Heart Of Adult Wistar Rats Research Articles

Anti-cancer tyrosine kinase inhibitors increase oxidative stress in primary cardiac fibroblasts

Anti-cancer tyrosine kinase inhibitors (TKI) are known to exert cardiotoxicity that can result in heart failure. Here we have used primary adult rat cardiac fibroblasts (CFs) to examine whether imatinib and sunitinib (both TKIs) increase oxidative stress and cause increased oxidation and activation of calcium/calmodulin dependent protein kinase II (CaMKII). Activation of CaMKII is known to be important for cellular function but excessive activation is linked to cardiac pathology. CFs, isolated from adult Wistar rat hearts, were maintained in culture and treated over 24 hour with the TKIs sunitinib and imatinib (0.1–20 µM) in serum-free medium (DMEM). Cell phenotype and growth was monitored using a Nikon Eclipse (TE300) inverted microscope and ROS assays performed using a specially designed assay kit (Abcam, UK) which uses DCFDA to measure hydroxyl, peroxyl and other ROS activity. Quantitative immunoblotting was used to assess ox-CaMKII protein levels. Treatment with both TKIs caused a dose-dependent decrease in cell viability. Sunitinib exerted more toxic effects at lower concentrations than imatinib. Decreased cell viability was evident at concentrations as low as 1 µM for sunitinib with dramatic cell loss (>50%) evident at 10–20 µM. Significantly increased ROS levels (using the DFCDA-based assay) were observed in the presence of both imatinib (4956±102 vs 1984±358 (imatinib vs vehicle (a.u.), n=3, p In conclusion, both imatinib and sunitinib cause increased oxidative stress in adult primary CFs with resultant activation of CaMKII. These effects are evident at lower concentrations of sunitinib than for imatinib and reflect the increased cardiotoxicity of this TKI reported in the patient.

Optogenetic termination of ventricular arrhythmias in the whole heart: towards biological cardiac rhythm management.

AimsCurrent treatments of ventricular arrhythmias rely on modulation of cardiac electrical function through drugs, ablation or electroshocks, which are all non-biological and rather unspecific, irreversible or traumatizing interventions. Optogenetics, however, is a novel, biological technique allowing electrical modulation in a specific, reversible and trauma-free manner using light-gated ion channels. The aim of our study was to investigate optogenetic termination of ventricular arrhythmias in the whole heart.Methods and resultsSystemic delivery of cardiotropic adeno-associated virus vectors, encoding the light-gated depolarizing ion channel red-activatable channelrhodopsin (ReaChR), resulted in global cardiomyocyte-restricted transgene expression in adult Wistar rat hearts allowing ReaChR-mediated depolarization and pacing. Next, ventricular tachyarrhythmias (VTs) were induced in the optogenetically modified hearts by burst pacing in a Langendorff setup, followed by programmed, local epicardial illumination. A single 470-nm light pulse (1000 ms, 2.97 mW/mm2) terminated 97% of monomorphic and 57% of polymorphic VTs vs. 0% without illumination, as assessed by electrocardiogram recordings. Optical mapping showed significant prolongation of voltage signals just before arrhythmia termination. Pharmacological action potential duration (APD) shortening almost fully inhibited light-induced arrhythmia termination indicating an important role for APD in this process.ConclusionBrief local epicardial illumination of the optogenetically modified adult rat heart allows contact- and shock-free termination of ventricular arrhythmias in an effective and repetitive manner after optogenetic modification. These findings could lay the basis for the development of fundamentally new and biological options for cardiac arrhythmia management.

Differential expression and regulation of anti-hypertrophic genes Npr1 and Npr2 during β-adrenergic receptor activation-induced hypertrophic growth in rats

We sought to determine the effect of chronic activation of β-adrenergic receptor (β-AR) on the left ventricular (LV) expression profile of Npr1 and Npr2 (coding for NPR-A and NPR-B, respectively) genes, and the functional activity of these receptors in adult Wistar rat hearts. The Npr1 gene expression was markedly reduced (3.5-fold), while the Npr2 gene expression was up regulated (4-fold) in Isoproterenol (ISO)-treated heart as compared with controls. A gradual reduction in NPR-A protein (3-fold), cGMP levels (75%) and a steady increased expression of NPR-B protein (4-fold), were noticed in ISO hearts. Further, in-vitro membranes assay shows that NPR-A dependent guanylyl cyclase (GC) activity was down-regulated (2-fold), whereas NPR-B dependent GC activity was increased (5-fold) in ISO treated hearts. Atenolol treatment normalized the altered expression of Npr1 and Npr2 genes. In conclusion, the chronic β-AR activation differentially regulates Npr1 and Npr2 genes in the heart. Npr1 down regulation is positively associated with the development of left ventricular hypertrophy (LVH) in ISO rats.

