Distinct Cardiac Connexin-43 Expression in Hypertrophied and Atrophied Myocardium May Impact the Vulnerability of the Heart to Malignant Arrhythmias. A Pilot Study.

Our and other studies suggest that myocardial hypertrophy in response to hypertension and hyperthyroidism increases propensity of the heart to malignant arrhythmias, while these are rare in conditions of hypothyroidism or type-1 diabetes mellitus associated with myocardial atrophy. One of the crucial factors impacting the susceptibility of the heart to life-threatening arrhythmias is gap junction channel protein connexin-43 (Cx43), which ensure cell-to-cell coupling for electrical signal propagation. Therefore, we aimed to explore Cx43 protein abundance and its topology in hypertrophic and hypotrophic cardiac phenotype. Analysis were performed in left ventricular tissue of adult male spontaneously hypertensive rat (SHR), Wistar Kyoto rats treated for 8-weeks with L-thyroxine, methimazol or strepotozotocin to induce hyperthyroid, hypothyroid and type-1 diabetic status as well as non-treated animals. Results showed that comparing to healthy rats there was a decrease of total myocardial Cx43 and its variant phosphorylated at serine368 in SHR and hyperthyroid rats. Besides, enhanced localization of Cx43 was demonstrated on lateral sides of hypertrophied cardiomyocytes. In contrast, total Cx43 protein and its serine368 variant were increased in atrophied left ventricle of hypothyroid and type-1 diabetic rats. It was associated with less pronounced alterations in Cx43 topology. In parallel, the abundance of PKCepsilon, which phosphorylates Cx43 at serine368 that stabilize Cx43 function and distribution was reduced in hypertrophied heart while enhanced in atrophied once. Findings suggest that differences in the abundance of cardiac Cx43, its variant phosphorylated at serine368 and Cx43 topology may explain, in part, distinct propensity of hypertrophied and atrophied heart to malignant arrhythmias.

Red palm oil supplementation does not increase blood glucose or serum lipids levels in Wistar rats with different thyroid status.

Red palm oil (RPO) is a rich natural source of antioxidant vitamins, namely carotenes, tocopherols and tocotrienols. However, it contains approximately 50 % saturated fatty acids the regular consumption of which could negatively modify lipid profile. The aim of our study was to test whether 7 weeks of RPO supplementation (1 g/kg body weight/day) would affect blood glucose and lipid metabolism in adult male Wistar rats with altered thyroid status. We induced hypothyroidism and hyperthyroidism in rats by oral administration of either methimazole or mixture of thyroid hormones. Different thyroid status (EU - euthyroid, HY - hypothyroid and HT - hyperthyroid) was characterized by different serum thyroid hormones levels (total and free thyroxine and triiodothyronine), changes in the activity of a marker enzyme of thyroid status - liver mitochondrial glycerol-3-phosphate dehydrogenase, and altered absolute and relative heart weights. Fasting blood glucose levels were higher in HT rats in comparison with EU and HY rats, but the changes caused by RPO supplementation were not significant. The achievement of the HY status significantly increased serum levels of total cholesterol, as well as with high-density lipoprotein-cholesterol and low-density lipoprotein-cholesterol: 2.43+/-0.15, 1.48+/-0.09, 0.89+/-0.08 mmol/l, compared to EU: 1.14+/-0.06, 0.77+/-0.06, 0.34+/-0.05 mmol/l and HT: 1.01+/-0.06, 0.69+/-0.04, 0.20+/-0.03 mmol/l, respectively. RPO supplementation did not increase significantly levels of blood lipids but tended to increase glutathione levels in the liver. In conclusion, RPO supplementation did not induce the presumed deterioration of glucose and lipid metabolism in rats with three well-characterized alterations in thyroid status.

Modulation of systemic and aortic nitric oxide by melatonin and n-3 polyunsaturated fatty acids in isoproterenol affected spontaneously hypertensive and normotensive Wistar rats.

