Jana Radosinska

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.

Epicatechin Prevents Blood Pressure Increase and Reduces Locomotor Hyperactivity in Young Spontaneously Hypertensive Rats

This study investigated the effects of subchronic (−)-epicatechin (Epi) treatment on locomotor activity and hypertension development in young spontaneously hypertensive rats (SHR). Epi was administered in drinking water (100 mg/kg/day) for 2 weeks. Epi significantly prevented the development of hypertension (138 ± 2 versus 169 ± 5 mmHg, p < 0.001) and reduced total distance traveled in the open-field test (22 ± 2 versus 35 ± 4 m, p < 0.01). In blood, Epi significantly enhanced erythrocyte deformability, increased total antioxidant capacity, and decreased nitrotyrosine concentration. In the aorta, Epi significantly increased nitric oxide (NO) synthase (NOS) activity and elevated the NO-dependent vasorelaxation. In the left heart ventricle, Epi increased NOS activity without altering gene expressions of nNOS, iNOS, and eNOS. Moreover, Epi reduced superoxide production in the left heart ventricle and the aorta. In the brain, Epi increased nNOS gene expression (in the brainstem and cerebellum) and eNOS expression (in the cerebellum) but had no effect on overall NOS activity. In conclusion, Epi prevented the development of hypertension and reduced locomotor hyperactivity in young SHR. These effects resulted from improved cardiovascular NO bioavailability concurrently with increased erythrocyte deformability, without changes in NO production in the brain.

Extracellular Vesicles in Cardiovascular Theranostics

Extracellular vesicles (EVs) are small bilayer lipid membrane vesicles that can be released by most cell types and detected in most body fluids. EVs exert key functions for intercellular communication via transferring their bioactive cargos to recipient cells or activating signaling pathways in target cells. Increasing evidence has shown the important regulatory effects of EVs in cardiovascular diseases (CVDs). EVs secreted by cardiomyocytes, endothelial cells, fibroblasts, and stem cells play essential roles in pathophysiological processes such as cardiac hypertrophy, cardiomyocyte survival and apoptosis, cardiac fibrosis, and angiogenesis in relation to CVDs. In this review, we will first outline the current knowledge about the physical characteristics, biological contents, and isolation methods of EVs. We will then focus on the functional roles of cardiovascular EVs and their pathophysiological effects in CVDs, as well as summarize the potential of EVs as therapeutic agents and biomarkers for CVDs. Finally, we will discuss the specific application of EVs as a novel drug delivery system and the utility of EVs in the field of regenerative medicine.

Acute dark chocolate ingestion is beneficial for hemodynamics via enhancement of erythrocyte deformability in healthy humans

Erythrocyte deformability is an important property of erythrocytes that considerably affects blood flow and hemodynamics. The high content of polyphenols present in dark chocolate has been reported to play a protective role in functionality of erythrocytes. We hypothesized that chocolate might influence erythrocytes not only after repeated chronic intake, but also immediately after its ingestion. Thus, we determined the acute effect of dark chocolate and milk (with lower content of biologically active substances) chocolate intake on erythrocyte deformability. We also focused on selected factors that may affect erythrocyte deformability, specifically nitric oxide production in erythrocytes and total antioxidant capacity of plasma. We determined posttreatment changes in the mentioned parameters 2hours after consumption of chocolate compared with their levels before consumption of chocolate. In contrast to milk chocolate intake, the dark chocolate led to a significantly higher increase in erythrocyte deformability. Nitric oxide production in erythrocytes was not changed after dark chocolate intake, but significantly decreased after milk chocolate. The plasma total antioxidant capacity remained unaffected after ingestion of both chocolates. We conclude that our hypothesis was confirmed. Single ingestion of dark chocolate improved erythrocyte deformability despite unchanged nitric oxide production and antioxidant capacity of plasma. Increased deformability of erythrocytes may considerably improve rheological properties of blood and thus hemodynamics in humans, resulting in better tissue oxygenation.

Therapeutic Potential of Hematopoietic Stem Cell-Derived Exosomes in Cardiovascular Disease

As other stem cells, hematopoietic stem cells (HSCs) are able to produce extracellular vesicles (EVs) including exosomes and microvesicles. This chapter summarizes the knowledge about the production of EVs by the HSCs, their role in the intercellular communication, and will discuss the cargo of these EVs as well as protective effects of HSCs-derived exosomes and microvesicles in cardiovascular diseases (CVD). Available data showed that cardioprotective action of injected HSCs could not be explained by direct transdifferentiationof injected cells into the cardiomyocytes, this effect is suggested to be mediated via paracrine communication (by EVs) between donor and recipient cells. Among the cargo molecules of HSCs-derived vesicles several miRNAs, and pro-angiogenic and anti-apoptotic proteins are proposed to be the mediators of heart regeneration, mostly via neovascularization. However, the direct evidence of cardioprotective effects of HSCs-derived exosomes and microvesicles is still lacking in the literature. On the other hand, EVs produced in HSCs-derived cells, specifically dendritic cells and endothelial progenitor cells, have been shown to provide direct cardioprotective effects in CVD. Anyway, further studies are needed to be performed to assess the therapeutic potential of HSCs-derived EVs-based cardiac regenerative therapies.

