Barbara Szeiffova Bacova

Dietary omega-3 fatty acids attenuate myocardial arrhythmogenic factors and propensity of the heart to lethal arrhythmias in a rodent model of human essential hypertension.

Objective: Hypertension-induced myocardial remodeling is known to be associated with increased risk for malignant arrhythmias and alterations in electrical coupling protein, connexin-43 (Cx43), may be involved. We investigated whether omega-3 fatty acids intake affects abnormalities of Cx43 as well as protein kinase C (PKC) signaling and myosin heavy chain (MyHC) profile at the early and late stage of hypertension in the context of the hearts susceptibility to ventricular fibrillation and ability to restore sinus rhythm. Methods: Untreated young and old male spontaneously hypertensive rats (SHRs) and age-matched normotensive rats were compared with animals supplemented by omega-3 (eicosapentaneoic acid + docosahexaneoic acid, 200 mg/kg body weight/day) for 2 months. Left ventricular tissues were taken for examination of subcellular integrity of gap junctions, Cx43 mRNA and protein expression, PKC&egr; and PKC&dgr; as well as MyHC determination. Electrically inducible ventricular fibrillation and sinus rhythm restoration (SRR) were examined on Langedorff-perfused heart preparation. Results: Omega-3 intake significantly reduced cardiovascular risk factors, suppressed inducible ventricular fibrillation, and facilitated SRR in hypertensive rats. Supplementation attenuated lateralization and internalization of Cx43, suppressed elevated Cx43 mRNA, enhanced total Cx43 protein expression and/or expression of its functional phosphorylated forms as well as the expression of cardioprotective PKC-&egr; and suppressed pro-apoptotic PKC-&dgr; isoform. Moreover, the omega-3 diet normalized MyHC profiles in SHR at early stage of disease and old nonhypertensive rats, but failed to do so in old SHR at late stage of disease. Conclusion: Findings suggest that amelioration of myocardial Cx43-related abnormalities, positive modulation of PKC pathways, and normalization of MyHC can significantly contribute to the antiarrhythmic effects of omega-3 in rat model mimicking human essential hypertension. Our results support the prophylactic use of omega-3 to minimize cardiovascular risk and sudden arrhythmic death.

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.

Can we protect from malignant arrhythmias by modulation of cardiac cell-to-cell coupling?

Defects in intercellular coupling in the heart play a key role in the initiation and persistence of malignant arrhythmias. Such disorders result from abnormal expression and distribution of connexins, the major constituents of cardiac gap junction channels. The alterations of myocardial connexin are well established as a consistent feature of both human and animal heart disease and aging. Following these facts, the modulation of connexin mediated intercellular coupling is suggested as a new antiarrhythmic approach. This review provides recent data supporting this concept. It can be challenging for the development of new antiarrhythmic drugs. Moreover, findings point out the implication of some endogenous compounds in protection from life-threatening arrhythmias via preservation of myocardial connexin.

Intake of n-3 Polyunsaturated Fatty Acids Increases Omega-3 Index in Aged Male and Female Spontaneously Hypertensive Rats.

The purpose of this study was to examine whether n-3 PUFA intake affects n-3 and n-6 FA levels in plasma and red blood cells as well as omega-3 index in old male and female spontaneously hypertensive (SHR) and healthy rats. Plasma linoleic acid and eicosapentaenoic acid increased due to n-3 PUFA intake in SHR and healthy rats. Comparing to healthy rats the levels of PUFA in red blood cells of SHR were lower in males and higher in females with exception of arachidonic acid, which was high in males and low in females. Feeding of rats with n-3 PUFA resulted in increase of red blood cells levels of eicosapentaenoic acid and/or docosahexaenoic acid in a sex- and strain-dependent manner. Moreover, n-3 PUFA intake decreased arachidonic acid in healthy female rats but increased it in SHR and did not affect it in males. Omega-3 index was lower in SHR comparing to healthy rats and it increased due to the consumption of n-3 PUFA. Results point out sex- and strain-related differences in red blood cells levels of n-3 and n-6 PUFA in basal conditions as well as in response to n-3 PUFA intake.

