Esma N. Okatan

ß-Blocker Timolol Prevents Arrhythmogenic Ca2+ Release and Normalizes Ca2+ and Zn2+ Dyshomeostasis in Hyperglycemic Rat Heart

Defective cardiac mechanical activity in diabetes results from alterations in intracellular Ca2+ handling, in part, due to increased oxidative stress. Beta-blockers demonstrate marked beneficial effects in heart dysfunction with scavenging free radicals and/or acting as an antioxidant. The aim of this study was to address how β-blocker timolol-treatment of diabetic rats exerts cardioprotection. Timolol-treatment (12-week), one-week following diabetes induction, prevented diabetes-induced depressed left ventricular basal contractile activity, prolonged cellular electrical activity, and attenuated the increase in isolated-cardiomyocyte size without hyperglycemic effect. Both in vivo and in vitro timolol-treatment of diabetic cardiomyocytes prevented the altered kinetic parameters of Ca2+ transients and reduced Ca2+ loading of sarcoplasmic reticulum (SR), basal intracellular free Ca2+ and Zn2+ ([Ca2+]i and [Zn2+]i), and spatio-temporal properties of the Ca2+ sparks, significantly. Timolol also antagonized hyperphosphorylation of cardiac ryanodine receptor (RyR2), and significantly restored depleted protein levels of both RyR2 and calstabin2. Western blot analysis demonstrated that timolol-treatment also significantly normalized depressed levels of some [Ca2+]i-handling regulators, such as Na+/Ca2+ exchanger (NCX) and phospho-phospholamban (pPLN) to PLN ratio. Incubation of diabetic cardiomyocytes with 4-mM glutathione exerted similar beneficial effects on RyR2-macromolecular complex and basal levels of both [Ca2+]i and [Zn2+]i, increased intracellular Zn2+ hyperphosphorylated RyR2 in a concentration-dependent manner. Timolol also led to a balanced oxidant/antioxidant level in both heart and circulation and prevented altered cellular redox state of the heart. We thus report, for the first time, that the preventing effect of timolol, directly targeting heart, seems to be associated with a normalization of macromolecular complex of RyR2 and some Ca2+ handling regulators, and prevention of Ca2+ leak, and thereby normalization of both [Ca2+]i and [Zn2+]i homeostasis in diabetic rat heart, at least in part by controlling the cellular redox status of hyperglycemic cardiomyocytes.

Enhancement of Cellular Antioxidant-Defence Preserves Diastolic Dysfunction via Regulation of Both Diastolic Zn2+ and Ca2+ and Prevention of RyR2-Leak in Hyperglycemic Cardiomyocytes

We examined whether cellular antioxidant-defence enhancement preserves diastolic dysfunction via regulation of both diastolic intracellular free Zn2+ and Ca2+ levels ([Zn2+]i and [Ca2+]i) levels N-acetyl cysteine (NAC) treatment (4 weeks) of diabetic rats preserved altered cellular redox state and also prevented diabetes-induced tissue damage and diastolic dysfunction with marked normalizations in the resting [Zn2+]i and [Ca2+]i. The kinetic parameters of transient changes in Zn2+ and Ca2+ under electrical stimulation and the spatiotemporal properties of Zn2+ and Ca2+ sparks in resting cells are found to be normal in the treated diabetic group. Biochemical analysis demonstrated that the NAC treatment also antagonized hyperphosphorylation of cardiac ryanodine receptors (RyR2) and significantly restored depleted protein levels of both RyR2 and calstabin2. Incubation of cardiomyocytes with 10 µM ZnCl2 exerted hyperphosphorylation in RyR2 as well as higher phosphorphorylations in both PKA and CaMKII in a concentration-dependent manner, similar to hyperglycemia. Our present data also showed that a subcellular oxidative stress marker, NF-κB, can be activated if the cells are exposed directly to Zn2+. We thus for the first time report that an enhancement of antioxidant defence in diabetics via directly targeting heart seems to prevent diastolic dysfunction due to modulation of RyR2 macromolecular-complex thereby leading to normalized [Ca2+]i and [Zn2+]i in cardiomyocytes.

