Anti-Fibrotic Potential of Angiotensin (1-7) in Hemodynamically Overloaded Rat Heart.

The extracellular matrix (ECM) is a highly dynamic structure controlling the proper functioning of heart muscle. ECM remodeling with enhanced collagen deposition due to hemodynamic overload impairs cardiomyocyte adhesion and electrical coupling that contributes to cardiac mechanical dysfunction and arrhythmias. We aimed to explore ECM and connexin-43 (Cx43) signaling pathways in hemodynamically overloaded rat heart as well as the possible implication of angiotensin (1-7) (Ang (1-7)) to prevent/attenuate adverse myocardial remodeling. Male 8-week-old, normotensive Hannover Spraque-Dawley rats (HSD), hypertensive (mRen-2)27 transgenic rats (TGR) and Ang (1-7) transgenic rats (TGR(A1-7)3292) underwent aortocaval fistula (ACF) to produce volume overload. Five weeks later, biometric and heart tissue analyses were performed. Cardiac hypertrophy in response to volume overload was significantly less pronounced in TGR(A1-7)3292 compared to HSD rats. Moreover, a marker of fibrosis hydroxyproline was increased in both ventricles of volume-overloaded TGR while it was reduced in the Ang (1-7) right heart ventricle. The protein level and activity of MMP-2 were reduced in both ventricles of volume-overloaded TGR/TGR(A1-7)3292 compared to HSD. SMAD2/3 protein levels were decreased in the right ventricle of TGR(A1-7)3292 compared to HSD/TGR in response to volume overload. In parallel, Cx43 and pCx43 implicated in electrical coupling were increased in TGR(A1-7)3292 versus HSD/TGR. It can be concluded that Ang (1-7) exhibits cardio-protective and anti-fibrotic potential in conditions of cardiac volume overload.

Melatonin receptor activation protects against low potassium-induced ventricular fibrillation by preserving action potentials and connexin-43 topology in isolated rat hearts.

Hypokalemia prolongs the QRS and QT intervals, deteriorates intercellular coupling, and increases the risk for arrhythmia. Melatonin preserves gap junctions and shortens action potential as potential antiarrhythmic mechanisms, but its properties under hypokalemia remain unknown. We hypothesized that melatonin protects against low potassium-induced arrhythmias through the activation of its receptors, resulting in action potential shortening and connexin-43 preservation. After stabilization in Krebs-Henseleit solution (4.5 mEq/L K+ ), isolated hearts from Wistar rats underwent perfusion with low-potassium (1 mEq/L) solution and melatonin (100 µmol/L), a melatonin receptor blocker (luzindole, 5 µmol/L), melatonin + luzindole or vehicle. The primary endpoint of the study was the prevention of ventricular fibrillation. Electrocardiography was used, and epicardial action potentials and heart function were measured and analyzed. The ventricular expression, dephosphorylation, and distribution of connexin-43 were examined. Melatonin reduced the incidence of low potassium-induced ventricular fibrillation from 100% to 59%, delayed the occurrence of ventricular fibrillation and induced a faster recovery of sinus rhythm during potassium restitution. Melatonin prevented QRS widening, action potential activation delay, and the prolongation of action potential duration at 50% of repolarization. Other ECG and action potential parameters, the left ventricular developed pressure, and nonsustained ventricular arrhythmias did not differ among groups. Melatonin prevented connexin-43 dephosphorylation and its abnormal topology (lateralization). Luzindole abrogated the protective effects of melatonin on electrophysiological properties and connexin-43 misdistribution. Our results indicate that melatonin receptor activation protects against low potassium-induced ventricular fibrillation, shortens action potential duration, preserves ventricular electrical activation, and prevents acute changes in connexin-43 distribution. All of these properties make melatonin a remarkable antifibrillatory agent.

Altered thyroid status affects myocardial expression of connexin-43 and susceptibility of rat heart to malignant arrhythmias that can be partially normalized by red palm oil intake.