We aimed to explore the effects of melatonin and n-3 polyunsaturated fatty acids (PUFA) supplementation on plasma and aortic nitric oxide (NO) levels in isoproterenol (Iso) affected spontaneously hypertensive (SHR) and Wistar rats. Untreated control rats were compared with Iso injected (118 mg/kg, s.c.) rats, and Iso injected plus supplemented with melatonin (10 mg/kg, p.o.) or PUFA (1.68 g/kg, p.o.) for two months. Plasma and aortic basal, L-NAME inhibited, adrenaline and acetylcholine stimulated NO were determined using Griess method. Plasma NO levels were lower in SHR versus Wistar rats. Iso decreased NO in Wistar while not in SHR. PUFA but not melatonin intake of Iso treated SHR increased plasma NO along with a decrease in systolic blood pressure. Basal aortic NO level was higher in SHR than Wistar rats and not altered by Iso. Intake of melatonin increased but PUFA decreased basal NO levels in Wistar+Iso and did not affect in SHR+Iso rats. Acetylcholine and adrenaline induced aortic NO release was significantly increased in Wistar+Iso but not SHR+Iso group. Melatonin intake increased Ach induced aortic NO in Wistar+Iso and SHR+Iso groups, whereas there was no effect of PUFA intake. Findings suggest that PUFA modulates plasma and melatonin aortic NO levels of isoproterenol affected rats in a strain-dependent manner.

Preventive and therapeutic application of molecular hydrogen in situations with excessive production of free radicals.

Excessive production of oxygen free radicals has been regarded as a causative common denominator of many pathological processes in the animal kingdom. Hydroxyl and nitrosyl radicals represent the major cause of the destruction of biomolecules either by a direct reaction or by triggering a chain reaction of free radicals. Scavenging of free radicals may act preventively or therapeutically. A number of substances that preferentially react with free radicals can serve as scavengers, thus increasing the internal capacity/activity of endogenous antioxidants and protecting cells and tissues against oxidative damage. Molecular hydrogen (H(2)) reacts with strong oxidants, such as hydroxyl and nitrosyl radicals, in the cells, that enables utilization of its potential for preventive and therapeutic applications. H(2) rapidly diffuses into tissues and cells without affecting metabolic redox reactions and signaling reactive species. H(2) reduces oxidative stress also by regulating gene expression, and functions as an anti-inflammatory and anti-apoptotic agent. There is a growing body of evidence based on the results of animal experiments and clinical observations that H(2) may represent an effective antioxidant for the prevention of oxidative stress-related diseases. Application of molecular hydrogen in situations with excessive production of free radicals, in particular, hydroxyl and nitrosyl radicals is relatively simple and effective, therefore, it deserves special attention.

Disordered myocardial Ca(2+) homeostasis results in substructural alterations that may promote occurrence of malignant arrhythmias.

We aimed to determine the impact of Ca(2+)-related disorders induced in intact animal hearts on ultrastructure of the cardiomyocytes prior to occurrence of severe arrhythmias. Three types of acute experiments were performed that are known to be accompanied by disturbances in Ca(2+) handling. Langedorff-perfused rat or guinea pig hearts subjected to K(+)-deficient perfusion to induce ventricular fibrillation (VF), burst atrial pacing to induce atrial fibrillation (AF) and open chest pig heart exposed to intramyocardial noradrenaline infusion to induce ventricular tachycardia (VT). Tissue samples for electron microscopic examination were taken during basal condition, prior and during occurrence of malignant arrhythmias. Cardiomyocyte alterations preceding occurrence of arrhythmias consisted of non-uniform sarcomere shortening, disruption of myofilaments and injury of mitochondria that most likely reflected cytosolic Ca(2+) disturbances and Ca(2+) overload. These disorders were linked with non-uniform pattern of neighboring cardiomyocytes and dissociation of adhesive junctions suggesting defects in cardiac cell-to-cell coupling. Our findings identified heterogeneously distributed high [Ca(2+)](i)-induced subcellular injury of the cardiomyocytes and their junctions as a common feature prior occurrence of VT, VF or AF. In conclusion, there is a link between Ca(2+)-related disorders in contractility and coupling of the cardiomyocytes pointing out a novel paradigm implicated in development of severe arrhythmias.

Protection of cardiac cell-to-cell coupling attenuate myocardial remodeling and proarrhythmia induced by hypertension.

