Ludmila Okruhlicova

Rosmarinic acid administration attenuates diabetes-induced vascular dysfunction of the rat aorta

Oxidative stress as well as inflammation processes are engaged in diabetic vascular complications. Rosmarinic acid, a natural phenol antioxidant carboxylic acid, was found to have multiple biological activity, including anti‐inflammatory and antitumour effects, which are a consequence of its inhibition of the inflammatory processes and of reactive oxygen species scavenging. The aim of this work was to study effects of rosmarinic acid administration on vascular impairment induced by experimental diabetes in rats.

Ultrastructure and histochemistry of rat myocardial capillary endothelial cells in response to diabetes and hypertension.

ABSTRACTInsufficient growth and rarefaction of capillaries, followed by endothelial dysfunction may represent one of the most critical mechanisms involved in heart damage. In this study we examined histochemical and ultrastructural changes in myocardial capillary endothelium in two models of heart failure streptozotocin-induced diabetes mellitus (STZ) and NO-deficient hypertension in male Wistar rats. Diabetes was induced by a single i.v. dose of STZ (45 mg/kg) and chronic 9-week stage was analysed. To induce NO-deficient hypertension, animals were treated with inhibitor of NO synthase L-nitroarginine methylester (L-NAME) (40 mg/kg) for 4 weeks. Left ventricular tissue was processed for enzyme catalytic histochemistry of capillary alkaline phosphatase (AlPh), dipeptidyl peptidase IV (DPP IV), and endothelial NO synthase/NADPH-diaphorase (NOS) and for ultrastructural analysis. In diabetic and hypertensive rats, lower/absent AlPh and DPP IV activities were found in focal micro-areas. NOS activity was significantly reduced and persisted only locally. Quantitative evaluation demonstrated reduction of reaction product intensity of AlPh, DPP and NOS by 49.50%, 74.36%, 20.05% in diabetic and 62.93%, 82.71%, 37.65% in hypertensive rats. Subcellular alterations of endothelial cells were found in heart of both groups suggesting injury of capillary function as well as compensatory processes. Endothelial injury was more significant in diabetic animals, in contrast the adaptation was more evident in hypertensive ones. Concluding: both STZ-induced diabetes- and NO-deficient hypertension-related cardiomyopathy were accompanied by similar features of structural remodelling of cardiac capillary network manifested as angiogenesis and angiopathy. The latter was however, predominant and may accelerate disappearance of capillary endothelium contributing to myocardial dysfunction.

Hibernating myocardium: pathophysiology, diagnosis, and treatment

Comprehensive management of patients with chronic ischemic disease is a critically important component of clinical practice. Cardiac myocytes have the potential to adapt to limited flow conditions by adjusting contractile function, reducing metabolism, conserving resources, and preserving myocardial integrity to cope with an oxygen and (or) nutrition shortage. A prime metabolic feature of cardiac myocytes affected by chronic ischemia is the return to a fetal gene pattern with predominance of carbohydrates as the substrate for energy. Structural adaptation with multiple intracellular changes is part of the remodeling process in hibernating myocardium. Transmural heterogeneity, which defines the pattern of injury in ventricular cardiomyocytes and the response to chronic ischemia, is a multifactorial process originating from functional, metabolic, and flow differences in subendocardial and subepicardial regions. Autophagy is typically activated in hibernating myocardium and has been identified as a prosurvival mechanism. Chronic ischemia is associated with changes in the number, size, and distribution of gap junctions and may give rise to conduction disturbances and arrhythmogenesis. Differentiation between viable and nonviable myocardium by assessing sensitivity of inotropic reserve is a crucial diagnostic tool that is correlated with the prognosis and outcome for improved contractility after restoration of blood perfusion in afflicted myocardium.Reliable and accurate diagnosis of ischemic, scar, and viable tissues is critical for recover strategies. Although early surgical reinstitution of blood flow is most effective in restoring physiologic function of the hibernating myocardium, several new approaches offer promising alternatives. Among others, vascular endothelial growth factor and fibroblast growth factor-2 (FGF-2), especially its lo-FGF-2 isoform, have been shown to be effective in rapid neovascularization. Substances such as statins, resveratrol, some hormones, and omega-3 fatty acids can improve recovery effect in chronically underperfused hearts. For patients with drug-refractory ischemia, intramyocardial transplantation of stem cells into predefined areas of the heart can enhance vascularization and have beneficial effects on cardiac function. This review of ischemic injury, its heterogeneity, accurate diagnosis, and newer methods of treatment, shows there is much information and tremendous hope for better management of patients with coronary heart disease.

Hypertension-related intermyocyte junction remodelling is associated with a higher incidence of low-K(+)-induced lethal arrhythmias in isolated rat heart.