Cardioprotective effect of selenium via modulation of cardiac ryanodine receptor calcium release channels in diabetic rat cardiomyocytes through thioredoxin system

Increased oxidative stress contributes to heart dysfunction via impaired Ca(2+) homeostasis in diabetes. Abnormal RyR2 function related with altered cellular redox state is an important factor in the pathogenesis of diabetic cardiomyopathy, while its underlying mechanisms remain poorly understood. In the present study, we used a streptozotocin-induced rat model of diabetic cardiomyopathy and tested a hypothesis that diabetes-related alteration in RyR2 function is related with ROS-induced posttranslational modifications. For this, we used heart preparations from either a diabetic rat or a sodium selenate (NaSe)-treated (0.3 mg/kg for 4 weeks) diabetic rat as well as either NaSe- (100 nmol/L) or thioredoxin (Trx; 5 μmol/L)-incubated (30 min) diabetic cardiomyocytes. Experimental approaches included imaging of intracellular free-Ca(2+) ([Ca(2+)]i) under both electrically stimulated and resting Fluo-3-loaded cardiomyocytes. RyR2-mediated SR-Ca(2+) leak was significantly enhanced in diabetic cardiomyocytes, resulting in reduced amplitude and prolonged time courses of [Ca(2+)]i transients compared to those of controls. Both SR-Ca(2+) leak and [Ca(2+)]i transients were normalized by treating diabetic rats with NaSe or by incubating diabetic myocytes with NaSe or Trx. Moreover, exposure of diabetic cardiomyocytes to antioxidants significantly improved [Ca(2+)]i handling factors such as phosphorylation/protein levels of RyR2, amount of RyR2-bound FKBP12.6 and activities of both protein kinase A and CaMKII. NaSe treatment also normalized the oxidative stress/antioxidant defense biomarkers in plasma as well as Trx activity and nuclear factor-κB phosphorylation in the diabetic rat heart. Collectively, these findings suggest that redox modification through Trx-system besides the glutathione system contributes to abnormal function of RyR2s in hyperglycemic cardiomyocytes, presenting a potential therapeutic target for treating diabetics to preserve cardiac function.

Interplay Between Cytosolic Free Zn(2+) and Mitochondrion Morphological Changes in Rat Ventricular Cardiomyocytes.

The Zn2+ in cardiomyocytes is buffered by structures near T-tubulus and/or sarcoplasmic/endoplasmic reticulum (S(E)R) while playing roles as either an antioxidant or a toxic agent, depending on the concentration. Therefore, we aimed first to examine a direct effect of ZnPO4 (extracellular exposure) or Zn2+ pyrithione (ZnPT) (intracellular exposure) application on the structure of the mitochondrion in ventricular cardiomyocytes by using histological investigations. The light microscopy data demonstrated that Zn2+ exposure induced marked increases on cellular surface area, an indication of hypertrophy, in a concentration-dependent manner. Furthermore, a whole-cell patch-clamp measurement of cell capacitance also supported the hypertrophy in the cells. We observed marked increases in mitochondrial matrix/cristae area and matrix volume together with increased lysosome numbers in ZnPO4- or ZnPT-incubated cells by using transmission electron microscopy, again in a concentration-dependent manner. Furthermore, we observed notable clustering and vacuolated mitochondrion, markedly disrupted and damaged myofibrils, and electron-dense small granules in Zn2+-exposed cells together with some implications of fission-fusion defects in the mitochondria. Moreover, we observed marked depolarization in mitochondrial membrane potential during 1-μM ZnPT minute applications by using confocal microscopy. We also showed that 1-μM ZnPT incubation induced significant increases in the phosphorylation levels of GSK3β (Ser21 and Ser9), Akt (Ser473), and NFκB (Ser276 and Thr254) together with increased expression levels in ER stress proteins such as GRP78 and calregulin. Furthermore, a new key player at ER-mitochondria sites, promyelocytic leukemia protein (PML) level, was markedly increased in ZnPT-incubated cells. As a summary, our present data suggest that increased cytosolic free Zn2+ can induce marked alterations in mitochondrion morphology as well as depolarization in mitochondrion membrane potential and changes in some cytosolic signaling proteins as well as a defect in ER-mitochondria cross talk.

Profiling of cardiac β-adrenoceptor subtypes in the cardiac left ventricle of rats with metabolic syndrome: Comparison with streptozotocin-induced diabetic rats.