Gap junction connexin channels are important determinants of myocardial conduction and synchronization that is crucial for coordinated heart function. One of the main risk factors for cardiovascular events that results in heart attack, congestive heart failure, stroke as well as sudden arrhythmic death is hypertension. Mislocalization and/or dysfunction of specific connexin-43 channels due to hypertension-induced myocardial remodeling have been implicated in the occurrence of life-threatening arrhythmias and heart failure in both, humans as well as experimental animals. Recent studies suggest that down-regulation of myocardial connexin-43, its abnormal distribution and/or phosphorylation might be implicated in this process. On the other hand, treatment of hypertensive animals with cardioprotective drugs (e.g. statins) or supplementation with non-pharmacological compounds, such as melatonin, omega-3 fatty acids and red palm oil protects from lethal arrhythmias. The antiarrhythmic effects are attributed to the attenuation of myocardial connexin-43 abnormalities associated with preservation of myocardial architecture and improvement of cardiac conduction. Findings uncover novel mechanisms of cardioprotective (antihypertensive and antiarrhythmic) effects of compounds that are used in clinical settings. Well-designed trials are needed to explore the antiarrhythmic potential of these compounds in patients suffering from hypertension.

Cardiac connexin-43 and PKC signaling in rats with altered thyroid status without and with omega-3 fatty acids intake.

Thyroid hormones are powerful modulators of heart function and susceptibility to arrhythmias via both genomic and non-genomic actions. We aimed to explore expression of electrical coupling protein connexin-43 (Cx43) in the heart of rats with altered thyroid status and impact of omega-3 polyunsaturated fatty acids (omega-3) supplementation. Adult male Lewis rats were divided into following six groups: euthyroid controls, hyperthyroid (treated with T(3)) and hypothyroid (treated with methimazol) with or without six-weeks lasting supplementation with omega-3 (20 mg/100 g/day). Left and right ventricles, septum and atria were used for immunoblotting of Cx43 and protein kinase C (PKC). Total expression of Cx43 and its phosphorylated forms were significantly increased in all heart regions of hypothyroid rats compared to euthyroid controls. In contrast, the total levels of Cx43 and its functional phosphorylated forms were decreased in atria and left ventricle of hyperthyroid rats. In parallel, the expression of PKC epsilon that phosphorylates Cx43, at serine 368, was increased in hypothyroid but decreased in hyperthyroid rat hearts. Omega-3 intake did not significantly affect either Cx43 or PKC epsilon alterations. In conclusion, there is an inverse relationship between expression of cardiac Cx43 and the levels of circulating thyroid hormones. It appears that increased propensity of hyperthyroid while decreased of hypothyroid individuals to malignant arrhythmias may be in part attributed to the changes in myocardial Cx43.

Ageing related down-regulation of myocardial connexin-43 and up-regulation of MMP-2 may predict propensity to atrial fibrillation in experimental animals.

Mechanisms underlying atrial fibrillation (AF), the most common cardiac arrhythmia, particularly in aged population, are not fully elucidated. We have previously shown an increased propensity of old guinea pigs (GPs) heart to inducible AF when comparing to young animals. This study aimed to verify our hypothesis that susceptibility of aged heart to AF may be attributed to abnormalities in myocardial connexin-43 (Cx43) and extracellular matrix that affect cardiac electrical properties. Experiments were conducted on male and female 4-week-old and 24-week-old GPs. Atrial tissue was processed for analysis of Cx43 topology using immunohistochemistry, expression of Cx43 protein using immunobloting, and expression of mRNA of Cx43 and extracellular matrix metalloproteinase-2 (MMP-2) using real time PCR. Immunohistochemistry revealed uniform Cx43 distribution predominantly on lateral sides of the cardiomyocytes of young male and female GP atria. In contrast, non-uniform distribution, mislocalization and reduced immunolabeling of Cx43 were detected in atria of old GPs. In parallel, the atrial tissue levels of Cx43 mRNA were significantly decreased, while mRNA expression of MMP-2 was significantly increased in old versus young GPs. The changes were more pronounced in old GPs males comparing to females. Findings indicate that age-related down-regulation of atrial Cx43 and up-regulation of MMP-2 as well as disordered Cx43 distribution can facilitate development of AF in old guinea pig hearts.

