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Introduction: Ventricular unloading during prolonged bed rest, mechanical circulatory support or microgravity has repeatedly been linked to potentially life-threatening arrhythmias. It is unresolved, whether this arrhythmic phenotype is caused by the reduction in cardiac workload or rather by underlying diseases or external stimuli. We hypothesized that the reduction in cardiac workload alone is sufficient to impair ventricular repolarization and to induce arrhythmias in hearts., Methods: Rat hearts were unloaded using the heterotopic heart transplantation. The ECG of unloaded and of control hearts were telemetrically recorded over 56 days resulting in >5 × 10 6 cardiac cycles in each heart. Long-term electrical remodeling was analyzed using a novel semi-automatic arrhythmia detection algorithm., Results: 56 days of unloading reduced left ventricular weight by approximately 50%. While unloading did not affect average HRs, it markedly prolonged the QT interval by approximately 66% and induced a median tenfold increase in the incidence of ventricular arrhythmias in comparison to control hearts., Conclusion: The current study provides direct evidence that the previously reported hypertrophic phenotype of repolarization during cardiac unloading translates into an impaired ventricular repolarization and ventricular arrhythmias in vivo . This supports the concept that the reduction in cardiac workload is a causal driver of the development of arrhythmias during ventricular unloading., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Schwoerer, Biermann and Ehmke.)

Type 1 diabetes mellitus (T1DM) is the most severe form of diabetes, which is characterized by absolute insulin deficiency induced by the destruction of pancreatic beta cells. The aim of this study was to evaluate the effect of a structural analogue of apelin-12 ((NαMe)Arg-Pro-Arg-Leu-Ser-His-Lys-Gly-Pro-Nle-Pro-Phe-OH, metilin) on hyperglycemia, mitochondrial (MCh) respiration in permeabilized cardiac left ventricular (LV) fibers, the myocardial energy state, and cardiomyocyte membranes damage in a model of streptozotocin (STZ) diabetes in rats. Metilin was prepared by solid-phase synthesis using the Fmoc strategy and purified using HPLC. Four groups of animals were used: initial state (IS); control (C), diabetic control (D) and diabetic animals additionally treated with metilin (DM). The following parameters have been studied: blood glucose, MCh respiration in LV fibers, the content of cardiac ATP, ADP, AMP, phosphocreatine (PCr) and creatine (Cr), the activity of creatine kinase-MB (CK-MB) and lactate dehydrogenase (LDH) in blood plasma. Administration of metilin to STZ-treated rats decreased blood glucose, increased state 3 oxygen consumption, the respiratory control ratio in MCh of permeabilized LV fibers, and increased the functional coupling of mitochondrial CK (mt-CK) to oxidative phosphorylation compared with these parameters in group D. In STZ-treated animals metilin administration caused an increase in the PCr content and prevention of the loss of total creatine (ΣCr=PCr+Cr) in the diabetic hearts, as well as restoration of the PCr/ATP ratio in the myocardium and a decrease in the activity of CK-MB and LDH in plasma to initial values. Thus, metilin prevented energy disorders disturbances in cardiomyocytes of animals with experimental T1DM.

Objectives: The aim of this work was to elucidate the role of GalR2 receptor activation in protecting the rat heart in vivo from ischemia/reperfusion (I/R) damage by a pharmacological peptide agonist WTLNSAGYLLGPβAH-OH (G1) and full-length rat galanin GWTLNSAGYLLGPHAIDNHRSFSDKHGLT-NH2 (G2) using M871, a selective inhibitor of GalR2., Methods: The peptides were prepared by the automatic solid-phase synthesis using the Fmoc-strategy and purified by high-performance liquid chromatography (HPLC). A 40-min left anterior descending (LAD) coronary artery occlusion followed by a 60-min reperfusion was performed. The criteria for damage/protection of the heart were the infarct size (IS) and plasma activity of creatine kinase-MB (CK-MB) at the end of reperfusion., Results: Intravenous injection of G1 or G2 at an optimal dose of 1 mg/kg at the fifth minute of reperfusion significantly reduced the IS (by 35% and 32%, respectively) and activity of CK-MB at the end of reperfusion (by 43% and 38%, respectively) compared with the control. Administration of M871 (8 mg/kg) 5 min before the onset of reperfusion abolished the effects of G1 on IS and CK-MB activity, returning them to control values. Co-administration of M871 (8 mg/kg) with G2 attenuated protective effect of G2 on both IS and plasma СK-MB activity. However, differences in these parameters between the M871+G2 and G2 groups did not reach statistical significance (P = 0.139 and P = 0.121, respectively)., Conclusion: Thus, GalR2 is the principal receptor subtype that transduces the protective effects of galanin and ligand G1 in myocardial I/R injury. This suggests that GalR2-specific peptide agonists could be used as drug candidates for treating ischemic heart disease., (© 2023 Société Française de Pharmacologie et de Thérapeutique. Published by John Wiley & Sons Ltd.)