We aimed to study the impact of altered thyroid status on myocardial expression of electrical coupling protein connexin-43 (Cx43), the susceptibility of rats to ventricular fibrillation (VF) and the effects of antioxidant-rich red palm oil (RPO). Adult male and female euthyroid, hyperthyroid (treated with T3/T4), hypothyroid (treated with methimazole) Wistar rats supplemented or not with RPO for 6 weeks were used. Function of isolated perfused heart and VF threshold were determined. Left ventricular tissue was used for assessment of mRNA and protein levels of Cx43, its phosphorylated forms and topology. Protein kinase C signaling (PKC) and gene transcripts of some proteins related to cardiac arrhythmias were assessed. Hyperthyroid state resulted in decrease of total and phosphorylated forms of Cx43 and suppression of PKC-ε expression in males and females, decrease of Cx43 mRNA in females, decrease of VF threshold and increase of functional parameters in male rat hearts. In contrast, hypothyroid status resulted in the increase of total and phosphorylated forms of Cx43, enhancement PKC-ε expression in males and females, increase of Cx43 mRNA in females, increase of VF threshold and decrease of functional parameters in male rat hearts. Function of the heart was partially normalized by RPO intake, which also enhanced myocardial Cx43 and PKC-ε expression as well as increased VF threshold in hyperthyroid male rats. We conclude that there is an inverse relationship between myocardial expression of Cx43, including its functional phosphorylated forms, and susceptibility of male rat hearts to VF in condition of altered thyroid status. RPO intake partly ameliorated adverse changes caused by excess of thyroid hormones.

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.

Quercetin improves postischemic recovery of heart function in doxorubicin-treated rats and prevents doxorubicin-induced matrix metalloproteinase-2 activation and apoptosis induction.

Quercetin (QCT) is flavonoid that possesses various biological functions including anti-oxidative and radical-scavenging activities. Moreover, QCT exerts some preventive actions in treatment of cardiovascular diseases. The aim of present study was to explore effects of prolonged administration of QCT on changes induced by repeated application of doxorubicin (DOX) in rat hearts. We focused on the ultrastructure of myocardium, matrix metalloproteinases (MMPs), biometric parameters, and apoptosis induction. Our aim was also to examine effects of QCT on ischemic tolerance in hearts exposed to chronic effects of DOX, and to determine possible mechanisms underlying effects of QCT. Our results showed that QCT prevented several negative chronic effects of DOX: (I) reversed DOX-induced blood pressure increase; (II) mediated improvement of deleterious effects of DOX on ultrastructure of left ventricle; (III) prevented DOX-induced effects on tissue MMP-2 activation; and (iv) reversed effects of DOX on apoptosis induction and superoxide dismutase inhibition. Moreover, we showed that rat hearts exposed to effects of QCT were more resistant to ischemia/reperfusion injury. Effects of QCT on modulation of ischemic tolerance were linked to Akt kinase activation and connexin-43 up-regulation. Taken together, these results demonstrate that prolonged treatment with QCT prevented negative chronic effects of DOX on blood pressure, cellular damage, MMP-2 activation, and apoptosis induction. Moreover, QCT influenced myocardial responses to acute ischemic stress. These facts bring new insights into mechanisms of QCT action on rat hearts exposed to the chronic effects of DOX.

Chronic cardiotoxicity of doxorubicin involves activation of myocardial and circulating matrix metalloproteinases in rats.