New aspects of pathogenesis of atrial fibrillation: remodelling of intercalated discs.

This review deals with the understanding of the role of key factors of ageing, oxidative stress and inflammation, in relation to development of age-related cardiovascular disease, atrial fibrillation. Increased production of reactive oxygen species and systemic inflammation promote cardiac structural and electrophysiologic remodeling that is crucial with respect to development and sustaining of atrial fibrillation. Data suggest that alterations in atrial connexin-43 and/or connexin-40 expression, phosphorylation and distribution affect cell-to-cell electrical coupling and molecular signalling that is proarrhythmogenic. However, studies showing causal relationship in the context of pathogenesis of atrial fibrillation are still scarce. Nevertheless, gap junctional connexin channels are considered as targets for arrhythmia prevention and therapeutic interventions aimed at mitochondria-related reactive oxygen species appear to be challenging. In addition, ageing is accompanied by abnormalities in adhesive junctions that most likely promote asynchronous contraction and arrhythmias. It is consistent with recent data that highlight a new perspective in regulation of intercalated disc function via adhesive junctions, fascia adherens and desmosomes. The crosstalk between adhesive and gap junctions is suggested to be implicated in pathogenesis of arrhythmias. On the other hand, modulation of adhesive proteins, N-cadherin and catenin may be promising tool aimed to synchronize heart function. Despite the progress in this field many questions remain to be answered by further research.

Modulation of cardiac connexin-43 by omega-3 fatty acid ethyl-ester supplementation demonstrated in spontaneously diabetic rats.

Previous data suggest that type 1 diabetes mellitus leads to the deterioration of myocardial intercellular communication mediated by connexin-43 (Cx43) channels. We therefore aimed to explore Cx43, PKC signaling and ultrastructure in non-treated and omega-3 fatty acid (omega-3) treated spontaneously diabetic Goto-Kakizaki (GK) rats considered as type 2 diabetes model. Four-week-old GK and non-diabetic Wistar-Clea rats were fed omega-3 (200 mg/kg/day) for 2 months and compared with untreated rats. Real-time PCR and immunoblotting were performed to determine Cx43, PKC-epsilon and PKC-delta expression. In situ Cx43 was examined by immunohistochemistry and subcellular alterations by electron microscopy. Omega-3 intake reduced blood glucose, triglycerides, and cholesterol in diabetic rats and this was associated with improved integrity of cardiomyocytes and capillaries in the heart. Myocardial Cx43 mRNA and protein levels were higher in diabetic versus non-diabetic rats and were further enhanced by omega-3. The ratio of phosphorylated (functional) to non-phosphorylated Cx43 was lower in diabetic compared to non-diabetic rats but was increased by omega-3, in part due to up-regulation of PKC-epsilon. In addition, pro-apoptotic PKC-delta expression was decreased. In conclusion, spontaneously diabetic rats at an early stage of disease benefit from omega-3 intake due to its hypoglycemic effect, upregulation of myocardial Cx43, and preservation of cardiovascular ultrastructure. These findings indicates that supplementation of omega-3 may be beneficial also in the management of diabetes in humans.

Acute anti-fibrillating and defibrillating potential of atorvastatin, melatonin, eicosapentaenoic acid and docosahexaenoic acid demonstrated in isolated heart model.