Histomorphological Study of Effects of Oral Administration of Cadmium Nitrate on Heart of Adult Wistar Rats

Histomorphological Study of Effects of Oral Administration of Cadmium Nitrate on Heart of Adult Wistar Rats

High-density lipoproteins reduce palmitate-induced cardiomyocyte apoptosis in an AMPK-dependent manner

Palmitate has been implicated in the induction of cardiomyocyte apoptosis via reducing the activity of 5′ AMP-activated protein kinase (AMPK). We sought to evaluate whether high-density lipoproteins (HDLs), known for their cardioprotective features and their potential to increase AMPK activity, can reduce palmitate-induced cardiomyocyte apoptosis and whether this effect is AMPK-dependent. Therefore, cardiomyocytes were isolated from adult Wistar rat hearts via perfusion on a Langendorff-apparatus and cultured in free fatty acid-free BSA control medium or 0.5 mM palmitate medium in the presence or absence of HDL (5 μg protein/ml) with or without 0.1 μM of the AMPK-inhibitor compound S for the analysis of Annexin V/propidium, genes involved in apoptosis and fatty acid oxidation, and cardiomyocyte contractility. We found that HDLs decreased palmitate-induced cardiomyocyte apoptosis as indicated by a reduction in Annexin V-positive cardiomyocytes and an increase in Bcl-2 versus Bax ratio. Concomitantly, HDLs increased the palmitate-impaired expression of genes involved in fatty acid oxidation. Furthermore, HDLs improved the palmitate-impaired cardiomyocyte contractility. All effects were mediated in an AMPK-dependent manner, concluding that HDLs reduce palmitate-induced cardiomyocyte apoptosis, resulting in improved cardiomyocyte contractility through a mechanism involving AMPK.

Effect of Apple (Malus domestica) on Na+/K+-ATPase Activity in Liver, kidney and Heart of Adult Wistar Rats

Effect of Apple (Malus domestica) on Na+/K+-ATPase Activity in Liver, kidney and Heart of Adult Wistar Rats

Electrical Propagation of Three-Dimensional Engineered Hearts using Decellularized Extracellular Matrix

[Background] Construction of three-dimensional cardiac tissues using decellularized extracellular matrix could be a new technique to create an “organ-like” structure of the heart. To engineer functional artificial hearts comparable for orthotopic hearts, however, much remain to be solved including electrical stability for efficient contraction. To elucidate the points, we examined electrophysiological properties of recellularized heart tissues. [Methods] Entire hearts of adult Wistar rats were decellularized using 0.5% SDS and 1% triton-X, and then recellularized with enzymatically-dispersed neonatal rat cardiac cells (1x10⊥8 cells) through antegrade coronary circulation. Three and seven days later after cell seeding, we observed excitation of spontaneous and pacing-induced beatings of recellularized heart tissues expressing Ca2+-indicating protein (GCaMP2) using high resolution cameras. We also conducted immunofluorescence staining to examine morphological aspects of engineered tissues. [Results] Live tissue fluorescence imaging revealed that GFP-labeled-isolated cardiac cells were dispersed into interstitial spaces through extravasation from coronary arteries. Engineered hearts seeded with GCaMP2-expressing cardiac cells started showing spontaneous beating, and were subjected to further electrophysiological experiments using optical imaging system. Both in spontaneous and pacing-induced beating hearts, we observed well-organized conduction of stable excitation in substantial areas of the engineered heart tissues, whereas we also recorded disorganized propagation of asynchronous excitation with multiple origins. Conduction velocity was markedly decreased (∼5 cm/sec) during early stages (up to nine days) after recellularization. Immunofluorescence study revealed randomly-mixed alignment of cardiomyocytes, endothelial cells and smooth muscle cells, stained with alpha-actinin, CD31 and sm-actin, respectively. Recellularized heart tissues also showed disarray of cardiomyocytes and markedly decreased expression of connexin43. [Conclusion] Three-dimensional engineered hearts showed electrical heterogeneity and proarrhythmic propensity during early stages of culture. These findings may be due to, at least in part, disorganized alignment of microstructure and immature formation of gap junction.

Protective effect of quercetin on atrazine-induced oxidative stress in the liver, kidney, brain, and heart of adult wistar rats.

