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Despite significant advances in the diagnosis and treatment of cardiovascular diseases, recent calls have emphasized the unmet need to improve precision-based approaches in cardiovascular disease. Although some studies provide preliminary evidence of the diagnostic and prognostic potential of circulating coding and non-coding RNAs, the complex RNA biology and lack of standardization have hampered the translation of these markers into clinical practice. In this position paper of the CardioRNA COST action CA17129, we provide recommendations to standardize the RNA development process in order to catalyse efforts to investigate novel RNAs for clinical use. We list the unmet clinical needs in cardiovascular disease, such as the identification of high-risk patients with ischaemic heart disease or heart failure who require more intensive therapies. The advantages and pitfalls of the different sample types, including RNAs from plasma, extracellular vesicles, and whole blood, are discussed in the sample matrix, together with their respective analytical methods. The effect of patient demographics and highly prevalent comorbidities, such as metabolic disorders, on the expression of the candidate RNA is presented and should be reported in biomarker studies. We discuss the statistical and regulatory aspects to translate a candidate RNA from a research use only assay to an in-vitro diagnostic test for clinical use. Optimal planning of this development track is required, with input from the researcher, statistician, industry, and regulatory partners., peer-reviewed

Cardiometabolic diseases (CMDs) are metabolic diseases (e.g., obesity, diabetes, atherosclerosis, rare genetic metabolic diseases, etc.) associated with cardiac pathologies. Pathophysiology of most CMDs involves increased production of reactive oxygen species and impaired antioxidant defense systems, resulting in cardiac oxidative stress (OxS). To alleviate OxS, various antioxidants have been investigated in several diseases with conflicting results. Here we review the effect of CMDs on cardiac redox homeostasis, the role of OxS in cardiac pathologies, as well as experimental and clinical data on the therapeutic potential of natural antioxidants (including resveratrol, quercetin, curcumin, vitamins A, C, and E, coenzyme Q10, etc.), synthetic antioxidants (including N-acetylcysteine, SOD mimetics, mitoTEMPO, SkQ1, etc.), and promoters of antioxidant enzymes in CMDs. As no antioxidant indicated for the prevention and/or treatment of CMDs has reached the market despite the large number of preclinical and clinical studies, a sizeable translational gap is evident in this field. Thus, we also highlight potential underlying factors that may contribute to the failure of translation of antioxidant therapies in CMDs.

Cardiovascular diseases (CVDs) are the top cause of death worldwide, and arterial hypertension per se remains the major preventable cause of CVDs [...]

The association between chronic inflammatory diseases (CIDs) and increased cardiovascular (CV) risk is well documented and can be a most threatening complication in these patients. However, the pathogenetic mechanisms underlying increased CV risk remain elusive, especially in their cellular and biochemical pathways. Using animal models to understand mechanisms underlying cardiac involvement are limited. Additionally, treatments may influence cardiovascular events through different outcomes. Some drugs used to treat CIDs can negatively affect cardiac function by a direct toxicity, whereas others may protect the myocardium. In the present article, we focus on the cardiac manifestations and risk factors, the pathogenetic mechanisms, and the effect of treatments on myocardial function and cardioprotection for five common worldwide CIDs (rheumatoid arthritis, systemic lupus erythematosus, systemic sclerosis, psoriasis and inflammatory bowel disease). We also give recommendations in order to evaluate common targets between CID and CV disease (CVD) and to design therapies to alleviate CID-related CVD. LINKED ARTICLES: This article is part of a themed issue on Risk factors, comorbidities, and comedications in cardioprotection. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v177.23/issuetoc.

Helium inhalation induces cardioprotection against ischemia/reperfusion injury, the cellular mechanism of which remains not fully elucidated. Extracellular vesicles (EVs) are cell-derived, nano-sized membrane vesicles which play a role in cardioprotective mechanisms, but their function in helium conditioning (HeC) has not been studied so far. We hypothesized that HeC induces fibroblast-mediated cardioprotection via EVs. We isolated neonatal rat cardiac fibroblasts (NRCFs) and exposed them to glucose deprivation and HeC rendered by four cycles of 95% helium + 5% CO2 for 1 h, followed by 1 h under normoxic condition. After 40 h of HeC, NRCF activation was analyzed with a Western blot (WB) and migration assay. From the cell supernatant, medium extracellular vesicles (mEVs) were isolated with differential centrifugation and analyzed with WB and nanoparticle tracking analysis. The supernatant from HeC-treated NRCFs was transferred to naïve NRCFs or immortalized human umbilical vein endothelial cells (HUVEC-TERT2), and a migration and angiogenesis assay was performed. We found that HeC accelerated the migration of NRCFs and did not increase the expression of fibroblast activation markers. HeC tended to decrease mEV secretion of NRCFs, but the supernatant of HeC or the control NRCFs did not accelerate the migration of naïve NRCFs or affect the angiogenic potential of HUVEC-TERT2. In conclusion, HeC may contribute to cardioprotection by increasing fibroblast migration but not by releasing protective mEVs or soluble factors from cardiac fibroblasts.