Melatonin has been reported to cause myocardial electrophysiological changes and prevent ventricular tachycardia or fibrillation (VT/VF) in ischemia and reperfusion. We sought to identify electrophysiological targets responsible for the melatonin antiarrhythmic action and to explore whether melatonin receptor-dependent pathways or its antioxidative properties are essential for these effects. Ischemia was induced in anesthetized rats given a placebo, melatonin, and/or luzindole (MT1/MT2 melatonin receptor blocker), and epicardial mapping with reperfusion VT/VFs assessment was performed. The oxidative stress assessment and Western blotting analysis were performed in the explanted hearts. Transmembrane potentials and ionic currents were recorded in cardiomyocytes with melatonin and/or luzindole application. Melatonin reduced reperfusion VT/VF incidence associated with local activation time in logistic regression analysis. Melatonin prevented ischemia-related conduction slowing and did not change the total connexin43 (Cx43) level or oxidative stress markers, but it increased the content of a phosphorylated Cx43 variant (P-Cx43 368 ). Luzindole abolished the melatonin antiarrhythmic effect, slowed conduction, decreased total Cx43, protein kinase Cε and P-Cx43 368 levels, and the IK1 current, and caused resting membrane potential (RMP) depolarization. Neither melatonin nor luzindole modified INa current. Thus, the antiarrhythmic effect of melatonin was mediated by the receptor-dependent enhancement of impulse conduction, which was associated with Cx43 phosphorylation and maintaining the RMP level.

The present study aimed to examine the biological activity and cardioprotective potential of Trametes versicolor heteropolysaccharides (TVH) in a rat model of metabolic syndrome (MetS). This study included 40 Wistar rats divided into 5 groups: CTRL-healthy non-treated rats; MetS-non-treated rats; and H-TV, M-TV and L-TV-rats with MetS treated with either 300, 200 or 100 mg/kg TVH per os for 4 weeks. After finishing the treatment, we conducted an oral glucose tolerance test (OGTT), hemodynamic measurements and the animals were sacrificed, hearts isolated and subjected to the Langendorff technique. Blood samples were used for the determination of oxidative stress parameters, lipid status and insulin levels. We showed that α-amylase inhibition was not the mode of TVH antidiabetic action, while TVH showed a moderate inhibition of pathogenic microorganisms' growth (MIC 8.00 mg·mL -1 ; MBC/MFC 16.00 mg·mL -1 ). H-TV and M-TV significantly reduced the level of prooxidants (O 2 - , H 2 O 2 , TBARS; p < 0.05), increased antioxidants activity (SOD, CAT, GSH; p < 0.05), reduced blood pressure ( p < 0.05), improved glucose homeostasis in the OGTT test ( p < 0.05), and ejection fraction ( p < 0.05) and cardiac contractility ( p < 0.05) compared to MetS ( p < 0.05). Moreover, TVH treatment normalized the lipid status and decreased insulin levels compared to MetS rats ( p < 0.05). The obtained results demonstrated that the TVH may be considered a useful agent for cardioprotection in MetS conditions.