Background:The conflicting roles of quercetin against tissue pathologies associated with oxidative stress are known.Objective:To evaluate the effect of quercetin at doses of 5 mg/kg (Q5) or 10 mg/kg (Q10) against atrazine (120 mg/kg, ATZ)-induced oxidative stress in various tissues of rats.Materials and Methods:Adult male albino Wistar rats were administered ATZ, Q5, and Q10 alone or in combination for 16 days. At the end of the 16th day, the animals were sacrificed by cervical dislocation; and the blood, heart, brain, kidney and liver were collected and used for biochemical determinations and histopathological examination.Results:Q10 but not Q5 attenuated ATZ-induced increase in the levels of serum enzyme markers sorbitol dehydrogenase (SDH), acid phosphatase (ACP), alkaline phosphatase (ALP), and aspartate aminotransferase (AST). The heart was less susceptible to ATZ-induced oxidative stress than the liver, kidney, and brain of treated animals, and there were tendencies for synergistic effects in the heart and liver of Q5 + ATZ-treated rats. Oxidative stress-induced by ATZ in terms of increased lipid peroxidation level and superoxide dismutase (SOD) activity was decreased in the brain of the Q5 + ATZ-treated rats but not that of the Q10 + ATZ-treated rats. Conversely, histopathological changes and oxidative stress-induced by ATZ in terms of elevated lipid peroxidation level, decreased SOD, and catalase (CAT) activities were prevented in the kidney and liver of the Q10 + ATZ-treated rats but not that of the Q5 + ATZ-treated rats.Conclusion:Quercetin at the investigated doses and especially the low dose may not protect against ATZ-induced oxidative stress in rat tissues in an overall sense.

Effect of nitric oxide donors S‐nitroso‐N‐acetyl‐dl‐penicillamine, spermine NONOate and propylamine propylamine NONOate on intracellular pH in cardiomyocytes

1. Previous studies suggest that exogenous nitric oxide (NO) and NO-dependent signalling pathways modulate intracellular pH (pH(i)) in different cell types, but the role of NO in pH(i) regulation in the heart is poorly understood. Therefore, in the present study we investigated the effect of the NO donors S-nitroso-N-acetyl-DL-penicillamine, spermine NONOate and propylamine propylamine NONOate on pH(i) in rat isolated ventricular myocytes. 2. Cells were isolated from the hearts of adult Wistar rats and pH(i) was monitored using the pH-sensitive fluorescent indicator 5-(and-6)-carboxy seminaphtharhodafluor (SNARF)-1 (10 μmol/L) and a confocal microscope. To test the effect of NO donors on the Na⁺/H⁺ exchanger (NHE), basal pH(i) in Na⁺-free buffer and pH(i) recovery from intracellular acidosis after an ammonium chloride (10 mmol/L) prepulse were monitored. The role of carbonic anhydrase was tested using acetazolamide (50 μmol/L). 4,4-Diisothiocyanatostilbene-2,2'-disulphonic acid (0.5 mmol/L; DIDS) was used to inhibit the Cl⁻/OH⁻ and Cl⁻/HCO₃-exchangers. Acetazolamide and DIDS were applied via the superfusion system 1 and 5 min before the NO donors. 3. All three NO donors acutely decreased pH(i) and this effect persisted until the NO donor was removed. In Na⁺-free buffer, the decrease in basal pH(i) was increased, whereas inhibition of carbonic anhydrase and Cl⁻/OH⁻ and Cl⁻/HCO₃⁻ exchangers did not alter the effects of the NO donors on pH(i). After an ammonium preload, pH(i) recovery was accelerated in the presence of the NO donors. 4. In conclusion, exogenous NO decreases basal pH(i), leading to increased NHE activity. Carbonic anhydrase and chloride-dependent sarcolemmal HCO₃⁻ and OH⁻ transporters are not involved in the NO-induced decrease in pH(i) in rat isolated ventricular myocytes.

Some Morphological Findings on the Heart of Adult Wistar Rats Following Experimental Artesunate Administration

Some Morphological Findings on the Heart of Adult Wistar Rats Following Experimental Artesunate Administration

Differences in the Pattern of Ventricular Activation in Small Rodents Determined by Morphological Organization of the Cardiac Ventricular Conduction System

Ventricular activation of the mouse heart differs significantly compared to activation in larger mammals. Knowledge of structural and functional characteristics of laboratory animals is essential for evaluation of results obtained from experiments. The present study was performed to evaluate whether the different pattern of activation is common to small rodents or unique for mice. Hearts of adult Wistar rats were isolated and Langendorff perfused. After removing the right and left ventricular free wall, extracellular activity of the septum and bundle branches (BB) was determined using a multi-terminal electrode harboring 247 terminals. Immunolabeling on cryosections was performed to assess expression and distribution of the gap junction proteins Connexin40 (Cx40), Cx43, Cx45, contractile (Desmin, alpha-actinin) and intercalated disk-related (N-cadherin, beta-catenin) proteins. Collagen distribution was assessed by Sirius Red staining. Reconstruction of the left and right bundle branch (LBB and RBB) using immuno-labeling revealed that the LBB spreads all over the septal surface. The RBB too is broad, albeit to a lesser extend than LBB. A sheet of connective tissue electrically separates the common bundle and proximal BB from the septal working myocardium. Immunolabeling revealed clear differences between the conduction system and the working myocardium with respect to expression level and distribution of the different proteins analyzed. The morphological organization of the area resulted in an electrical activation pattern of the septum comparable to what is common in larger mammals: earliest activation at the midseptum via the bundle branches. From our data we conclude that the pattern of ventricular activation in the rat heart and the structure of the conduction system fit to data described for larger mammals and differ from the different pattern previously found in mouse heart.

