Antiarrhythmic Effects of Melatonin and Omega-3 Are Linked with Protection of Myocardial Cx43 Topology and Suppression of Fibrosis in Catecholamine Stressed Normotensive and Hypertensive Rats.

Cardiac ß-adrenergic overstimulation results in oxidative stress, hypertrophy, ischemia, lesion, and fibrosis rendering the heart vulnerable to malignant arrhythmias. We aimed to explore the anti-arrhythmic efficacy of the anti-oxidative and anti-inflammatory compounds, melatonin, and omega-3, and their mechanisms of actions in normotensive and hypertensive rats exposed to isoproterenol (ISO) induced ß-adrenergic overdrive. Eight-month-old, male SHR, and Wistar rats were injected during 7 days with ISO (cumulative dose, 118 mg/kg). ISO rats were either untreated or concomitantly treated with melatonin (10 mg/kg/day) or omega-3 (Omacor, 1.68 g/kg/day) until 60 days of ISO withdrawal and compared to non-ISO controls. Findings showed that both melatonin and omega-3 increased threshold current to induce ventricular fibrillation (VF) in ISO rats regardless of the strain. Prolonged treatment with these compounds resulted in significant suppression of ISO-induced extracellular matrix alterations, as indicated by reduced areas of diffuse fibrosis and decline of hydroxyproline, collagen-1, SMAD2/3, and TGF-ß1 protein levels. Importantly, the highly pro-arrhythmic ISO-induced disordered cardiomyocyte distribution of electrical coupling protein, connexin-43 (Cx43), and its remodeling (lateralization) were significantly attenuated by melatonin and omega-3 in Wistar as well as SHR hearts. In parallel, both compounds prevented the post-ISO-related increase in Cx43 variant phosphorylated at serine 368 along with PKCε, which are known to modulate Cx43 remodeling. Melatonin and omega-3 increased SOD1 or SOD2 protein levels in ISO-exposed rats of both strains. Altogether, the results indicate that anti-arrhythmic effects of melatonin and omega-3 might be attributed to the protection of myocardial Cx43 topology and suppression of fibrosis in the setting of oxidative stress induced by catecholamine overdrive in normotensive and hypertensive rats.

Cardiac Cx43 and ECM Responses to Altered Thyroid Status Are Blunted in Spontaneously Hypertensive versus Normotensive Rats.

Heart function and its susceptibility to arrhythmias are modulated by thyroid hormones (THs) but the responsiveness of hypertensive individuals to thyroid dysfunction is elusive. We aimed to explore the effect of altered thyroid status on crucial factors affecting synchronized heart function, i.e., connexin-43 (Cx43) and extracellular matrix proteins (ECM), in spontaneously hypertensive rats (SHRs) compared to normotensive Wistar Kyoto rats (WKRs). Basal levels of circulating THs were similar in both strains. Hyperthyroid state (HT) was induced by injection of T3 (0.15 mg/kg b.w. for eight weeks) and hypothyroid state (HY) by the administration of methimazol (0.05% for eight weeks). The possible benefit of omega-3 polyunsaturated fatty acids (Omacor, 200 mg/kg for eight weeks) intake was examined as well. Reduced levels of Cx43 in SHRs were unaffected by alterations in THs, unlike WKRs, in which levels of Cx43 and its phosphorylated form at serine368 were decreased in the HT state and increased in the HY state. This specific Cx43 phosphorylation, attributed to enhanced protein kinase C-epsilon signaling, was also increased in HY SHRs. Altered thyroid status did not show significant differences in markers of ECM or collagen deposition in SHRs. WKRs exhibited a decrease in levels of profibrotic transforming growth factor ß1 and SMAD2/3 in HT and an increase in HY, along with enhanced interstitial collagen. Short-term intake of omega-3 polyunsaturated fatty acids did not affect any targeted proteins significantly. Key findings suggest that myocardial Cx43 and ECM responses to altered thyroid status are blunted in SHRs compared to WKRs. However, enhanced phosphorylation of Cx43 at serine368 in hypothyroid SHRs might be associated with preservation of intercellular coupling and alleviation of the propensity of the heart to malignant arrhythmias.