Cardioprotective medications are still unmet clinical needs. We have previously identified several cardioprotective microRNAs (termed ProtectomiRs), the mRNA targets of which may reveal new drug targets for cardioprotection. Here we aimed to identify key molecular targets of ProtectomiRs and confirm their association with cardioprotection in a translational pig model of acute myocardial infarction (AMI). By using a network theoretical approach, we identified 882 potential target genes of 18 previously identified protectomiRs. The Rictor gene was the most central and it was ranked first in the protectomiR-target mRNA molecular network with the highest node degree of 5. Therefore, Rictor and its targeting microRNAs were further validated in heart samples obtained from a translational pig model of AMI and cardioprotection induced by pre- or postconditioning. Three out of five Rictor-targeting pig homologue of rat ProtectomiRs showed significant upregulation in postconditioned but not in preconditioned pig hearts. Rictor was downregulated at the mRNA and protein level in ischemic postconditioning but not in ischemic preconditioning. This is the first demonstration that Rictor is the central molecular target of ProtectomiRs and that decreased Rictor expression may regulate ischemic postconditioning-, but not preconditioning-induced acute cardioprotection. We conclude that Rictor is a potential novel drug target for acute cardioprotection.

Catechins represent a group of polyphenols that possesses various beneficial effects in the cardiovascular system, including protective effects in cardiac ischemia-reperfusion (I/R) injury, a major pathophysiology associated with ischemic heart disease, myocardial infarction, as well as with cardioplegic arrest during heart surgery. In particular, catechin, (−)-epicatechin, and epigallocatechin gallate (EGCG) have been reported to prevent cardiac myocytes from I/R-induced cell damage and I/R-associated molecular changes, finally, resulting in improved cell viability, reduced infarct size, and improved recovery of cardiac function after ischemic insult, which has been widely documented in experimental animal studies and cardiac-derived cell lines. Cardioprotective effects of catechins in I/R injury were mediated via multiple molecular mechanisms, including inhibition of apoptosis; activation of cardioprotective pathways, such as PI3K/Akt (RISK) pathway; and inhibition of stress-associated pathways, including JNK/p38-MAPK; preserving mitochondrial function; and/or modulating autophagy. Moreover, regulatory roles of several microRNAs, including miR-145, miR-384-5p, miR-30a, miR-92a, as well as lncRNA MIAT, were documented in effects of catechins in cardiac I/R. On the other hand, the majority of results come from cell-based experiments and healthy small animals, while studies in large animals and studies including comorbidities or co-medications are rare. Human studies are lacking completely. The dosages of compounds also vary in a broad scale, thus, pharmacological aspects of catechins usage in cardiac I/R are inconclusive so far. Therefore, the aim of this focused review is to summarize the most recent knowledge on the effects of catechins in cardiac I/R injury and bring deep insight into the molecular mechanisms involved and dosage-dependency of these effects, as well as to outline potential gaps for translation of catechin-based treatments into clinical practice.

Several mechanisms may contribute to cardiovascular pathology associated with diabetes, including dysregulation of matrix metalloproteinases (MMPs). Quercetin (QCT) is a substance with preventive effects in treatment of cardiovascular diseases and diabetes. The aim of the present study was to explore effects of chronic QCT administration on changes in heart function in aged lean and obese Zucker Diabetic Fatty (ZDF) rats and that in association with MMPs. Signaling underlying effects of diabetes and QCT were also investigated. In the study, we used one-year-old lean and obese ZDF rats treated for 6 weeks with QCT. Results showed that obesity worsened heart function and this was associated with MMP-2 upregulation, MMP-28 downregulation, and inhibition of superoxide dismutases (SODs). Treatment with QCT did not modulate diabetes-induced changes in heart function and MMPs. However, QCT activated Akt kinase and reversed effects of diabetes on SODs inhibition. In conclusion, worsened heart function due to obesity involved changes in MMP-2 and MMP-28 and attenuation of antioxidant defense by SOD. QCT did not have positive effects on improvement of heart function or modulation of MMPs. Nevertheless, its application mediated activation of adaptive responses against oxidative stress through Akt kinase and prevention of diabetes-induced negative effects on antioxidant defense by SODs.

Iron is an essential mineral participating in different functions of the organism under physiological conditions. Numerous biological processes, such as oxygen and lipid metabolism, protein production, cellular respiration, and DNA synthesis, require the presence of iron, and mitochondria play an important role in the processes of iron metabolism. In addition to its physiological role, iron may be also involved in the adaptive processes of myocardial “conditioning”. On the other hand, disorders of iron metabolism are involved in the pathological mechanisms of the most common human diseases and include a wide range of them, such as type 2 diabetes, obesity, and non-alcoholic fatty liver disease, and accelerate the development of atherosclerosis. Furthermore, iron also exerts potentially deleterious effects that may be manifested under conditions of ischemia/reperfusion (I/R) injury, myocardial infarction, heart failure, coronary artery angioplasty, or heart transplantation, due to its involvement in reactive oxygen species (ROS) production. Moreover, iron has been recently described to participate in the mechanisms of iron-dependent cell death defined as “ferroptosis”. Ferroptosis is a form of regulated cell death that is distinct from apoptosis, necroptosis, and other types of cell death. Ferroptosis has been shown to be associated with I/R injury and several other cardiac diseases as a significant form of cell death in cardiomyocytes. In this review, we will discuss the role of iron in cardiovascular diseases, especially in myocardial I/R injury, and protective mechanisms stimulated by different forms of “conditioning” with a special emphasis on the novel targets for cardioprotection.