Myocardial connexin-43 is upregulated in response to acute cardiac injury in rats1

We aimed to explore whether myocardial intercellular channel protein connexin-43 (Cx43) along with PKCε and MMP-2 might be implicated in responses to acute cardiac injury induced by 2 distinct sublethal interventions in Wistar rats. Animals underwent either single chest irradiation at dose of 25 Gy or subcutaneous injection of isoproterenol (ISO, 120 mg/kg) and were compared with untreated controls. Forty-two days post-interventions, the hearts were excised and left ventricles were used for analysis. The findings showed an increase of total as well as phosphorylated forms of myocardial Cx43 regardless of the type of interventions. Enhanced phosphorylation of Cx43 coincided with increased PKCε expression in both models. Elevation of Cx43 was associated with its enhanced distribution on lateral surfaces of the cardiomyocytes in response to both interventions, while focal areas of fibrosis without Cx43 were found in post-ISO but not post-irradiated rat hearts. In parallel, MMP-2 activity was decreased in the former while increased in the latter. Cardiac function was maintained and the susceptibility of the hearts to ischemia or malignant arrhythmias was not deteriorated 42 days after interventions when compared with controls. Altogether, the findings indicate that myocardial Cx43 is most likely implicated in potentially salutary responses to acute heart injury.

Myocardial Connexin-43 is Implicated in the Prevention of Malignant Arrhythmia in Rats Suffering from Essential Hypertension

Gap-junction connexin (Cx) channels are important determinants of myocardial conduction and synchronization that is crucial for heart function. Hypertensioninduced structural remodeling is associated with an increased risk of life-threatening arrhythmias and heart failure in both humans and experimental animals. Recent studies suggest that abnormal distribution and/or downregulation of Cx43 accompanied with altered protein kinase C (PKC)ε signaling in spontaneously hypertensive rats were linked with increased propensity to ventricular fibrillation compared to normotensive rats. By contrast, the long-term treatment of hypertensive rats with cardioprotective compounds such as melatonin, omega-3 fatty acids, or red palm oil resulted in protection from lethal arrhythmia. Their antiarrhythmic effect was attributed to the attenuation of abnormal Cx43 topology and modulation of Cx43 mRNA as well as protein expression and its functional phosphorylated forms. The latter might be attributed to upregulation of PKCε. It appears that maladaptive consequences of hypertension resulting in abnormal myocardial distribution of Cx43 and its downre‐ gulation can contribute to arrhythmogenesis and occurrence of malignant arrhyth‐ mias. On the other hand, the attenuation of myocardial Cx43 abnormalities by treatment with melatonin, omega-3 fatty acids, or red palm oil confers arrhythmia protection in rodent model of essential hypertension. Findings uncover novel mechanisms of cardioprotective effects of melatonin, omega-3 fatty acids, and red palm oil. Welldesigned clinical trials are needed to explore antiarrhythmic potential of these compounds in human essential hypertension.

SPONTANEOUSLY DIABETIC RATS IN PRE-HYPERTENSIVE STAGE BENEFIT FROM OMEGA-3 FATTY ACIDS SUPPLEMENTATION: PP.17.141

Background: The prevalence of hypertension is frequent in patients who have type-2 diabetes (D2), while both events can result in cardiac dysfunction and occurrence of severe arrhythmias, such as ventricular fibrillation (VF). The latter, is known to be facilitated by impairment of myocardial cell-to-cell electrical coupling ensured by connexin (Cx) channels. Goal of this study was to investigate whether myocardial Cx43 mRNA and protein expression and/or phosphorylation are altered in D2 rats known to develop hypertension over time and whether these rats may benefit from omega-3 fatty acids (omega-3) supplementation. Design and Methods: Four week-old male Goto-Kakizaki rats at early stage of diabetes prior development of hypertension (∼ ten week later) and age-matched healthy rats were divided into un-treated and treated for 2-month with omega-3 (Vesteralens, Norway, 200 mg/kg/day). Left ventricles were used for determination of Cx43 mRNA and protein expression using real time PCR and immunobloting and for expression of PKC-epsilon, which phosphorylates Cx43. Myocardial ultrastructure and susceptibility of isolated perfused heart to aconitine-induced VF were examined as well. Results showed that omega-3 reduced elevated blood glucose, cholesterol and triglycerides in diabetic rats. Myocardial Cx43 mRNA and protein level was higher in diabetic than non-diabetic rats and further increased due to omega-3. On the other hand, ratio of phosphorylated to non-phosphorylated Cx43 was lower in diabetic than healthy rats, while enhanced upon omega-3. It was accompanied by increased expression of PKC-epsilon. Diabetic rat heart was much prone to VF compared to healthy and omega-3 treated rat hearts, which exhibited preserved integrity of cardiomyocytes and their junctions. Conclusions: Spontaneously diabetic rats in pre-hypertensive stage benefit from omega-3 fatty acids supplementation due to suppression of cardiovascular risk markers and up-regulation of Cx43 linked with decreased arrhythmia susceptibility. Findings challenge to know possible beneficial effects of omega-3 fatty acids supplementation in patients at early stages of type-2 diabetes.