The aim of this study was to characterise the arrhythmogenic mechanisms involved in hypokalaemia‐induced sustained ventricular fibrillation (SVF), in hypertensive rats. The hearts from rats with hypertension induced by the nitric oxide synthase inhibitor L‐NAME, and age‐matched normotensive controls, were perfused in Langendorff mode with oxygenated Krebs‐Henseleit solution followed by a K+‐deficient solution. In additional experiments, free intracellular Ca2+ concentration ([Ca2+]i) was measured using fura‐2 in conjunction with an epicardial optical probe. The epicardial electrocardiogram was continuously monitored during all experiments. The gap junction protein connexin‐43 and the ultrastructure of the cardiomyocytes were examined, and selected enzyme activities were measured in situ. There was a higher incidence of low‐K+‐induced SVF in the hearts of hypertensive compared to normotensive rats (83% vs. 33%, P < 0.05). Perfusion with a low‐K+‐containing solution lead to elevation of diastolic [Ca2+]i that was accompanied by premature beats, bigeminy, ventricular tachycardia and transient ventricular fibrillation. These events occurred earlier with increased incidence and duration in the hearts of hypertensive rats (arrhythmia scores: hypertensive, 4.9 ± 0.7; normotensive, 3.1 ± 0.1; P < 0.05), which exhibited apparent remodelling accompanied by a significant decrease in the density of connexin‐43‐positive gap junctions. Moreover, low‐K+‐related myocardial changes, including local impairment of intermyocyte junctions, ultrastructural alterations due to Ca2+ overload and intercellular uncoupling, and decreased enzyme activities were more pronounced and more dispersed in hypertensive than normotensive rats. In conclusion, nitric oxide‐deficient hypertension is associated with decreased myocardial coupling at gap junctions. The further localised deterioration of junctional coupling, due to low‐K+‐induced Ca2+ disturbances, as well as spatial heterogeneity of myocardial alterations including interstitial fibrosis, probably provide the mechanisms for re‐entry and sustaining ventricular fibrillation.

Gap junction remodelling is involved in the susceptibility of diabetic rats to hypokalemia-induced ventricular fibrillation.

The objective of the present study was to examine the susceptibility of diabetic rats with cardiomyopathy to hypokalemia-induced ventricular fibrillation and to localize gap junction protein connexin-43 as well as subcellular changes that may be involved in the development of severe arrhythmia. Our results showed a significantly higher incidence of sustained ventricular fibrillation in diabetic hearts as compared with control hearts, 80% vs 20%, respectively. Diabetic cardiomyopathy itself was accompanied by a distinct decrease in connexin-43-immunopositive gap junctions. Moreover, interstitial fibrosis and subcellular alterations to various degrees were observed in diabetic hearts, and a further deterioration of the ultrastructure and impairment of intercellular junctions, and a stronger local decrease in connexin-43 levels due to hypokalemia were found. These changes were heterogeneously distributed throughout the myocardium and occurred earlier and were more pronounced in diabetic hearts than control hearts. In conclusion, our results indicate that diabetic cardiomyopathy is associated with down-regulation of gap junction proteins and may account for the higher vulnerability of diabetic rats to ventricular fibrillation in combination with impairment of intercellular communication due to hypokalemia.

Cytochemical distribution of cyclic AMP-dependent 3′,5′-nucleotide phosphodiesterase in the rat myocardium

SummaryThe cytochemical localization of cAMP-dependent phosphodiesterase was studied in the rat myocardium. Slices 40 μm thick from perfusion-fixed rat hearts were incubated in the medium with cAMP as a substrate and Pb ions as a capture metal of the reaction product. After the incubation in the basic medium the specific precipitate of cAMP phosphodiesterase was localized on the sarcolemma of cardiomyocytes. In addition, it was localized on the plasmalemma of endothelia cells of capillaries and small coronary arteries as well as on the membrane of smooth muscle cells. Using selective inhibitors SK&F 94120 for phosphodiesterase III and Rolipram for the IV isoenzyme, both isoforms were detected on the membrane of smooth muscle cell. In addition, phosphodiesterase III was localized on the sarcolemma only and phosphodiesterase IV on the sarcolemma of cardiomyocytes and the plasmalemma of endothelial cells.