Cardioprotection against ischemia/reperfusion injury is still an unmet clinical need. The transient activation of Toll-like receptors (TLRs) has been implicated in cardioprotection, which may be achieved by treatment with blood-derived extracellular vesicles (EVs). However, since the isolation of EVs from blood takes considerable effort, the aim of our study was to establish a cellular model from which cardioprotective EVs can be isolated in a well-reproducible manner. EV release was induced in HEK293 cells with calcium ionophore A23187. EVs were characterized and cytoprotection was assessed in H9c2 and AC16 cell lines. Cardioprotection afforded by EVs and its mechanism were investigated after 16 h simulated ischemia and 2 h reperfusion. The induction of HEK293 cells by calcium ionophore resulted in the release of heterogenous populations of EVs. In H9c2 and AC16 cells, stressEVs induced the downstream signaling of TLR4 and heme oxygenase 1 (HO-1) expression in H9c2 cells. StressEVs decreased necrosis due to simulated ischemia/reperfusion injury in H9c2 and AC16 cells, which was independent of TLR4 induction, but not that of HO-1. Calcium ionophore-induced EVs exert cytoprotection by inducing HO-1 in a TLR4-independent manner.

Search for new cardioprotective therapies is of great importance since no cardioprotective drugs are available on the market. In line with this need, several natural biomolecules have been extensively tested for their potential cardioprotective effects. Previously, we have shown that biglycan, a member of a diverse group of small leucine-rich proteoglycans, enhanced the expression of cardioprotective genes and decreased ischemia/reperfusion-induced cardiomyocyte death via a TLR-4 dependent mechanism. Therefore, in the present study we aimed to test whether decorin, a small leucine-rich proteoglycan closely related to biglycan, could exert cardiocytoprotection and to reveal possible downstream signaling pathways. Methods: Primary cardiomyocytes isolated from neonatal and adult rat hearts were treated with 0 (Vehicle), 1, 3, 10, 30 and 100 nM decorin as 20 h pretreatment and maintained throughout simulated ischemia and reperfusion (SI/R). In separate experiments, to test the mechanism of decorin-induced cardio protection, 3 nM decorin was applied in combination with inhibitors of known survival pathways, that is, the NOS inhibitor L-NAME, the PKG inhibitor KT-5823 and the TLR-4 inhibitor TAK-242, respectively. mRNA expression changes were measured after SI/R injury. Results: Cell viability of both neonatal and adult cardiomyocytes was significantly decreased due to SI/R injury. Decorin at 1, 3 and 10 nM concentrations significantly increased the survival of both neonatal and adult myocytes after SI/R. At 3nM (the most pronounced protective concentration), it had no effect on apoptotic rate of neonatal cardiac myocytes. No one of the inhibitors of survival pathways (L-NAME, KT-5823, TAK-242) influenced the cardiocytoprotective effect of decorin. MYND-type containing 19 (Zmynd19) and eukaryotic translation initiation factor 4E nuclear import factor 1 (Eif4enif1) were significantly upregulated due to the decorin treatment. In conclusion, this is the first demonstration that decorin exerts a direct cardiocytoprotective effect possibly independent of NO-cGMP-PKG and TLR-4 dependent survival signaling.

A number of pregnant women all over the world suffer from depression and are treated during gestation with antidepressants, mostly with selective serotonin reuptake inhibitors or selective serotonin and norepinephrine reuptake inhibitors. Exposure to prenatal stress is also a great risk factor for a developing fetus and could be responsible for altered fetal development or various neurobehavioral disturbances of a child. Administration of selective serotonin and norepinephrine reuptake inhibitor venlafaxine is associated with various cardiovascular adverse effects, such as tachycardia, increased blood pressure, arrhythmias and hypertensive crisis. The aim of this study was to focus on the effect of pre-gestational chronic mild unpredictable stress and/or administration of antidepressant venlafaxine (10 mg/kg/day, p. o.) on specific parameters, determining the function of the cardiovascular system of male and female rat offspring. Blood pressure and standard ECG were recorded in the offspring. Exposure to pre-gestational stress did not cause significant changes in the systolic, diastolic blood pressure and pulse frequency either in males or in females, compared to the unexposed control animals. Pre-gestational stress caused the shortening of QT interval and prolongation of QRS complex duration in males. On the other hand, in females, the effects of pre-gestational stress were potentiated by the administration of venlafaxine and resulted in elevated systolic and diastolic blood pressure, prolonged QT interval and shortened QRS complex duration, compared to the control. In conclusion, the female rat offspring are more sensitive to exposure to pre-gestational, to chronic mild unpredictable stress and venlafaxine.