Myocardial ischemic adenosine production decreases in subsequent events that may blunt its protective functions. To test the relation between total or mitochondrial cardiac adenine nucleotide pool (TAN) on the energy status with adenosine production, Langendorff perfused rat hearts were subjected to three protocols: 1 min ischemia at 40 min, 10 min ischemia at 50 min, and 1 min ischemia at 85 min in Group I; additional infusion of adenosine (30 µM) for 15 min after 10 min ischemia in Group I-Ado, and 1 min ischemia at 40 and 85 min in the controls (Group No I). A 31 P NMR and an HPLC were used for the analysis of nucleotide and catabolite concentrations in the heart and coronary effluent. Cardiac adenosine production in Group I measured after 1 min ischemia at 85 min decreased to less than 15% of that at 40 min in Group I, accompanied by a decrease in cardiac ATP and TAN to 65% of the initial results. Adenosine production at 85 min was restored to 45% of that at 40 min in Group I-Ado, accompanied by a rebound of ATP and TAN by 10% vs. Group I. Mitochondrial TAN and free AMP concentrations paralleled that of total cardiac TAN. Changes in energy equilibrium or mitochondrial function were minor. This study highlights that only a fraction of the cardiac adenine nucleotide pool is available for adenosine production, but further studies are necessary to clarify its nature.

Background: Lactoferrin, an iron-binding glycoprotein, is known to have protective effects against intestinal and cerebral ischemia-reperfusion (IR) injuries; however, its cardioprotective effects against the stunned myocardium are unknown. This study aimed to test the hypothesis that lactoferrin has cardioprotective effects against stunned myocardium., Methods: Using isolated rat hearts (Langendorff system), we determined the effects of lactoferrin administered enterally and by direct cardiac perfusion. Rat hearts were perfused using the Langendorff system, and two experiments were performed. In experiment 1, the hearts were divided into the enteral lactoferrin (E-LF) 7.5 m, 15 m, 30 m, and 60 m groups, where lactoferrin (1000 mg/kg) was administered enterally 7.5, 15, 30, and 60 min, respectively, before perfusion; and a control group, where saline was administered 30 min before perfusion. In experiment 2, hearts were allocated to the perfusate lactoferrin (P-LF) 15 and 100 groups, where 15 mg/L and 100 mg/L lactoferrin were respectively added to the perfusate, and a control group. Each group was perfused for 20 min prior to 15 min of no-flow ischemia with pacing, followed by 20 min of reperfusion. The primary outcome was the maximum left ventricular derivative of pressure development (LV dP/dt max) 15 min after reperfusion. Myocardial phospho-protein kinase B (p-Akt) was assayed using western blotting., Results: The LV dP/dt max 15 min after reperfusion in the E-LF 15 and 30 m groups was significantly higher than that in the control group. However, the effects disappeared in the E-LF 60 m group. In the second experiment, there were no significant differences in LV dP/dt max. Myocardial p-Akt was not significantly activated in any lactoferrin group., Conclusion: Cardioprotection was observed 15-30 min after enteral lactoferrin but not by direct cardiac perfusion with lactoferrin. Myocardial p-Akt was not associated with the cardioprotective effect. The cardioprotective effect may be induced by enteral lactoferrin-induced substances., (© 2022. The Author(s).)

We aimed to investigate the cardioprotective effects of ethanolic Melissa officinalis L. extract (ME) in the rat model of myocardial ischemia/reperfusion (I/R) injury. Thirty-two Wistar rats were randomly divided into a CTRL non-treated control group with myocardial I/R injury and three experimental groups of rats treated with 50, 100, or 200 mg/kg of ME for 7 days per os. Afterward, hearts were isolated, and cardiodynamic function was assessed via the Langendorff model of global 20 min ischemia and 30 min reperfusion. Oxidative stress parameters were determined spectrophotometrically from the samples of coronary venous effluent (O 2 - , H 2 O 2 , TBARS, and NO 2 - ,) and heart tissue homogenate (TBARS, NO 2 - , SOD, and CAT). H/E and Picrosirius red staining were used to examine cardiac architecture and cardiac collagen content. ME improved cardiodynamic parameters and achieved to preserve cardiac architecture after I/R injury and to decrease fibrosis, especially in the ME200 group compared to CTRL. ME200 and ME100 markedly decreased prooxidants TBARS, O 2 - , and H 2 O 2 while increasing NO 2 - . Hereby, we confirmed the ME`s ability to save the heart from I/R induced damage, even after short-term preconditioning in terms of preserving cardiodynamic alterations, cardiac architecture, fibrosis, and suppressing oxidative stress, especially in dose of 200 mg/kg.