Localization of α1,2,3-subunit isoforms of Na,K-ATPase in cultured neonatal and adult rat myocardium: The immunofluorescence and immunocytochemical study

By indirect immunofluorescence and preembedding peroxidase-diaminobenzidine technique the localization of polyclonal and monoclonal antibodies against α1, α2 and α3 isoforms of the Na,K-ATPase were studied in rat myocardium.The α1-subunit was identified predominantly on sarcolemma of cultured myocytes, neonatal, as well as adult cardiocytes. The α2 signal was localized around nuclei of cultured cardiocytes, very weak signals were seen in neonatal and more intense signal, were dispersed throughout the adult myocytes. The α3-subunit immunoreactivity was weak and localized in cell processes connecting individual cultured cells, on sarcolemma and intercalated discs of neonatal cells and very weak in adult working myocytes. Cytochemically demonstrated ouabain resistant Na,K-ATPase localized in junctional sarcoplasmic reticulum may represent α1 isoenzyme which is directly involved in modulation of action potential fluxes.

Mechanisms of cardiac radiation injury and potential preventive approaches.

In addition to cytostatic treatment and surgery, the most common cancer treatment is gamma radiation. Despite sophisticated radiological techniques however, in addition to irradiation of the tumor, irradiation of the surrounding healthy tissue also takes place, which results in various side-effects, depending on the absorbed dose of radiation. Radiation either damages the cell DNA directly, or indirectly via the formation of oxygen radicals that in addition to the DNA damage, react with all cell organelles and interfere with their molecular mechanisms. The main features of radiation injury besides DNA damage is inflammation and increased expression of pro-inflammatory genes and cytokines. Endothelial damage and dysfunction of capillaries and small blood vessels plays a particularly important role in radiation injury. This review is focused on summarizing the currently available data concerning the mechanisms of radiation injury, as well as the effectiveness of various antioxidants, anti-inflammatory cytokines, and cytoprotective substances that may be utilized in preventing, mitigating, or treating the toxic effects of ionizing radiation on the heart.

Omega-3 fatty acids and atorvastatin affect connexin 43 expression in the aorta of hereditary hypertriglyceridemic rats.

Statins and omega-3 polyunsaturated fatty acids (n-3 PUFA) reduce cardiovascular disease incidence during hypertriglyceridemia (HTG). To elucidate possible cardioprotective mechanisms, we focused on gap junction protein connexin 43 (Cx43). Its expression is disturbed during atherogenesis, but little information is available on its expression during HTG. Experiments were performed on adult male hereditary HTG (hHTG) rats treated with n-3 PUFA (30 mg/day) and atorvastatin (0.5 mg/100 g body weight per day) for 2 months. Cx43 expression and distribution in the aorta were investigated by using Western blotting and immunolabeling, followed by quantitative analysis. Transmission electronmicroscopy was used to study ultrastructure of endothelial contact sites. In contrast to age-matched Wistar, Cx43 expression in aorta of hHTG rats was significantly higher (p < 0.05), and prominent Cx43 immunospots were seen in tunica media and less in endothelium of hHTG rats. Changes in Cx43 expression were accompanied by local qualitative subcellular alterations of interendothelial connections. Treatment of hHTG rats with n-3 PUFA and atorvastatin markedly lowered Cx43 expression in aorta and modified connexin distribution in endothelium and media (p < 0.05 vs. untreated hHTG). The protective effect of treatment of HTG was observed on the structural integrity of the endothelium and was readily visible at the molecular level. Results indicate the involvement of altered Cx43 expression in vascular pathophysiology during HTG and during HTG treatment.

Supplementation with n-3 polyunsaturated fatty acids to lipopolysaccharide-induced rats improved inflammation and functional properties of renal Na,K-ATPase

Measurements of enzyme kinetics of renal Na, K-ATPase were used for characterization of ATP- and Na⁺-binding sites in rats that were subjected to 10 days of moderate inflammation that was induced by a single dose of Escherichia coli lipopolysaccharides (LPSs) at a dose of 1 mg kg⁻¹ body weight. We hypothesized that LPSs might initiate a malfunction of renal Na, K-ATPase, which is a key enzyme involved in regulation of sodium homeostasis in the organism. We also investigated the potential effect that fish oil (FO) has in the prevention of Na, K-ATPase alterations by administering FO daily at a dose of 30 mg kg⁻¹. Alone, LPS elevated the level of C-reactive protein by more than 500% and free radicals by 36% in plasma, as indicated by an increased level of malondialdehyde. The Na, K-ATPase was slightly altered in the vicinity of the ATP-binding site as suggested by the 9% increase of the concentration of ATP necessary for half-maximal activation of the enzyme, thus indicating a deteriorated binding of ATP as a consequence of inflammation. Daily supplementation of FO partly attenuated LPS-induced injury, as observed by a significant decrease in the plasma levels of C-reactive protein and free radicals, hence maintaining the activity of renal Na, K-ATPase to the level of healthy control animals. In conclusion, our findings showed that FO prevented an excessive malondialdehyde production in LPS-treated animals and stabilized renal Na, K-ATPase.