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.

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.

Background: Quercetin (QCT) was shown to exert beneficial cardiovascular effects in young healthy animals. The aim of the present study was to determine cardiovascular benefits of QCT in older, 6-month and 1-year-old Zucker diabetic fatty (ZDF) rats (model of type 2 diabetes). Methods: Lean (fa/+) and obese (fa/fa) ZDF rats of both ages were treated with QCT for 6 weeks (20 mg/kg/day). Isolated hearts were exposed to ischemia-reperfusion (I/R) injury (30 min/2 h). Endothelium-dependent vascular relaxation was measured in isolated aortas. Expression of selected proteins in heart tissue was detected by Western blotting. Results: QCT reduced systolic blood pressure in both lean and obese 6-month-old rats but had no effect in 1-year-old rats. Diabetes worsened vascular relaxation in both ages. QCT improved vascular relaxation in 6-month-old but worsened in 1-year-old obese rats and had no impact in lean controls of both ages. QCT did not exert cardioprotective effects against I/R injury and even worsened post-ischemic recovery in 1-year-old hearts. QCT up-regulated expression of eNOS in younger and PKC&epsilon, expression in older rats but did not activate whole PI3K/Akt pathway. Conclusions: QCT might be beneficial for vascular function in diabetes type 2, however, increasing age and/or progression of diabetes may confound its vasculoprotective effects. QCT seems to be inefficient in preventing myocardial I/R injury in type 2 diabetes and/or higher age. Impaired activation of PI3K/Akt kinase pathway might be, at least in part, responsible for failing cardioprotection in these subjects.

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.

The present study was oriented to gender specificity of Na,K-ATPase in cerebellum, the crucial enzyme maintaining the intracellular homeostasis of Na ions in healthy and diabetic Wistar rats. The effects of diabetes on properties of the Na,K-ATPase in cerebellum derived from normal and streptozotocin (STZ)-diabetic rats of both genders were investigated. The samples were excised at different time intervals of diabetes induced by STZ (65 mg kg−1) for 8 days and 16 weeks. In acute 8-day-lasting model of diabetes, Western blot analysis showed significant depression of α1 isoform of Na,K-ATPase in males only. On the other hand, concerning the activity, the enzyme seems to be resistant to the acute model of diabetes in both genders. Prolongation of diabetes to 16 weeks was followed by increasing the number of active molecules of Na,K-ATPase exclusively in females as indicated by enzyme kinetic studies. Gender specificity was observed also in nondiabetic animals revealing higher Na,K-ATPase activity in control males probably caused by higher number of active enzyme molecules as indicated by increased value of V max when comparing to control female group. This difference seems to be age dependent: at the age of 16 weeks, the V max value in females was higher by more than 90%, whereas at the age of 24 weeks, this difference amounted to only 28%. These data indicate that the properties of Na,K-ATPase in cerebellum, playing crucial role in maintaining the Na+ and K+ gradients, depend on gender, age, and duration of diabetic impact.

Although incidence and prevalence of prediabetes are increasing, little is known about its cardiac effects. Therefore, our aim was to investigate the effect of prediabetes on cardiac function and to characterize parameters and pathways associated with deteriorated cardiac performance. Long-Evans rats were fed with either control or high-fat chow for 21 wk and treated with a single low dose (20 mg/kg) of streptozotocin at week 4. High-fat and streptozotocin treatment induced prediabetes as characterized by slightly elevated fasting blood glucose, impaired glucose and insulin tolerance, increased visceral adipose tissue and plasma leptin levels, as well as sensory neuropathy. In prediabetic animals, a mild diastolic dysfunction was observed, the number of myocardial lipid droplets increased, and left ventricular mass and wall thickness were elevated; however, no molecular sign of fibrosis or cardiac hypertrophy was shown. In prediabetes, production of reactive oxygen species was elevated in subsarcolemmal mitochondria. Expression of mitofusin-2 was increased, while the phosphorylation of phospholamban and expression of Bcl-2/adenovirus E1B 19-kDa protein-interacting protein 3 (BNIP3, a marker of mitophagy) decreased. However, expression of other markers of cardiac auto- and mitophagy, mitochondrial dynamics, inflammation, heat shock proteins, Ca2+/calmodulin-dependent protein kinase II, mammalian target of rapamycin, or apoptotic pathways were unchanged in prediabetes. This is the first comprehensive analysis of cardiac effects of prediabetes indicating that mild diastolic dysfunction and cardiac hypertrophy are multifactorial phenomena that are associated with early changes in mitophagy, cardiac lipid accumulation, and elevated oxidative stress and that prediabetes-induced oxidative stress originates from the subsarcolemmal mitochondria. Listen to this article's corresponding podcast http://ajpheart.podbean.com/e/myocardial-dysfunction-in-prediabetes/ .