Effect of metabolic syndrome on neural plasticity and morphology of the hippocampus: correlations of neurological deficits with physiological status of the rat.

Fat-rich diet (FRD) triggers health complications like hypertension, dyslipidemia, hyperglycemia, insulin resistance and non-alcoholic fatty liver disease, known as the risk factors of metabolic syndrome (MetS), which may result in neurological deficits. The impact of MetS on neuronal functions and brain morphology are poorly understood. We induced MetS-like conditions by exposing hypertriacylglycerolemic (HTG) rats to FRD for eight weeks with the aim to study possible neurological dysfunctions. HTG-FRD rats were compared to HTG rats and Wistar rats on standard diet. The physiological status of the animals was monitored by body, liver and kidney weight. Morphology of the liver, vessel wall and hippocampus were investigated. Basal neurotransmission and synaptic plasticity were measured in the hippocampus ex-vivo. A marked increase of liver weight with marks of steatosis was found in the HTG-FRD group. FRD induced an increase of aortic intima-media thickness. Extracellular recording revealed FRD-induced impairment of long-term potentiation (LTP) at Cornu Ammonis (CA)3-CA1 synapse, contrary to increased presynaptic fiber volley (pV). Reduced thickness of pyramidal cell layer at the CA1 area was found morphometrically. LTP was directly associated with kidney weight and inversely associated with liver weight, pV directly correlated with liver weight, liver/body wt ratio and aortic intima-media thickness. Our results suggest correlations between altered physiological status due to MetS-like conditions and neurological deficits, which may be related with consecutive development of so-called metabolic cognitive syndrome.

Myocardial connexin-43 and PKC signalling are involved in adaptation of the heart to irradiation-induced injury: Implication of miR-1 and miR-21.

Intercellular connexin-43 (Cx43) channels are essential for electrical coupling and direct cardiac cell to cell communication to ensure heart function. Expression of Cx43 is altered due to stressful conditions and also affected by the alterations in extracellular matrix. We aimed to explore the effect of chest irradiation on myocardial expression of Cx43 and miR-1 which regulates GJA1 gene transcription for Cx43. Implication of miR-21 that regulates expression of extracellular matrix proteins and PKC signalling that may affect Cx43-mediated coupling was examined as well. Western blot and real-time PCR analyses revealed that six weeks after the exposure of healthy Wistar rats chest to single irradiation of 25 Gy significant myocardial alterations were observed: 1)/ increase of total Cx43 protein expression and its functional phosphorylated forms; 2) suppressed levels of miR-1; 3) enhanced expression of PKCε which phosphorylates Cx43; 4) increase of miR-21 levels; 5) increase of PKCδ expression. These results suggest that irradiation causes post-transcriptional regulation of myocardial Cx43 expression by miR-1 possibly through miR-21 and PKC signalling. We conclude that single dose of irradiation has the potential to enhance myocardial intercellular communication that might be beneficial for the heart that needs to be investigated in details in further studies.

Melatonin attenuates hypertension-related proarrhythmic myocardial maladaptation of connexin-43 and propensity of the heart to lethal arrhythmias.

We hypothesized that the pineal hormone melatonin, which exhibits cardioprotective effects, might affect myocardial expression of cell-to-cell electrical coupling protein connexin-43 (Cx43) and protein kinase C (PKC) signaling, and hence, the propensity of the heart to lethal ventricular fibrillation (VF). Spontaneously hypertensive (SHR) and normotensive Wistar rats fed a standard rat chow received melatonin (40 µg/mL in drinking water during the night) for 5 weeks, and were compared with untreated rats. Melatonin significantly reduced blood pressure and normalized triglycerides in SHR, whereas it decreased body mass and adiposity in Wistar rats. Compared with healthy rats, the threshold to induce sustained VF was significantly lower in SHR (18.3 ± 2.6 compared with 29.2 ± 5 mA; p < 0.05) and increased in melatonin-treated SHR and Wistar rats to 33.0 ± 4 and 32.5 ± 4 mA. Melatonin attenuated abnormal myocardial Cx43 distribution in SHR, and upregulated Cx43 mRNA, total Cx43 protein, and its functional phosphorylated forms in SHR, and to a lesser extent, in Wistar rat hearts. Moreover, melatonin suppressed myocardial proapoptotic PKCδ expression and increased cardioprotective PKCε expression in both SHR and Wistar rats. Our findings indicate that melatonin protects against lethal arrhythmias at least in part via upregulation of myocardial Cx43 and modulation of PKC-related cardioprotective signaling.