Little is known about metabolic syndrome (MetS)-associated cardiomyopathy, especially in relation to the role and contribution of beta-adrenoceptor (β-AR) subtypes. Therefore, we examined the roles of β-AR subtypes in the cardiac function of rats with MetS (MetS group) and compared it with that of rats with streptozotocin (STZ)-induced diabetes (STZ group). Compared with the normal control rats, the protein levels of cardiac β1- and β2-AR in the MetS group were significantly decreased and with no changes in their mRNA levels, whereas the protein levels of β3-AR were similar to those of the controls. However, as shown previously, the protein levels of cardiac β1- and β2-AR in the STZ group were decreased, whereas the β3-AR levels were significantly increased by comparison with the controls. Additionally, the mRNA levels of β2- and β3-AR were increased, but β1-AR mRNA was decreased in the STZ group. Furthermore, left ventricular developed pressure responses to β3-AR agonist BRL37344 were increased in the STZ group but not in the MetS group, whereas for both groups, the responses to noradrenaline were not different from those of the controls. However, the response to stimulation with high concentrations of fenoterol was depressed in the MetS group, compared with the controls, but not in the STZ group. Consequently, our data suggest that the contribution of the β-AR system to cardiac dysfunction in the rats with MetS is not the same as that in the STZ group, although they have similar cardiac dysfunction with similar ultrastructural changes to the myocardium.

High-carbohydrate diet-induced myocardial remodelling in rats

BACKGROUND: A high-carbohydrate diet leads to the metabolic syndrome (MetS), which leads to an increased risk for cardiovascular dysfunction; however, the effect of high-carbohydrate diets on systemic metabolism has not yet been fully determined. It has been observed that abnormal fatty acid metabolism and increased oxidative stress play a role in the pathogenesis of MetS-related cardiovascular diseases. OBJECTIVE: To examine the effects of high sucrose intake on left ventricular contractility and structure of heart tissue. METHODS: MetS was induced in male rats with drinking water supplemented with 32% sucrose for 16 weeks. Oral glucose tolerance test results and parameters related to insulin resistance were used to validate MetS. RESULTS: Body weight and blood glucose levels were higher in the MetS group compared with age-matched controls. The increased serum leptin and triglyceride levels and decreased ghrelin levels further supported the existence of MetS in the MetS group. The ratio of total oxidant status to total antioxidant status measured in serum was also higher in the MetS group compared with the control group. The hemodynamic parameters of the MetS group, such as heart rate, and systolic and diastolic blood pressure levels, were markedly higher in the MetS group, while left ventricular developed pressure was significantly diminished with prolonged time course. Moreover, these functional alterations in cardiac preparations were further supported with structural changes such as significant increases in myofibril undulation and increased lipid droplets. CONCLUSIONS: These data highlight the link between high carbohydrate intake, MetS and cardiac dysfunction, in part due to increased systemic oxidative stress and lipid deposition in the heart tissue.

Contributions of phosphodiesterases to type-2 diabetes induced cardiac dysfunction