Matrix metalloproteinases (MMPs) are important in the pathogenesis of numerous diseases. The present study aimed to monitor the activation of MMP-2 and MMP-9 in spontaneously hypertensive rats (SHR) and their normotensive counterparts&mdash, Wistar-Kyoto rats (WKY). The animals were divided according to age (7, 20, and 52 weeks) and phenotype into: WKY-7, WKY-20, WKY-52, SHR-7, SHR-20 and SHR-52 groups. MMP plasma activities were determined by gelatine zymography. We monitored selected parameters of oxidative stress and antioxidant status. N-terminal pro-brain natriuretic peptide (NT-proBNP) was determined as a marker of heart function and neurohumoral activation. SHR-7 showed higher MMP-2 activity compared with WKY-7, while SHR-52 showed lower MMP-2 and MMP-9 activities compared with WKY-52. Examining age-dependent changes in MMP activities, we found a decrease in MMP-2 activity and increase in MMP-9 activity with increasing age in both phenotypes. Parameters of oxidative stress and antioxidant status as well as NT-proBNP levels were not significantly worsened due to aging in SHR. Our results suggest that hypertension is accompanied by varying MMP activation during aging. The results of our study may indicate that MMP-2 inhibition is therapeutically applicable during the development of hypertension, while in developed, stabilized and uncomplicated hypertension, systemic MMP-2 and MMP-9 inhibition may not be desirable.

Quercetin (QCT) is a natural polyphenolic compound enriched in human food, mainly in vegetables, fruits and berries. QCT and its main derivatives, such as rhamnetin, rutin, hyperoside, etc., have been documented to possess many beneficial effects in the human body including their positive effects in the cardiovascular system. However, clinical implications of QCT and its derivatives are still rare. In the current paper we provide a complex picture of the most recent knowledge on the effects of QCT and its derivatives in different types of cardiac injury, mainly in ischemia-reperfusion (I/R) injury of the heart, but also in other pathologies such as anthracycline-induced cardiotoxicity or oxidative stress-induced cardiac injury, documented in in vitro and ex vivo, as well as in in vivo experimental models of cardiac injury. Moreover, we focus on cardiac effects of QCT in presence of metabolic comorbidities in addition to cardiovascular disease (CVD). Finally, we provide a short summary of clinical studies focused on cardiac effects of QCT. In general, it seems that QCT and its metabolites exert strong cardioprotective effects in a wide range of experimental models of cardiac injury, likely via their antioxidant, anti-inflammatory and molecular pathways-modulating properties; however, ageing and presence of lifestyle-related comorbidities may confound their beneficial effects in heart disease. On the other hand, due to very limited number of clinical trials focused on cardiac effects of QCT and its derivatives, clinical data are inconclusive. Thus, additional well-designed human studies including a high enough number of patients testing different concentrations of QCT are needed to reveal real therapeutic potential of QCT in CVD. Finally, several negative or controversial effects of QCT in the heart have been reported, and this should be also taken into consideration in QCT-based approaches aimed to treat CVD in humans.

The interest in non-coding RNAs, which started more than a decade ago, has still not weakened. A wealth of experimental and clinical studies has suggested the potential of non-coding RNAs, especially the short-sized microRNAs (miRs), to be used as the new generation of therapeutic targets and biomarkers of cardiovascular disease, an ever-growing public health issue in the modern world. Among the hundreds of miRs characterized so far, microRNA-1 (miR-1) and microRNA-21 (miR-21) have received some attention and have been associated with cardiac injury and cardioprotection. In this review article, we summarize the current knowledge of the function of these two miRs in the heart, their association with cardiac injury, and their potential cardioprotective roles and biomarker value. While this field has already been extensively studied, much remains to be done before research findings can be translated into clinical application for patient’s benefit.

l-Alpha-glycerylphosphorylcholine (GPC) is a widely used food supplement. GPC has been shown to exert beneficial effects in several organs; however, the cardiac effects of GPC have yet to be investigated. The aim of the present study was therefore to map out the effects of GPC on cardiac myocytes, with or without ischemia–reperfusion insult. Neonatal rat cardiac myocytes were treated with GPC at 1, 10, 80, and 100 µM concentrations for 15 min, 3 h, or 24 h, respectively. Cell viability by calcein assay and the degree of oxidative stress by DHE (superoxide level) and H2DCF (total ROS accumulation) staining were measured. In separate experiments, cardiomyocytes were pre-treated with the optimal concentration of GPC for 3 h and then cells were exposed to 4 h of simulated ischemia followed by 2 h of reperfusion (SI/R). Cell viability was measured at the end of the SI/R protocol. In normoxic conditions, the 15-min and the 3-h GPC treatment did not affect cell viability, total ROS, and superoxide levels. Under SI/R conditions, the 3-h GPC treatment protected the cardiac myocytes from SI/R-induced cell death and did not alter the level of oxidative stress. The 24-h GPC treatment in normoxic conditions resulted in significant cell death and increased oxidative stress at each concentration. Here we provide the first evidence for the cytoprotective effect of short-term GPC treatment. However, long-term administration of GPC may exert cytotoxicity in a wide concentration range in cardiac myocytes. These results may draw attention to a comprehensive cardiac safety protocol for the testing of GPC. Electronic supplementary material The online version of this article (10.1007/s11010-019-03580-1) contains supplementary material, which is available to authorized users.