Up-regulation of myocardial connexin-43 in spontaneously hypertensive rats fed red palm oil is most likely implicated in its anti-arrhythmic effects.

The purpose of this study was to test our hypothesis that red palm oil (RPO) intake may affect abnormalities of myocardial connexin-43 (Cx43) and protein kinase Cε (PKCε) signaling, and consequently the propensity of the spontaneously hypertensive rat heart (SHR) heart to arrhythmias. SHR and Wistar-Kyoto (WKY) rats fed a standard rat chow plus red palm oil (200 µL/day) for 5 weeks were compared with untreated rats. Cytosolic but not particulate PKCε expression as well as Cx43-mRNA, total Cx43 proteins, and its phoshorylated forms were increased, and disordered localization of Cx43 was attenuated in the left ventricle of RPO-fed SHR compared with untreated rats. These alterations were associated with suppression of early post-ischemic-reperfusion-related ventricular tachycardia and electrically inducible ventricular fibrillation. However, the treatment dose of RPO caused down-regulation of myocardial Cx43, but did not alter its cell membrane distribution or overall PKCε expression in WKY rats. It was, however, associated with poor arrhythmia protection, suggesting overdosing. Results indicate that SHR benefit from RPO intake, particularly because of its apparent anti-arrhythmic effects. This protection can be, in part, attributed to the preservation of cell-to-cell communication via up-regulation of myocardial Cx43, but not with PKCε activation.

Impact of radiotherapy on laryngeal intrinsic muscles.

Ionizing radiation as a cancer therapy is associated with a variety of undesirable side effects. Consequently, radiotherapy can negatively affect neuromuscular function. Clinical observations have identified problems with swallowing and voice function. Our study aims to evaluate the impact of radiotherapy on laryngeal soft tissues using image analysis to quantify its effect on the structure of the vocalis and thyroarytenoid muscles. Case control study, retrospective analysis. We collected total laryngectomy specimens from six patients with persistent or recurrent cancer who had received preoperative radiotherapy (60-66 Gy). The control group consisted of total laryngectomy specimens from six patients who underwent surgery as primary treatment. Sampling of the specimens only included non-cancerous laryngeal tissue. Laryngeal histological slices were evaluated using digital morphometric analysis system. Percentage of fibrosis and density of muscle fibers within the thyroarytenoid muscle were evaluated in both groups. We found no significant quantitative differences in muscle fibrosis (7.92% vs. 7.52%, P > 0.1). Changes were rather qualitative and included changes in the organization of the muscular fibers. A significant reduction in muscle fibers, however, was observed in the samples from irradiated larynges (66.45% vs. 42.03%, P < 0.01). Our analysis suggests that radiotherapy is responsible for a significant reduction in muscle fibers in the thyroarytenoid muscle and that these changes occur during treatment or relatively early after its completion. Loss of muscle mass after irradiation correlates with clinical observations of muscle weakness and decreased function in patients who undergo radiotherapy.

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.

Distinct lethal arrhythmias susceptibility is associated with sex-related difference in myocardial connexin-43 expression.

OBJECTIVES: To elucidate gender-related differences in occurrence of sudden cardiac death the myocardial connexin-43 (Cx43) and the susceptibility of male and female rat hearts to ventricular fibrillation (VF) were investigated. METHODS AND RESULTS: Ventricular tissues taken from male and female normotensive Wistar and spontaneously hypertensive (SHR) rats were processed for immuno-fluorescence and immuno-blotting of Cx43. Susceptibility to ventricular fibrillation was examined in isolated heart preparation using either electrical stimulation or low K+ perfusion. Results showed that VF susceptibility of male either normotensive or hypertensive rats was significantly increased comparing to female counterparts. In correlation, ventricular expression of Cx43 was markedly lower in males of both normotensive and hypertensive rats comparing to females. SHR in addition exhibited abnormal myocardial Cx43 distribution due to structural remodelling. CONCLUSIONS: Findings indicate that higher level of myocardial Cx43 expression is linked with lower lethal arrhythmia susceptibility and vice versa. It appears that Cx43 can be involved in sex-related differences in incidence of life-threatening arrhythmias.

