantly decreased after losartan treatment. CONCLUSION: Blockage of AT1 receptor increased strength, mass and trabecular connections of ovariectomized rat femurs. Therefore, it is tempting to speculate that drugs, including AT1 receptor blockers, may be used for the treatment of osteoporosis or reduction of its detrimental effects in the future.

Shear stress activation of nitric oxide synthase and increased nitric oxide levels in human red blood cells.

Red blood cells (RBC) play an important role in the balance between generation and scavenging of nitric oxide (NO) and hence its local bioavailability and influence on vasomotor control. Previous studies have reported increased NO levels in RBC suspensions subsequent to exposure to shear forces; the present study was designed to further investigate changes in intracellular NO concentration and possible mechanisms involved for RBC exposed to well-controlled shear forces. Attached human RBC were subjected to shear stresses up to 0.1Pa in a parallel-plate flow channel; fluorescent methods were used to monitor changes in intracellular NO and calcium concentrations. Intracellular NO concentration, estimated by the fluorescence level of 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM), increased sharply within 30s following the application of shear stress between 0.013 and 0.1Pa. This increase was only partially prevented by the absence of l-arginine and by the presence of l-N-acetyl-methyl-arginine (L-NAME), strongly suggesting that this response was in part related to the activation of NO-synthase (NOS) enzyme. The increase in intracellular NO concentration under shear stress was also inhibited by calcium chelation in the suspending medium, indicating the role of calcium entry for NOS activation. Increases of intracellular calcium concentrations under the same shearing conditions were demonstrated by monitoring Fluo-3/AM fluorescence in RBC exposed to shear stress. Serine 1177 phosphorylated NOS protein, the activated form of the enzyme determined by immunohistochemistry, was found to be significantly increased following the exposure of RBC to 0.1Pa shear stress for 1min. These data confirm that RBC possess a NOS enzyme that is actively synthesizing NO and activated by effective shear forces. The data also suggest that there may be additional (e.g., non-enzymatic) NO generating mechanisms in RBC that are also enhanced under shear stress.

Sex-related effects on diabetes-induced alterations in calcium release in the rat heart.

The present study was designed to determine whether the properties of local Ca(2+) release and its related regulatory mechanisms might provide insight into the role of sex differences in heart functions of control and streptozotocin-induced diabetic adult rats. Left ventricular developed pressure, the rates of pressure development and decay (+/-dP/dt), basal intracellular Ca(2+) level ([Ca(2+)](i)), and spatiotemporal parameters of [Ca(2+)](i) transients were found to be similar in male and female control rats. However, spatiotemporal parameters of Ca(2+) sparks in cardiomyocytes isolated from control females were significantly larger and slower than those in control males. Diabetes reduced left ventricular developed pressure to a lower extent in females than in males, and the diabetes-induced depressions in both +dP/dt and -dP/dt were less in females than in males. Diabetes elicited a smaller reduction in the amplitude of [Ca(2+)](i) transients in females than in males, a smaller reduction in sarcoplasmic reticulum-Ca(2+) load, and less increase in basal [Ca(2+)](i). Similarly, the elementary Ca(2+) events and their control proteins were clearly different in both sexes, and these differences were more marked in diabetes. Diabetes-induced depression of the Ca(2+) spark amplitude was significantly less in females than in matched males. Levels of cardiac ryanodine receptors (RyR2) and FK506-binding protein 12.6 in control females were significantly higher than those shown in control males. Diabetes induced less RyR2 phosphorylation and FK506-binding protein 12.6 unbinding in females. Moreover, total and free sulfhydryl groups were significantly less reduced, and PKC levels were less increased, in diabetic females than in diabetic males. The present data related to local Ca(2+) release and its related proteins describe some of the mechanisms that may underlie sex-related differences accounting for females to have less frequent development of cardiac diseases.

Gender related differential effects of Omega-3E treatment on diabetes-induced left ventricular dysfunction.