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.

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.

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.

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.

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.

Remote ischemic conditioning of the heart (including pre-, per-, and post-conditioning) is a phenomenon where short episodes of non-lethal ischemia in the distant vessels within the heart or distant organs from the heart protects the myocardium against sustained ischemia/reperfusion injury. Several pathways have been proposed to be involved in the mechanisms of Remote ischemic conditioning. While triggers of Remote ischemic conditioning act in preconditioned areas, its mediators transduce protective signals via humoral or neuronal pathways to the heart. Remote ischemic conditioning is mediated via receptor and nonreceptor signaling through secondary mediators, which transfer the signal within the cardiomyocyte and activate cardioprotective pathways that lead to higher resistance of the heart to ischemia/reperfusion. Apparently, identification of endogenous signal molecules involved in the mechanisms of Remote ischemic conditioning have therapeutic implications in the management of patients suffering from myocardial ischemia through the development of diverse beneficial effects. Recently, different non-coding RNAs such as microRNAs or long non-coding RNAs have been identified as emerging factors that trigger protective mechanisms in the heart. These non-coding RNAs are transferred to the heart via extracellular vesicles that exert remote cardioprotection. This review is intended to summarize the existing knowledge about the potential role of extracellular vesicles as humoral transmitters of Remote ischemic conditioning and emphasize the involvement of non-coding RNAs in the mechanism of cardioprotection by Remote ischemic conditioning.

Background: Ischemia-reperfusion (I/R) injury of the heart as a consequence of myocardial infarction or cardiac surgery represents a serious clinical problem. One of the most prominent mechanisms of I/R injury is the development of oxidative stress in the heart. In this regard, I/R has been shown to enhance the production of reactive oxygen/nitrogen species in the heart which lead to the imbalance between the pro-oxidants and antioxidant capacities of the endogenous radical-scavenging systems. Objectives: Increasing the antioxidant capacity of the heart by the administration of exogenous antioxidants is considered beneficial for the heart exposed to I/R. N-acetylcysteine (NAC) and Nmercaptopropionylglycine (MPG) are two sulphur containing amino acid substances, which belong to the broad category of exogenous antioxidants that have been tested for their protective potential in cardiac I/R injury. Observations: Pretreatment of hearts with both NAC and MPG has demonstrated that these agents attenuate the I/R-induced alterations in sarcolemma, sarcoplasmic reticulum, mitochondria and myofibrils in addition to improving cardiac function. While experimental studies have revealed promising data suggesting beneficial effects of NAC and MPG in cardiac I/R injury, the results of clinical trials are not conclusive because both positive and no effects of these substances have been reported on the post-ischemic recovery of heart following cardiac surgery or myocardial infarction. Conclusion: It is concluded that both NAC and MPG exert beneficial effects in preventing the I/Rinduced injury; however, further studies are needed to establish their effectiveness in reversing the I/R-induced abnormalities in the heart.

Quercetin, a polyphenolic compound present in various types of food, has been shown to exert beneficial effects in different cardiac as well as non-cardiac ischemia/reperfusion (I/R) models in adult animals. However, there is no evidence about the effects of quercetin on I/R injury in non-mature animals, despite the fact that efficiency of some interventions against I/R is age-dependent. This study was aimed to investigate the effects of chronic quercetin treatment on I/R injury in juvenile and adult rat hearts. Juvenile (4-week-old) as well as adult (12-week-old) rats were treated with quercetin (20 mg/kg/day) for 4 weeks, hearts were excised and exposed to 25-min global ischemia followed by 40-min reperfusion. Functional parameters of hearts and occurrence of reperfusion arrhythmias were registered to assess the cardiac function. Our results have shown that quercetin improved post-ischemic recovery of LVDP, as well as recovery of markers of contraction and relaxation, +(dP/dt)max and –(dP/dt)max, respectively, in juvenile hearts, but not in adult hearts. Quercetin had no impact on incidence as well as duration of reperfusion arrhythmias in animals of both ages. We conclude that the age of rats plays an important role in heart response to quercetin treatment in the particular dose and duration of the treatment. Therefore, the age of the treated subjects should be taken into consideration when choosing the dose of quercetin and duration of its application in prevention and/or treatment of cardiovascular diseases.