Cardioprotective compounds such as atorvastatin, melatonin, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) exhibit antiarrhythmic potential in clinical and/or experimental conditions but underlying mechanisms are poorly understood. We have previously shown that protection from ventricular fibrillation (VF) due to prolonged treatment with these compounds was linked with modulation of myocardial connexin-43, which is responsible for myocardial electrical coupling and synchronisation. To elucidate further the antiarrhythmic potential of atorvastatin, melatonin, EPA and DHA we aimed to explore their acute anti-fibrillating effects and defibrillating efficacy. Experiments were conducted on isolated perfused heart preparation of adult male and female hypertriglyceridemic (HTG) rats when using atorvastatin, EPA and DHA, while melatonin was examined in hearts of old male and female guinea pigs. VF inducibility was tested in hearts pre-treated for 10 min with atorvastatin, EPA or DHA (15 µmol) or melatonin (50 µmol) and compared with non-pre-treated hearts. Sustained VF was induced in all untreated HTG rat hearts. In contrast, its incidence was reduced to 30% and 60% by atorvastatin, 70% and 75% by EPA, 60% and 60% by DHA in male or female rat hearts respectively. Moreover, bolus (150 µmol) of EPA and DHA administered directly to the fibrillating heart restored sinus rhythm in 6 of 6 hearts and atorvastatin in 4 of 6 hearts. Threshold to induce sustained VF was 21.7 ± 3.8 mA in male and 38.3 ± 2.9 mA in female guinea pig hearts. However, sustained VF was not possible to induce even by the strongest (50 mA) stimulus in the heart pre-treated with melatonin regardless the sex. In conclusion, atorvastatin, melatonin, EPA and DHA exhibit clear cut acute anti-fibrillating efficacy. Findings challenge to investigate expression of connexin-43, especially its phosphorylated status associated with connexin channel function, in acute conditions.

Lower omega-3 index is a marker of increased propensity of hypertensive rat heart to malignant arrhythmias.

Polyunsaturated omega-3 fatty acids (omega-3 PUFA) are important components of cell membrane affecting its function and their deficiency is deleterious to health. We have previously shown that spontaneously hypertensive rats (SHR) are prone to life-threatening arrhythmias that are reduced by omega-3 PUFA intake. Purpose of this study was to explore plasma and red blood cells (RBC) profile of omega-3 and omega-6 PUFA as well as to determine omega-3 index, a risk factor for sudden cardiac death, in aged SHR and the effect of omega-3 PUFA intake. Male and female 12-month-old SHR and age-matched Wistar rats fed with omega-3 PUFA (200 mg/kg BW/day/2 month) were compared with untreated rats. Composition of omega-3 PUFA: alpha linolenic acid, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) as well as omega-6 PUFA: linoleic acid and arachidonic acid was analyzed by gas chromatography. Results showed sex- and strain-related differences of basal omega-3 and omega-6 PUFA levels in plasma and RBC as well as in response to omega-3 PUFA intake. Comparing to Wistar rats omega-3 index, expressed as a percentage of EPA+DHA of total fatty acids, was lower in SHR and it increased due to consumption of omega-3 PUFA. Findings support our hypothesis that lower omega-3 index may be also a marker of increased propensity of the hypertensive rat heart to malignant arrhythmias.

N-3 polyunsaturated fatty acids supplementation does not affect changes of lipid metabolism induced in rats by altered thyroid status.

Epidemiological studies have demonstrated that n-3 polyunsaturated fatty acid (PUFA) consumption is associated with a reduced risk of atherosclerosis and hyperlipidemia. It is well known that lipid metabolism is also influenced by thyroid hormones. The aim of our study was to test whether n-3 PUFA supplementation (200 mg/kg of body weight/day for 6 weeks given intragastrically) would affect lipid metabolism in Lewis male rats with altered thyroid status. Euthyroid, hypothyroid, and hyperthyroid status of experimental groups was well defined by plasma levels of triiodothyronine, the activity of liver mitochondrial glycerol-3-phosphate dehydrogenase, and by relative heart weight. Fasting blood glucose levels were significantly higher in the hyperthyroid compared to the euthyroid and hypothyroid rats (5.0±0.2 vs. 3.7±0.4 and 4.4±0.2 mmol/l, respectively). In hyperthyroid animals, the concentration of plasma postprandial triglycerides was also increased compared to euthyroid and hypothyroid rats (0.9±0.1 vs. 0.5±0.1 and 0.4±0.1 mmol/l, respectively). On the other hand, hypothyroidism compared to euthyroid and hyperthyroid status was associated with elevated plasma levels of total cholesterol (2.6±0.2 vs. 1.5±0.1 and 1.6±0.1 mmol/l, respectively), LDL cholesterol (0.9±0.1 vs. 0.4±0.1 and 0.2±0.1 mmol/l, respectively) as well as HDL cholesterol (1.6±0.1 vs. 1.0±0.1 and 1.3±0.1 mmol/l, respectively). Supplementation of n-3 PUFA in the present study did not significantly modify either relative heart weight or glucose and lipid levels in any thyroid status.