The present study was designed to determine whether there are beneficial effects of intake of Omega-3E (containing 70% pure omega-3 and 2% natural vitamin E) in cardiac dysfunction of diabetic rats. We also examined whether there are gender-related differences in the responses to the intake of Omega-3E on the heart dysfunction. Experiments were performed by using Langendorff-perfused hearts from normal, diabetic (with 50 mg/kg streptozotocin), and Omega-3E (50 mg/kg body weight/day) treated diabetic 3-month-old Wistar rats. Omega-3E treatment of the diabetics caused small, but significant decrease (13% and 14% female versus male) in the blood glucose level. Omega-3E treatment of the diabetic female rats did not prevent diabetes-induced decrease in left ventricular developed pressure (LVDP) and increase in left ventricular end-diastolic pressure (LVEDP) with respect to the control female rats. On the other hand, the treatment of diabetic male rats caused significant recovery in depressed LVDP. Furthermore, such treatment of diabetic female and male rats caused significant recovery in depressed rates of changes of developed pressure. This effect was more significant in males. Besides, Omega-3E caused significant further lengthening in the diabetes-induced increased time to the peak of the developed pressure in females, while it normalized the lengthening in the relaxation of the developed pressure in diabetic males. In addition, Omega-3E treatment caused significant restorations in the diabetes-induced altered activities of antioxidant enzymes without any significant gender discrepancy. Present data show that there are gender related differences in diabetic heart dysfunction and the response to antioxidant treatment.

Restoration of diabetes-induced abnormal local Ca2+ release in cardiomyocytes by angiotensin II receptor blockade.

Stimulation of local renin-angiotensin system and increased levels of oxidants characterize the diabetic heart. Downregulation of ANG II type 1 receptors (AT(1)) and enhancement in PKC activity in the heart point out the role of AT(1) blockers in diabetes. The purpose of this study was to evaluate a potential role of an AT(1) blocker, candesartan, on abnormal Ca(2+) release mechanisms and its relationship with PKC in the cardiomyocytes from streptozotocin-induced diabetic rats. Cardiomyocytes were isolated enzymatically and then incubated with either candesartan or a nonspecific PKC inhibitor bisindolylmaleimide I (BIM) for 6-8 h at 37 degrees C. Both candesartan and BIM applied on diabetic cardiomyocytes significantly restored the altered kinetic parameters of Ca(2+) transients, as well as depressed Ca(2+) loading of sarcoplasmic reticulum, basal Ca(2+) level, and spatiotemporal properties of the Ca(2+) sparks. In addition, candesartan and BIM significantly antagonized the hyperphosphorylation of cardiac ryanodine receptor (RyR2) and restored the depleted protein levels of both RyR2 and FK506 binding protein 12.6 (FKBP12.6). Furthermore, candesartan and BIM also reduced the increased PKC levels and oxidized protein thiol level in membrane fraction of diabetic rat cardiomyocytes. Taken together, these data demonstrate that AT(1) receptor blockade protects cardiomyocytes from development of cellular alterations typically associated with Ca(2+) release mechanisms in diabetes mellitus. Prevention of these alterations by candesartan may present a useful pharmacological strategy for the treatment of diabetic cardiomyopathy.

Effects of diabetes on ryanodine receptor Ca release channel (RyR2) and Ca2+ homeostasis in rat heart.

The defects identified in the mechanical activity of the hearts from type 1 diabetic animals include alteration of Ca2+ signaling via changes in critical processes that regulate intracellular Ca2+ concentration. These defects result partially from a dysfunction of cardiac ryanodine receptor calcium release channel (RyR2). The present study was designed to determine whether the properties of the Ca2+ sparks might provide insight into the role of RyR2 in the altered Ca2+ signaling in cardiomyocytes from diabetic animals when they were analyzed together with Ca2+ transients. Basal Ca2+ level as well as Ca2+-spark frequency of cardiomyoctes isolated from 5-week streptozotocin (STZ)-induced diabetic rats significantly increased with respect to aged-matched control rats. Ca2+ transients exhibited significantly reduced amplitude and prolonged time courses as well as depressed Ca2+ loading of sarcoplasmic reticulum in diabetic rats. Spatio-temporal properties of the Ca2+ sparks in cardiomyocytes isolated from diabetic rats were also significantly altered to being almost parallel to the changes of Ca2+ transients. In addition, RyR2 from diabetic rat hearts were hyperphosphorylated and protein levels of both RyR2 and FKBP12.6 depleted. These data show that STZ-induced diabetic rat hearts exhibit altered local Ca2+ signaling with increased basal Ca2+ level.