Irradiation-Induced Cardiac Connexin-43 and miR-21 Responses Are Hampered by Treatment with Atorvastatin and Aspirin

Radiation of the chest during cancer therapy is deleterious to the heart, mostly due to oxidative stress and inflammation related injury. A single sub-lethal dose of irradiation has been shown to result in compensatory up-regulation of the myocardial connexin-43 (Cx43), activation of the protein kinase C (PKC) signaling along with the decline of microRNA (miR)-1 and an increase of miR-21 levels in the left ventricle (LV). We investigated whether drugs with antioxidant, anti-inflammatory or vasodilating properties, such as aspirin, atorvastatin, and sildenafil, may affect myocardial response in the LV and right ventricle (RV) following chest irradiation. Adult, male Wistar rats were subjected to a single sub-lethal dose of chest radiation at 25 Gy and treated with aspirin (3 mg/day), atorvastatin (0.25 mg/day), and sildenafil (0.3 mg/day) for six weeks. Cx43, PKCε and PKCδ proteins expression and levels of miR-1 as well as miR-21 were determined in the LV and RV. Results showed that the suppression of miR-1 was associated with an increase of total and phosphorylated forms of Cx43 as well as PKCε expression in the LV while having no effect in the RV post-irradiation as compared to the non-irradiated rats. Treatment with aspirin and atorvastatin prevented an increase in the expression of Cx43 and PKCε without change in the miR-1 levels. Furthermore, treatment with aspirin, atorvastatin, and sildenafil completely prevented an increase of miR-21 in the LV while having partial effect in the RV post irradiation. The increase in pro-apoptotic PKCδ was not affected by any of the used treatment. In conclusion, irradiation and drug-induced changes were less pronounced in the RV as compared to the LV. Treatment with aspirin and atorvastatin interfered with irradiation-induced compensatory changes in myocardial Cx43 protein and miR-21 by preventing their elevation, possibly via amelioration of oxidative stress and inflammation.

Omega-3 Index and Anti-Arrhythmic Potential of Omega-3 PUFAs

Omega-3 polyunsaturated fatty acids (PUFAs), namely eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are permanent subjects of interest in relation to the protection of cardiovascular health and the prevention of the incidence of both ventricular and atrial arrhythmias. The purpose of this updated review is to focus on the novel cellular and molecular effects of omega-3 PUFAs, in the context of the mechanisms and factors involved in the development of cardiac arrhythmias; to provide results of the most recent studies on the omega-3 PUFA anti-arrhythmic efficacy and to discuss the lack of the benefit in relation to omega-3 PUFA status. The evidence is in the favor of omega-3 PUFA acute and long-term treatment, perhaps with mitochondria-targeted antioxidants. However, for a more objective evaluation of the anti-arrhythmic potential of omega-3 PUFAs in clinical trials, it is necessary to monitor the basal pre-interventional omega-3 status of individuals, i.e., red blood cell content, omega-3 index and free plasma levels. In the view of evidence-based medicine, it seems to be crucial to aim to establish new approaches in the prevention of cardiac arrhythmias and associated morbidity and mortality that comes with these conditions.

Effects of Red Palm Oil on Myocardial Antioxidant Enzymes, Nitric Oxide Synthase and Heart Function in Spontaneously Hypertensive Rats

The purpose of this study was to investigate the effect of antioxidants rich red palm oil (RPO) supplementation on cardiac oxidative stress known as crucial factor deteriorating heart function in hypertension. 3-month-old, male spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto rats (WKY) were fed standard rat chow without or with RPO (0.2 mL/day/5 weeks). General characteristic of rats were registered. Left ventricular tissue (LV) was used to determine expression of superoxide dismutases (SOD1, SOD2) and glutathione peroxidases (Gpx) as well as activity of nitric oxide synthase (NOS). Functional parameters of the heart were examined during basal conditions and at the early-phase of post-ischemic reperfusion using Langendorff-perfused system. RPO intake significantly reduced elevated blood pressure and total NOS activity as well as increased lowered expression of mitochondrial SOD2 in SHR hearts during basal condition. Moreover, RPO supplementation resulted in suppression of elevated heart rate, increase of reduced coronary flow and enhancement of systolic and diastolic heart function at the early-phase of post-ischemic reperfusion. It is concluded that SHR benefit from RPO intake due to decrease of blood pressure, amelioration of oxidative stress and protection of heart function that was deteriorated by post-ischemic reperfusion.