Impaired cardiac ischemic tolerance in spontaneously hypertensive rats is attenuated by adaptation to chronic and acute stress.

Chronic hypertension may have a negative impact on the myocardial response to ischemia. On the other hand, intrinsic ischemic tolerance may persist even in the pathologically altered hearts of hypertensive animals, and may be modified by short- or long-term adaptation to different stressful conditions. The effects of long-term limitation of living space (ie, crowding stress [CS]) and brief ischemia-induced stress on cardiac response to ischemia/reperfusion (I/R) injury are not yet fully characterized in hypertensive subjects. The present study was designed to test the influence of chronic and acute stress on the myocardial response to I/R in spontaneously hypertensive rats (SHR) compared with their effects in normotensive counterparts. In both groups, chronic, eight-week CS was induced by caging five rats per cage in cages designed for two rats (200 cm(2)/rat), while controls (C) were housed four to a cage in cages designed for six animals (480 cm(2)/rat). Acute stress was evoked by one cycle of I/R (5 min each, ischemic preconditioning) before sustained I/R in isolated Langendorff-perfused hearts of normotensive and SHR rats. At baseline conditions, the effects of CS were manifested only as a further increase in blood pressure in SHR, and by marked limitation of coronary perfusion in normotensive animals, while no changes in heart mechanical function were observed in any of the groups. Postischemic recovery of contractile function, severity of ventricular arrhythmias and lethal injury (infarction size) were worsened in the hypertrophied hearts of C-SHR compared with normotensive C. However, myo-cardial stunning and reperfusion-induced ventricular arrhythmias were attenuated by CS in SHR, which was different from deterioration of I/R injury in the hearts of normotensive animals. In contrast, ischemic preconditioning conferred an effective protection against I/R in both groups, although the extent of anti-infarct and anti-arrhythmic effects was lower in SHR. Both forms of stress may improve the altered response to ischemia in hypertensive subjects. In contrast to short-term preconditioning stress, chronic psychosocial stress was associated with a higher risk of lethal arrhythmias and contractile failure in normotensive animals exposed to an acute ischemic challenge.

Gender specific influence of fish oil or atorvastatin on functional properties of renal Na,K-ATPase in healthy Wistar and hypertriglyceridemic rats.

For better understanding of pathophysiological processes leading to increased retention of sodium as a consequence of hyperlipidemia, the properties of renal Na,K-ATPase, a key enzyme involved in maintaining sodium homeostasis in the organism, were studied. Enzyme kinetics of renal Na,K-ATPase were used for characterization of ATP- and Na(+)-binding sites after administration of fish oil (FO) (30 mg·day(-1)) or atorvastatin (0.5 mg·100 g(-1)·day(-1)) to healthy Wistar rats and rats with hereditary hypertriglyceridemia of both genders. Untreated healthy Wistar and also hypertriglyceridemic female rats revealed higher Na,K-ATPase activity as compared to respective untreated male groups. Hypertriglyceridemia itself was accompanied with higher Na,K-ATPase activity in both genders. Fish oil improved the enzyme affinity to ATP and Na(+), as indicated by lowered values of K(m) and K(Na) in Wistar female rats. In Wistar male rats FO deteriorated the enzyme in the vicinity of the Na(+)-binding site as revealed from the increased K(Na) value. In hypertriglyceridemic rats FO induced a significant effect only in females in the vicinity of the sodium binding sites resulting in improved affinity as documented by the lower value of K(Na). Atorvastatin aggravated the properties of Na,K-ATPase in both genders of Wistar rats. In hypertriglyceridemic rats protection of Na,K-ATPase was observed, but this effect was bound to females only. Both treatments protected renal Na,K-ATPase in a gender specific mode, resulting probably in improved extrusion of excessive intracellular sodium out of the cell affecting thus the retention of sodium in hHTG females only.