Comparative studies of ATP sensitive potassium channels in heart and pancreatic beta cells using Vaughan-Williams class Ia antiarrhythmics.

Actions of cibenzoline and disopyramide, agents with Vaughan-Williams class Ia antiarrhythmic action, on ATP sensitive K+ (KATP) channels were examined in heart and pancreatic beta cells. Single ventricular myocytes and beta cells were prepared enzymatically from adult Wistar rat hearts and pancreatic islets. Using patch clamp techniques, KATP channel activities were recorded in whole cell and single channel modes. In whole cell experiments, myocytes were bathed with Tyrode's medium (34 degrees C); inside out patches were bathed with internal solutions (22-24 degrees C) containing 1 microM ATP and varying concentrations of cibenzoline or disopyramide. Myocytes were voltage clamped at -40 mV and glibenclamide blockade conductance was produced by cromakalim. Micromolar concentrations of both cibenzoline and disopyramide suppressed cromakalim induced conductance. When applied to the cytosolic surface of the cell membrane in inside out configuration, both drugs reversibly inhibited single KATP channel activities. Neither unitary conductance nor intraburst fast kinetics was affected by the compounds. At a holding potential of -40 mV under symmetrical approximately 150 mM K+ conditions, half maximum doses (IC50) were 0.9 microM [Hill coefficient (h) = 1.3] for cibenzoline induced block of cardiac KATP channels and 1.8 microM (h = 1.0) for disopyramide block. At +40 mV, IC50 for cibenzoline block was 1.4 microM (h = 0.9). Thus there was little voltage dependence in cibenzoline induced channel block. A similar IC50 value of 2.5 microM (h = 1.2 at -60 mV under symmetrical approximately 150 mM K+) was observed for cibenzoline induced block of KATP channels. Near therapeutic concentrations of cibenzoline and disopyramide inhibit KATP channel activities in both heart and pancreatic beta cells. This may be causally related to the fasting hypoglycaemia which is sometimes reported in patients receiving the drugs. These antiarrhythmic agents may also modulate myocardial electrical properties during hypoxia or ischaemia.

DNA synthesis in atrial myocytes of rats with aortic stenosis.

Indices of labeled myonuclei have been determined in hypertrophying hearts of adult Wistar rats by autoradiography after single-pulse or repeated [3H]thymidine administration. After single [3H]thymidine injections, only 1.36 +/- 0.66 and 1.32 +/- 0.87% labeled myonuclei were observed in the left and right atria, respectively. In the experiments with multiple [3H]thymidine administration, the first injection of this precursor was given on the seventh day after aortic constriction; thereafter, 30 or 42 injections of [3H]thymidine were given at 12-hr intervals up to the fourth postoperation week. Following 30 repeated [3H]thymidine injections, 29.75 +/- 4.65 and 16.78 +/- 3.33% labeled myonuclei were visible in left and right atrial muscle cells, respectively. The cumulative labeling index for left atrium myocytes clearly correlates (r = 0.65-0.73) with an increase in the weight of the heart. Increase in heart weight to more than 160% of controls corresponds to [3H]thymidine labeling of 38.06 +/- +/- and 21.67 +/- 4.16% in left and right atrial myocytes, respectively, whereas in hearts weighing less than 140% of controls, [3H]thymidine labels only 8.20 +/- 1.93% in the left atrium and 3.94 +/- 1.57% in the right one. In the ventricles, cumulative indices of myonuclear labeling do not exceed 0.217 +/- 0.11% even in hearts weighing nearly 180% of controls. Cumulative frequencies of labeling for AV system myocytes are almost ten times higher (1.97 +/- 0.38). These results, together with our data concerning mycocardial infarction (27-29,31), make it necessary to reconsider the role of cardiomyocyte hyperplasia in different experimental and pathological conditions, paying special attention to the proliferative behavior of the atrial muscle cells. DNA synthesis in atrial myocytes seems to be stimulated by heart hyperfunction.