Materials & Method: A representative population sample of men and women, 45-69 years old was examined in Novosibirsk in the year 2003 – 2005 (baseline survey, HAPIEE project). We conducted the data collection on new cases of type 2 diabetes in the selected cohort of 10 years based on 2 sources of information: The re-screening of the same sample from 2006 – 2008 and on the basis of analysis of a database of Novosibirsk Municipal Register of type 2 diabetes in 2003 – 2014 in a prospective study. DM was defined by epidemiological criteria in patients with established diabetes history and in individuals with fasting blood glucose level ≥7.0 mmol/l (WHO, 1999).T current monitoring procedure of blood glucose for diabetes patients is typically invasive and thus cause pain. In this respect, we propose a novel biosensor configuration enabling recurrent and relatively painless blood glucose detection required for critical and chronic health care. This new concept mainly derives from the fact that in recent years dental implantation have become a mature clinical procedure without any pain in teeth and bone marrow. Hence, within a certified implant fixture, we designed the intra-oral amperometric biosensor for more convenient glucose monitoring. In the abstract, proper methods of drug polymerization and glucose oxidase (GOD) degradation are first investigated to assess the life time of the biosensor. Second the calibration process of the biosensor is carried out to evaluate its sensitivity and stability via C-V voltammogram. Extensive works are completed for interface circuits and miniature GOD-coated electrodes that can measure glucose concentrations up to 400 mg/dl. Besides a prototype module integrating the amperometric biosensor with a low-power bluetooth 4.0 communication chip to transmit the measured data is also successfully developed and tested. Also, additional functionalisation experiments implement the use of graphene-combined GOD electrodes to enhance the biosensor’s sensitivity and selectivity. In summary, with constant improvements on such devices, the proposed innovational technical platform is able to bring about more regular and relatively painless blood glucose detection procedures for enormous diabetic or chronic patients worldwide, and more potential medi-care applications.Background & Aim: RVX-208 binds selectively to the second ligand domain of Bromodomain and Extra-Terminal (BET) proteins and inhibits their activity. Each BET protein has two bromodomains that bind acetylated lysine in histones. When a BET protein binds ligand, it recruits transcriptional machinery to DNA and thereby modifies gene activity. Analysis of pooled data from two phase trials: SUSTAIN and ASSURE revealed a 55% relative risk reduction (RRR) of MACE in RVX-208 treated patients (n=331) vs. placebo (n=168). But in those with DM, RVX-208 treatments lead to a 77% RRR of MACE vs. placebo. RVX-208 increased production of ApoA-I yielding more High-Density Lipoprotein (HDL) particles. While these effects should lower MACE, the magnitude was more than expected, prompting studies to identify properties of RVX-208 beyond its effects on ApoA-I/HDL. Methods: Plasma biomarkers from SUSTAIN and ASSURE trials were analyzed. Microarray data from RVX208 treated Primary Human Hepatocytes (PHH) or Human Whole Blood (HWB) were used to identify differentially expressed genes, and guide measurements of specific proteins in clinical samples to confirm key findings. Results: Biomarkers from the trials showed significant increases (p<0.05, unless specified) between RVX-208 vs. placebo in: HDL-c (+3 mg/dL), ApoA-I (+7.5 mg/dL), large HDL (+0.7 umol/L), HDL size (+0.1 nm), and total HDL particles (+1.8 umol/L, p<0.07). Glucose in all patients (n=499) or in those with DM (n=192) given RVX-208 or placebo was unchanged vs. baseline. In patients with DM (n=119) and low HDL (<40 mg/dL), RVX-208 reduced glucose by -0.3 mmol/L but in placebo it increased +0.9 mmol/L. These modest changes do not predict the MACE reductions. Thus microarrays were used to survey PHH and HWB exposed to RVX-208. In PHH, RVX-208 decreased expression of genes in pathways for “cholesterol and fatty acid synthesis”, innate immunity and glucose processing. Most profound were, effects on complement and coagulation pathways, where RVX-208 down regulated expression of 19/26 and 20/33 genes respectively. These data were confirmed by RT-PCR of key mRNAs. Furthermore, specific complement and coagulation proteins were found to be decreased in plasma from the trials (range 7-12% vs. baseline). Microarrays from HWB exposed ex-vivo to RVX-208 identified pathways with known roles in atherogenesis including: Pro-inflammatory signaling, cell-cell interactions and extracellular matrix organization. RVX-208 significantly down regulated several pro-atherogenic genes (43/56) but unregulated anti-atherogenic genes (9/17), that control monocyte recruitment, migration and activation, macrophage function, inflammatory signaling and plaque stability to suggest an overall anti-atherosclerotic benefit. Conclusion: RVX-208 treatment is associated with marked MACE reductions in SUSTAIN and ASSURE patients and especially in those with DM. RVX-208 modifies cellular epigenetic to impact multiple biological processes that underlie CVD. Combined effects of RVX-208 on reverse cholesterol transport, vascular inflammation, innate immunity, atherosclerosis and thrombosis may explain its efficacy in reducing MACE. These data provide the foundation for the recently initiated phase 3 on RVX-208.T 2 diabetes is one of the major causes of cardiovascular disease. Insulin stimulates the generation of anti-atherosclerotic signaling molecule like Nitric Oxide (NO). The effects of increasing or decreasing insulin sensitivity, specifically on the endothelium due to the bioavailability of NO and vascular function, has not been widely investigated. We studied different models of insulin sensitivity and its effects in mediating atherosclerosis and cardiovascular complications. Different experimental models of insulin resistance at a whole-body level and specific to the endothelium demonstrated that insulin resistance eventually leads to an increase in generation of reactive oxygen species and accelerated atherosclerosis via the insulin receptor signaling pathways and these subjected mice have also demonstrated impaired acetylcholine-induced aortic relaxation. This impairment could be reversed by NADPH oxidase inhibitors, suggesting a role of reactive oxygen species in mediating insulin resistance and endothelial dysfunction.