Omega-3 fatty acids and atorvastatin suppress ventricular fibrillation inducibility in hypertriglyceridemic rat hearts: implication of intracellular coupling protein, connexin-43.

Omega-3 fatty acids (omega-3 FA) and statins exhibit besides lipid-lowering effects the antiarrhythmic ability in clinic, while definite mechanisms are not yet elucidated. Our goal was to examine whether these compounds can modulate inducibility of hypertriglyceridemic (HTG) rat heart to ventricular fibrillation (VF) and myocardial cell-to-cell coupling protein connexin-43 (Cx43). HTG and healthy Wistar rats were orally treated with omega-3 FA(30 mg/100 g/day/2 mth) and atorvastatin (Ato, 0.5 mg/100 g/day/2 mth) and compared to untreated rats. Susceptibility of the heart to electrically-inducible VF and functional parameters were monitored using Langendorff-perfused isolated heart. Ventricular tissues from treated and untreated HTG and Wistar rat hearts were processed for ultrastructure examination as well as for analysis of myocardial Cx43 distribution and expression using antiCx43 MAB, immunofluorescence and immunoblotting. Both, omega-3 FA and atorvastatin reduced elevated blood pressure, triglycerides and heart rate in HTG rats. Compared to Wistar the threshold to induce VF was lower in HTG rat hearts, which exhibited abnormal Cx43 distribution, decreased immunostaining and elevated phosphorylated form of Cx43. In contrast, an enhancement of immunostaining of Cx43, suppression of hyperphosphorylation of Cx43 and improvement of cardiomyocyte and intercellular junction integrity by omega-3 FA and atorvastatin was associated with a significant increase of threshold for VF. Moreover, treatment resulted in up-regulation of myocardial Cx43 and increase of VF threshold in healthy rats that was associated with up-regulation of Cx43. Results indicate that antiarrhythmic effects of omega-3 FA and atorvastatin are linked with modulation of expression and/or phosphorylation of Cx43 and protection of cardiomyocyte and cell-to-cell junction integrity. As both compounds are ligands for PPAR, a possible regulation of Cx43 gene expression and pathways involved in Cx43 phosphorylation should be investigated.

Modulation of connexin-43 by omega-3 fatty acids in the aorta of old spontaneously hypertensive rats.

Hypertension alters expression of connexin-43 (Cx43) in cardiovascular system. The aim of the study was to investigate the effect of omega-3 polyunsaturated fatty acids (30 mg/day for 2 months) on expression of Cx43 in the aorta of 1-year-old male spontaneously hypertensive rats (SHR). Spatial distribution and expression of Cx43 in aortic wall of SHR and age-matched Lewis rats were determined by immunofluorescent method and Western blot. NO synthase (NOS) activity and endothelium-dependent relaxation of the aorta were measured as well. Immunofluorescent pattern of Cx43 was identified in endothelial and smooth muscle cells of the aorta of all experimental groups studied. However, local decrease in the number and intensity of fluorescent spots and reduced phosphorylation of Cx43 were observed in SHR in contrast to normotensive LEW. Omega-3 fatty acid diet increased Cx43 immunolabeling in endothelium and media of SHR comparing to untreated ones. Parallel, 3-fatty acids significantly elevated phosphorylation of Cx43 in the aorta of SHR (p<0.001). Despite the omega-3 fatty acids reduced blood pressure and stimulated aortic NOS activity in SHR, endothelium-dependent relaxation of the aorta did not significantly change. Results indicate that the aorta of old SHR might partially benefit from 3-PUFA supplementation due to increased Cx43 phosphorylation, NOS activity and decreased blood pressure.

Diabetes and thyroid hormones affect connexin-43 and PKC-epsilon expression in rat heart atria.