Matrix metalloproteinases (MMPs) play an important role in the pathogenesis of heart failure (HF). Our aim was to determine the activities of circulating MMP-2 and MMP-9 in patients with HF in respect of gender, comorbidities and treatment (n=51). We did not reveal any differences in circulating pro-MMP-2 and pro-MMP-9 activities between the patients with HF and without it. However, there was a decrease in activity of pro-MMP-2 in treated hypertensive participants versus healthy ones. In contrast, we observed increased pro-MMP-2 activity in hypertensive participants with coexistent HF versus hypertensive participants without HF. In addition, a decrease in pro-MMP-2 activity was shown in women suffering from HF versus men suffering from HF. In conclusion, potential inhibitory effect of antihypertensive treatment on pro-MMP-2 activity was found. Coexistent HF with hypertension probably reduces the inhibitory effect of antihypertensive treatment on pro-MMP-2 activity. Our data also suggest the role of potential cardioprotective factors influencing the activity of pro-MMP-2 in women.

Ischemic heart disease is caused by obstruction of the coronary arteries that reduces myocardial perfusion. The most efficient way to restore blood supply to the ischemic myocardium is reperfusion of the affected area. However, if reperfusion is initiated too late, it enhances ischemia-reperfusion damage to the heart. Oxidative stress is one of the major causes for the development of ischemia-reperfusion (I/R) injury to the heart, which is characterized by decreased functional performance, ultrastructural changes and metabolic alterations. Excessive formation of oxyradicals during the development of I/R injury has now been shown to produce a wide variety of abnormalities in cardiac subcellular organelles such as sarcolemma, sarcoplasmic reticulum, mitochondria and myofibrils. Different reactive oxygen species (ROS) have been shown to cause alterations in several proteins leading to their malfunction and changes in enzyme activities. Lipid peroxidation leads to depressed membrane fluidity and increased permeability, as well as changes in gene expression leading to impaired recovery of cardiac dysfunction due to I/R injury. Since the redox status of cardiomyocytes depends mainly on the balance between ROS production and availability of endogenous antioxidant defense systems, extensive efforts are made to maintain the redox status during I/R injury. Cardioprotection by ischemic conditioning as well as treatment of the heart with exogenous antioxidants have been observed to result in the increased availability of endogenous antioxidants and prevent oxidative damage to subcellular organelles. Thus antioxidant therapy is considered to be a promising strategy to decrease the I/R induced damage to the heart. Accordingly, this article is intended to describe the mechanisms of oxidative stress induced subcellular defects leading to cardiac dysfunction due to I/R injury and to outline the role of antioxidant defense mechanisms in the prevention as well as treatment of the ischemic heart disease.

Na+-K+ ATPase is primarily localized in the plasma membrane and occurs in the form of α-subunits (α-1, -2, and -3 isoforms) and β-subunits (β-1 and -2 isoforms) mainly. The inhibition of this enzyme by cardiac glycosides such as ouabain has been shown to raise the intracellular concentration of Na+ and promote the increase in cardiac contractile force as a consequence of increased Na+-Ca2+ exchange. Several studies have observed that Na+-K+ ATPase not only serves as a receptor for cardiac glycosides but also acts as a Na+-pump for maintaining the electrolyte homeostasis as well as a signal transducer for the formation of reactive oxygen radicals and cellular growth. Different hormones and endogenous factors such as marinobufagenin are considered to regulate the Na+-K+ ATPase activity through protein kinase-induced phosphorylation of phospholemman, a membrane protein which is tightly associated with this enzyme. Stimulation of Na+-K+ ATPase by catecholamines and phosphorylation of phospholemman seems to be an adaptive mechanism for the prevention of Ca2+-overload-induced arrhythmias whereas excessive increase in the plasma levels of marinobufagenin or high concentrations of cardiac glycosides are considered to induce cardiac dysfunction associated with depression of the Na+-K+ ATPase activity.

The plasma membrane Ca2+/Mg2+ ecto-ATPase is an acidic glycoprotein, which hydrolyzes different nucleoside triphosphates and is activated by millimolar concentrations of various divalent cations. Unlike transport ATPases, it does not require Mg-ATP as a substrate and is different from the mitochondrial, myofibrillar, and sarcoplasmic reticulum ATPases. This enzyme is present in all tissues of the body including liver, brain, heart, kidney, blood, platelets, endothelium, and smooth muscles. The Ca2+/Mg2+ ecto-ATPase is considered to play diverse physiological roles such as termination of purinergic transmission, regulation of extracellular ATP concentration, gating mechanism for Ca2+ and Mg2+ fluxes, ATP-driven proton pump, cell-to-cell communication as well as cellular differentiation and transformation in a tissue specific manner. The activity of Ca2+/Mg2+ ecto-ATPase is altered by a wide variety of physiological, pharmacological, and pathological interventions which change membrane fluidity and its composition with respect to cholesterol and phospholipid contents. The molecular weight of this enzyme varies from tissue to tissue in the range of 180–240 kDa with subunits of 90, 80, 67, 20, and 10 kDa. The cDNA sequence for the plasma membrane Ca2+/Mg2+ ecto-ATPase from different tissues show homology with different adhesion molecules including CD36, CD39, and CD70. The evidence in the existing literature suggests that the Ca2+/Mg2+ ecto-ATPase is a multifunctional adhesion molecule which exists in different isoforms in various tissues.

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.