AIM: To investigate the role of matrix metalloproteinases (MMPs) in the responses of rats to a prolonged doxorubicin (DOX) treatment. METHODS: Male Wistar rats were used. DOX was administered by intraperitoneal injections of seven doses (cumulative dose was 15 mg/kg). Control animals were treated with saline. Tissue or plasma samples were collected at four and eight weeks after the application of the last dose. Protein levels were determined by immunoblot assay, and MMP activities were measured by gelatin zymography. Superoxide content was analyzed using a lucigenin chemiluminescence assay and superoxide dismutase (SOD) activities with a SOD assay kit. Qualitative structural alterations of the heart were characterized by transmission electron microscopy. RESULTS: Systolic blood pressure was higher in DOX-treated rats as compared with the control rats at 8 weeks after treatment. In contrast, there were no differences in the heart rate between the control and DOX-treated rats. DOX treatment caused marked heterogeneous subcellular alterations of cardiomyocytes and structural disorganizations of the cardiac extracellular space. The effects of DOX were linked to a stimulation of plasma MMP-2 and MMP-9 activities that had already increased by 4 weeks after the end of the treatment. In the left ventricle, however, DOX only led to increased MMP-2 activation at 8 weeks after the end of treatment. These changes in tissue MMP-2 were connected with stimulation of Akt kinase activation, inhibition of SOD, an increase in superoxide levels, induction of iNOS protein expression and caspase-3 activation. CONCLUSION: Our results show that MMPs are involved in the chronic cardiotoxicity of DOX in rats. The data also suggest that reactive oxygen species (superoxide), NO production (iNOS) and the Akt kinase pathway can modulate MMP-2 activities in rat hearts influenced by DOX.

Biological Effects of Hydrogen Water on Subjects with NAFLD: A Randomized, Placebo-Controlled Trial.

Non-alcoholic fatty liver disease (NAFLD) is a liver pathology affecting around 25% of the population worldwide. Excess oxidative stress, inflammation and aberrant cellular signaling can lead to this hepatic dysfunction and eventual carcinoma. Molecular hydrogen has been recognized for its selective antioxidant properties and ability to attenuate inflammation and regulate cellular function. We administered hydrogen-rich water (HRW) to 30 subjects with NAFLD in a randomized, double-blinded, placebo-controlled manner for eight weeks. Phenotypically, we observed beneficial trends (p > 0.05) in decreased weight (≈1 kg) and body mass index in the HRW group. HRW was well-tolerated, with no significant changes in liver enzymes and a trend of improved lipid profile and reduced lactate dehydrogenase levels. HRW tended to non-significantly decrease levels of nuclear factor kappa B, heat shock protein 70 and matrix metalloproteinase-9. Interestingly, there was a mild, albeit non-significant, tendency of increased levels of 8-hydroxy-2'-deoxyguanosine and malondialdehyde in the HRW group. This mild increase may be indicative of the hormetic effects of molecular hydrogen that occurred prior to the significant clinical improvements reported in previous longer-term studies. The favorable trends in this study in conjunction with previous animal and clinical findings suggest that HRW may serve as an important adjuvant therapy for promoting and maintaining optimal health and wellness. Longer term studies focused on prevention, maintenance, or treatment of NAFLD and early stages of NASH are warranted.

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.

Automatic Quantification of Cardiomyocyte Dimensions and Connexin 43 Lateralization in Fluorescence Images.

Cardiomyocytes' geometry and connexin 43 (CX43) amount and distribution are structural features that play a pivotal role in electrical conduction. Their quantitative assessment is of high interest in the study of arrhythmias, but it is usually hampered by the lack of automatic tools. In this work, we propose a software algorithm (Myocyte Automatic Retrieval and Tissue Analyzer, MARTA) to automatically detect myocytes from fluorescent microscopy images of cardiac tissue, measure their morphological features and evaluate the expression of CX43 and its degree of lateralization. The proposed software is based on the generation of cell masks, contouring of individual cells, enclosing of cells in minimum area rectangles and splitting of these rectangles into end-to-end and middle compartments to estimate CX43 lateral-to-total ratio. Application to human ventricular tissue images shows that mean differences between automatic and manual methods in terms of cardiomyocyte length and width are below 4 µm. The percentage of lateral CX43 also agrees between automatic and manual evaluation, with the interquartile range approximately covering from 3% to 30% in both cases. MARTA is not limited by fiber orientation and has an optimized speed by using contour filtering, which makes it run hundreds of times faster than a trained expert. Developed for CX43 studies in the left ventricle, MARTA is a flexible tool applicable to morphometric and lateralization studies of other markers in any heart chamber or even skeletal muscle. This open-access software is available online.

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.

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 heart's 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, PKCε and PKCδ 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-ε and suppressed pro-apoptotic PKC-δ 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.