We have examined the changes of intercellular electrical coupling protein connexin-43 (Cx43) and of PKC-epsilon in heart atria of diabetic rats and/or after the treatment with triiodothyronine (T(3)). Diabetes was induced in Wistar-Kyoto rats by streptozotocin (50 mg/kg, i.v.) and atria were examined after 5 (acute stage) and 10 (chronic stage) weeks. T(3) (10 microg/100 g/day) was applied via a gastric tube for the last 10 days prior to the end of the experiments to non-diabetic and to the half of diabetic rats. Expression and phosphorylated status of Cx43, as well as expression of PKC-epsilon, were analyzed by Western blots using mouse monoclonal anti-Cx43 and rabbit polyclonal anti-PKC-epsilon antibodies. We found that the Cx43 expression was significantly increased after the treatment with T(3) and in the acute diabetes. Both in diabetes and after T(3) treatment the phosphorylation of Cx43 isoforms was markedly suppressed compared to the non-diabetic and T(3)-untreated controls. Such a down-regulation was less pronounced in diabetic rats after the T(3)-treatment. The expression of atrial PKC-epsilon was increased in diabetic rats. This increase was suppressed after T(3) administration and the expression was decreased in T(3)-treated non-diabetic rats. We suggest that the reduced Cx43 phosphorylation in diabetic and hyperthyroid rats can deteriorate a cell-to-cell coupling and consequently facilitate a development of atrial tachyarrhythmia in diabetic or hyperthyroid animals.

Thyroid hormones suppress epsilon-PKC signalling, down-regulate connexin-43 and increase lethal arrhythmia susceptibility in non-diabetic and diabetic rat hearts.

We examined whether thyroid hormones affect myocardial epsilon-PKC signalling, downstream target substrate, connexin-43 (Cx43) and arrhythmogenesis in non-diabetic and diabetic rats. Diabetes was induced by a single streptozotocin injection (50mg/kg, i.v.). Triiodothyronine (T(3)) was applied by gavage (1microg/kg of body weight for 10 days) to 4 weeks and 9 weeks diabetic and age-matched non-diabetic rats. Western blot analysis of Cx43 and epsilon-PKC, immunofluorescence of Cx43, ultrastructure of cardiomyocytes and myocardial conduction velocity were performed. Isolated perfused heart preparation was used to test ventricular fibrillation susceptibility. T(3) significantly decreased epsilon-PKC expression in non-diabetic and suppressed in diabetic rat heart ventricles. Decline of epsilon-PKC signalling was associated with decrease of Cx43 phosphorylation in diabetic and to a greater extent in non-diabetic rat hearts. However, conduction velocity was significantly decreased in diabetic while enhanced due to T(3) and increased in non-diabetic T(3)-treated rat heart ventricles compared to non-treated. T(3)-induced down-regulation of Cx43 was associated with increased cardiac propensity to ventricular fibrillation. Findings indicate that activation of epsilon-PKC signalling linked with phosphorylation of Cx43 is one of the mechanisms involved in the adaptation of the heart to hyperglycemia. Suppression of epsilon-PKC and Cx43 phosphorylation by T(3) abolish benefit of adaptation rendering the heart prone to lethal arrhythmias.

Thyroid hormones modulate occurrence and termination of ventricular fibrillation by both long-term and acute actions.

Thyroid hormones (TH) are powerful modulators of heart function, but their arrhythmogenic effects are less elucidated. We have examined both acute and long-term action of TH on the heart susceptibility to the ventricular fibrillation (VF) and on the heart ability to terminate VF and restore a sinus rhythm. Triiodothyronine (T3) was applied in the range of 10(-9)-10(-6) mol/l in acute experiments using isolated perfused aged (14-month-old) guinea pig hearts. L-thyroxine (T4) was applied in the dose of 50 microg/100g/day to young (3-month-old) and aged (20-month-old) rats for 2 weeks. The T4 treatment resulted in an increased susceptibility of young, but not adult rat hearts to a hypokalemia-induced VF and facilitated a spontaneous sinus rhythm (SSR) restoration in the latter group. The acute T3 administration in the range of 10(-9)-10(-7) mol/l significantly decreased the susceptibility of an isolated heart to an electrically induced VF and also facilitated the sinus rhythm restoration. The SSR restoration was, however, not affected by 10(-6) mol/l concentration of T3, which also led to an increased VF susceptibility. Results indicate that TH can affect the susceptibility of the heart to VF and its ability to restore the sinus rhythm via acute (non-genomic) and long-term (genomic) actions. Furthermore, an anti- and pro-arrhythmic potential of TH appears to be age- and dose-dependent.