Sex and social stress may represent risk factors in the etiology of hypertension and heart response to ischemia–reperfusion (I/R) injury. Phosphatidylinositol 3-kinase/protein kinase B (Akt) plays an important role in the processes associated with hypertension and myocardial tolerance to I/R, and may be involved in myocardial stress reaction. The impact of chronic stress on the response to I/R was investigated in the hearts of 7-week-old spontaneously hypertensive (SHR) and Wistar–Kyoto (WKY) rats of both sexes. Stress was induced by reducing living space to 70 cm2/100 g body mass of rat for 2 weeks, while the controls were kept at 200 cm2/100 g. Langendorff-perfused hearts, subjected to I/R, exhibited higher vulnerability to ventricular tachycardia in crowd-stressed SHR vs. the control rats, and this was more pronounced in the males. Myocardial infarction was not affected by crowding stress in any of the groups. Male and female SHR showed increased activation of cardiac Akt, whereas nitric oxide synthase activity (NOS) with pro-apoptotic signaling decreased in the males but was not altered in the females (vs. WKY rats). NOS was enhanced in the female SHR and WKY groups by comparison with the respective males. Stress only reduced NOS activity in the SHR groups, and without changes in apoptotic markers. In conclusion, we showed that stress in young SHR mainly affects the nonlethal markers for I/R, and has no impact on myocardial infarction and apoptosis, despite reduced NOS activity.

Although pleiotropy, which is defined as multiple effects derived from a single gene, was recognized many years ago, and considerable progress has since been achieved in this field, it is not very clear how much this feature of a drug is clinically relevant. During the last decade, beneficial pleiotropic effects from hypolipidemic drugs (as in, effects that are different from the primary ones) have been associated with reduction of cardiovascular risk. As with statins, the agonists of peroxisome proliferator-activated receptors (PPARs), niacin and fibrates, have been suggested to exhibit pleiotropic activity that could significantly modify the outcome of a cardiovascular ailment. This review examines findings demonstrating the impacts of treatment with hypolipidemic drugs on cardiac response to ischemia in a setting of acute ischemia–reperfusion, in relation to PPAR activation. Specifically, it addresses the issue of susceptibility to ischemia, with particular regard to the preconditioning-like cardioprotection conferred by hypolipidemic drugs, as well as the potential molecular mechanisms behind this cardioprotection. Finally, the involvement of PPAR activation in the mechanisms of non-metabolic cardioprotective effects from hypolipidemic drugs, and their effects on normal and pathologically altered myocardium (in the hearts of hypertensive rats) is also discussed.

Doxorubicin (DOX) is chemotherapeutic agent frequently used in treatment of many types of malignancies. Limitation of its use is toxicity on several organs. The aim of study was to investigate mechanisms involved in chronic effects of DOX on rat hearts and to search for effects of flavonoid quercetin (QCT) on DOX-induced changes. In the study, male Wistar rats were used. DOX was administered by i.p. injection of seven doses (cumulative dose 15 mg.kg-1). QCT (20 mg/kg/day) was administered 3 weeks during and 3 weeks after DOX treatment. DOX induced subcellular alterations of cardiomyocytes and disorganizations of cardiac extracellular space. Effects of DOX were associated with MMP-2 activation, decreased SOD activity, increased superoxide content, caspase-3 activation, and iNOS induction. QCT prevented the deleterious effects of DOX on ultrastructure of the tissue of left ventricle and reversed the DOX-induced effects on MMP-2 activation. QCT application also prevented effects of DOX on apoptosis induction and SOD inhibition. Moreover, QCT increased myocardial resistance to ischemia/reperfusion injury in DOX-treated rat hearts. These effects of QCT were linked to Akt kinase/GSK-3s/beta-catenin signaling and associated with connexin-43 up-regulation.

Time sequence study was performed to characterize the effects of diabetes mellitus type 1 on properties of the Na, K-ATPase in cerebral cortex derived from normal and streptozotocin (STZ)-diabetic rats of both genders. The samples were excised at varying time intervals of diabetes induced by STZ (65 mg kg−1) for 8 days, and 8 and 16 weeks. Expression of α1–3 isoforms of Na, K-ATPase was not altered in statistically significant level during all stages of diabetes neither in female nor in male rats as revealed from Western blot analysis. Studies of kinetic properties of the enzyme resulted in variations in active number of Na, K-ATPase molecules as well as its qualitative properties. Sixteen-week-old control male rats showed better affinity to substrate as indicated by 13 % decrease of K m value. The effect persisted also in males subjected to 8 days lasting diabetes; however, in males subjected to 8 weeks lasting diabetes, the effect was lost. In 25-week-old rats, the Na, K-ATPase revealed again altered properties in males and females but the mechanism of the variation was different. In females, the number of active molecules of Na, K-ATPase was higher by 32 % in controls and by 17 % in rats with chronic diabetes when comparing to respective male groups as suggested by increased value of V max. So the properties of Na, K-ATPase in cerebral cortex, playing crucial role in maintaining intracellular homeostasis of Na+ ions, depend on gender, age, and duration of diabetic insult.