Autoantibodies against G-protein-coupled receptors modulate heart mast cells.

Mast cells are believed to be involved in myocardial tissue remodelling under pathophysiological conditions. We examined the effects of autoantibodies against G-protein-coupled receptors in sera of patients with heart diseases on myocardial mast cells in the cultured neonatal Sprague-Dawley rat heart cells. Cells collected at day 3 and 10 of the culture were preincubated with autoantibodies against alpha1-adrenoceptor and angiotensin II AT1-receptor, agonist phenylephrine and angiotensin II, and control IgG. The pretreated cultured cells were stained for selected mast cell markers tryptase, chymase and TNF-alpha. The cultured cells were also processed for observation with electron microscopy. The autoantibodies-treatment of the 3-day cultured cells caused both increased intensity of immunofluorescence (p < 0.05) and their enlarged diameters of the mast cells when compared to age-matched ones. In contrast, the fluorescence of preincubated 10-day-old mast cells was decreased compared with controls (p < 0.01). In control samples, the fluorescence of 10-day-old mast cells was significantly higher than that of 3-day-old ones (p < 0.001). Results of electron microscopy examination demonstrated there was an increased granulation of treated 3-day-old mast cells, while a degranulation of mast cells at day 10 of application. The results suggest the modulation effect of the autoantibodies against G-protein-coupled receptors on mast cells, indicating a potential functional link between the autoantibodies against G-protein-coupled receptors and the mast cells in progression of heart disease.

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

Insufficient 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.

Enhanced connexin-43 and alpha-sarcomeric actin expression in cultured heart myocytes exposed to triiodo-L-thyronine.

This study examined whether triiodo-L-thyronine (T3) affects the expression of the major intercellular channel protein, connexin-43, and contractile protein alpha-sarcomeric actin. Cultured cardiomyocytes from newborn rats were treated on day three in culture with 10 or 100 nM T3 and examined 48 and 72 h thereafter. Treated and untreated cells were examined by immunofluorescence and electron microscopy. Expression levels of Cx43 and sarcomeric alpha-actin were monitored by Western blot analysis. Immunofluorescence labeling showed cell membrane location of Cx43 in punctuate gap junctions, whereby fluorescence signal area was significantly higher in cultured cardiomyocytes exposed to T3. This correlated with electron microscopical findings showing increased numbers and size of gap junction profiles, as well as with a significant dose-dependent increase of Cx43 expression detected by Western blot. Immunofluorescence of sarcomeric a-actin was enhanced and its expression increased dose- and time-dependently in T3-treated cultured heart myocytes. However, exposure to the higher dosage (100 nM) of T3 caused mild disintegration of sarcomeric a-actin in some myocytes, suggesting an over-dosage. The results indicate that T3 up-regulates Cx43 and accelerates gap junction formation in cultured neonatal cardiomyocytes. They suggest that thyroid status cannot only modulate the mechanical function of cardiomyocytes but also cell-to-cell communication essential for myocardial electrical and metabolic synchronizations.

Mast cell infiltration in the wall of varicose veins.

Varicose veins of the lower extremities are abnormally dilated, tortuous and elongated. The exact cause of vein dilatation has still not been established. Mast cells produce, store and release various types of vasoactive compounds (histamine, tryptase, prostaglandins, leukotrienes, and cytokines). Histamine enhances local vasopermeability and smooth muscle cell proliferation, leading to thickening of the intima. Tryptase can contribute to local vascular injury and subsequent weakness of the vascular wall causing varix formation. The aim of the present study was the comparison of mast cell infiltration in the wall of varicose and non-varicose veins. The mean mast cell density in the wall of varicose veins was 0.86 mast cell per mm2 and in healthy non-dilated vein walls, density was 1.23 mast cell per mm2. This difference was not statistically significant, therefore we could not confirm our hypothesis. Nevertheless, we suggest that mast cells could play an important role in the development of varices and the factor released by the mast cells should be further examined.