Effect of yeast biomass with high content of carotenoids on erythrocyte deformability, NO production and Na,K-ATPase activity in healthy and LPS treated rats

Measurements of red blood cell (RBC) deformability together with estimation of NO-synthase activity and Na,K-ATPase activity were used for characterization of RBC functionality in rats subjected to single dose of Escherichia coli lipopolysaccharides (LPS) at a dose of 1 mg/kg. We hypothesized that LPS might initiate a malfunction of RBC. We also investigated the potential effect of carotenoids (10 mg/kg/day) produced in red yeast biomass of Rhodotorula glutinis on RBC in LPS-challenged rats. LPS significantly reduced the deformability of RBC (by 14%) together with decrease of NO-synthase activity by 20%. Daily supplementation of carotenoids for 10 days attenuated the LPS-induced injury, as observed by 22% increase of RBC deformability and 23% increase of NO-synthase activity. The activity of Na,K-ATPase was also improved probably due to increased number of active enzyme molecules as indicated by 66% enhancement of Vmax value, hence maintaining the activity of erythrocyte Na,K-ATPase to the level even higher as compared with healthy control animals. It may be concluded that administration of yeast biomass with high content of carotenoids resulted in advanced function of erythrocytes as concerns their ability to squeeze through narrow capillaries of the circulation, better intrinsic production of NO and improvement of intracellular homeostasis of sodium.

Molecular changes induced by repeated restraint stress in the heart: the effect of oxytocin receptor antagonist atosiban.

Even though stress belongs to the most common lifestyle risk factors of cardiovascular diseases, there are only limited data on direct influence of stressors on the heart. The aim of the present study was to explore selected protein signaling pathways in response to repeated immobilization stress in the heart tissue. Effects of simultaneous treatment with atosiban, an oxytocin receptor antagonist, on stress-induced changes in the heart were also investigated. Male Wistar rats were exposed to repeated immobilization (2 h daily, lasting 2 weeks). The results showed increased phosphorylation of Akt kinase, enhanced levels of Bcl-2, and decreased levels of cleaved caspase-3 in the left ventricle in response to chronic stress independently of the treatment. Exposure to restraint led to the rise of HSP-90 and p53 in vehicle-treated rats only. Stress failed to modify MMP-2 activity and ultrastructure of the heart tissue. Treatment with the oxytocin/vasopressin receptor antagonist atosiban reversed stress-induced rise in HSP-90 and p53 proteins. In conclusion, our data demonstrate that repeated restraint stress induces Akt kinase activation and this is associated with elevation of anti-apoptotic proteins (Bcl-2) and down-regulation of pro-apoptotic proteins (cleaved caspase-3). These findings suggest that activation of pro-survival anti-apoptotic Akt kinase pathway plays an important role in molecular mechanisms underlying responses and adaptation of the rat heart to repeated stress exposure. The results further indicate a regulatory role of oxytocin/vasopressin in the control of stress-induced activation in HSP-90 and related proteins.

Role of cytokines and inflammation in heart function during health and disease

By virtue of their actions on NF-κB, an inflammatory nuclear transcription factor, various cytokines have been documented to play important regulatory roles in determining cardiac function under both physiological and pathophysiological conditions. Several cytokines including TNF-α, TGF-β, and different interleukins such as IL-1 IL-4, IL-6, IL-8, and IL-18 are involved in the development of various inflammatory cardiac pathologies, namely ischemic heart disease, myocardial infarction, heart failure, and cardiomyopathies. In ischemia-related pathologies, most of the cytokines are released into the circulation and serve as biological markers of inflammation. Furthermore, there is an evidence of their direct role in the pathogenesis of ischemic injury, suggesting cytokines as potential targets for the development of some anti-ischemic therapies. On the other hand, certain cytokines such as IL-2, IL-4, IL-6, IL-8, and IL-10 are involved in the post-ischemic tissue repair and thus are considered to exert beneficial effects on cardiac function. Conflicting reports regarding the role of some cytokines in inducing cardiac dysfunction in heart failure and different types of cardiomyopathies seem to be due to differences in the nature, duration, and degree of heart disease as well as the concentrations of some cytokines in the circulation. In spite of extensive research work in this field of investigation, no satisfactory anti-cytokine therapy for improving cardiac function in any type of heart disease is available in the literature.

MMP activity detection in zymograms

Matrix metalloproteinases (MMP) belong to a distinguished class of zinc-dependent endopeptidases. Zymography is a semi-quantitative tool for determining the activity of different MMP isoenzymes in a variety of biological samples. In substrate gel zymography, protein samples of different origin (tissue, cell lysates, plasma/serum, perfusates, other liquids) are separated in sodium dodecyl sulfate (SDS) polyacrylamide gels containing copolymerized substrate (gelatin, casein, elastin, etc.), and after incubation-enabling substrate cleavage by MMPs, MMP activities are detected after the gel staining as transparent bands against a dark-blue background. In situ zymography is a histological modification of substrate zymography in frozen sections, allowing detection of the localization of the MMP activities within the tissue. Here, we describe detailed experimental protocols of all abovementioned techniques and provide examples for several sample measurements.