The purpose of the article is to study the structure, innervation and state of the epicardial adipose tissue of the aortic-pulmonary region of the heart of rats at the age of 3-4 months and 18-23 months using neuroimmunohistochemical markers. Using a complex of histological and immunohistochemical methods, various nervous apparatus (ganglia, clusters of chromaffin cells, nerve trunks, nerve fibers, nerve plexuses, synaptic endings) with different mediators were identified in the white and brown adipose tissue of the base of the rat heart. It was found that parasympathetic and sympathetic postganglionic nerve fibers are involved in the innervation of white and brown adipose tissue. They penetrate into adipose tissue as part of Remak's cords of varicose axons along arterial vessels, form terminal synaptic plexuses of the en passant type, and are involved in the innervation of adipocytes of both types of epicardial adipose tissue. It was found that PGP 9.5+ cholinergic terminal nerve fibers predominate over catecholaminergic ones. During aging of rats, neurodegenerative, involutive (desimatization), and destructive pathological changes in white adipocytes were noted in epicardial adipose tissue.

Purpose : Heart-type fatty acid binding protein (H-FABP) is primary transporter of free fatty acid and plays an important role in myocardial metabolism, which is characterized by high specificity and rapid appearance under ischemic condition. The objective of this study was to clarify the usefulness of imaging study of targeting H-FABP appearance using radio-labeled antibody, and correlation with myocardial fatty acid metabolism and perfusion in acute reperfusion ischemia. Method : Wistar rats were allotted to sham-operated control group (sham; n=4), ischemia non-reperfused group (IG; n=5), and ischemia-reperfusion group (RG; n=5). Ligation of left coronary artery (LCA) was performed for IG and RG. 20 min of ischemia was followed by 60min of reperfusion for RG. 125 I labeled anti H-FABP antibody (anti H-FABP), BMIPP and 99m Tc-sestamibi (MIBI) was injected intravenously. Multi-tracer digital autoradiogram was performed using µ-imager ® . The ratio of radioactivity in LCA related (culprit) area to the inferior (remote) area (target uptake ratio=TUR) was generated. Results : In sham group, no visually detectable accumulation was observed for the anti H-FABP image, and TUR MIBI and TUR BMIPP were equivalent to 1. In IG, TUR MIBI and TUR BMIPP were remarkably low (0.12±0.01, 0.24±0.07). In RG, TUR MIBI was significantly lower (0.20±0.03, p<0.05 vs. other groups). However, TUR BMIPP was significantly higher (2.78±1.28, p<0.05) compared to the sham and IG, whereas anti H-FABP showed markedly higher ratio in the reperfused area compared to the sham and IG (3.43±0.73 vs. 0.31±0.13 and 1.09±0.07 for IG and sham; p<0.05, and <0.01, respectively). Conclusion : Anti H-FABP accumulated specifically in reperfused area under acute ischemia, and it accorded to the area where fatty acid metabolism was activated. This study has shown the future potential for clinical application in vivo imaging of acute coronary syndrome., Competing Interests: None., (2022, The Japanese Society of Nuclear Cardiology.)

Connexins (Cxs) are a family of membrane-spanning proteins, expressed in vertebrates and named according to their molecular weight. They are involved in tissue homeostasis, and they function by acting at several communication levels. Cardiac Cxs are responsible for regular heart function and, among them, Cx26 and Cx43 are widely expressed throughout the heart. Cx26 is present in vessels, as well as in cardiomyocytes, and its localization is scattered all over the cell aside from at the intercalated discs as is the case for the other cardiac Cxs. However, having been found in cardiomyocytes only recently, both its subcellular localization and its functional characterization in cardiomyocytes remain poorly understood. Therefore, in this study we aimed to obtain further data on the localization of Cx26 at the subcellular level. Our TEM immunogold analyses were performed on rat heart ventricles and differentiated H9c2 cardiac cell sections as well as on differentiated H9c2 derived extracellular vesicles. The results confirmed the absence of Cx26 at intercalated discs and showed the presence of Cx26 at the level of different subcellular compartments. The peculiar localization at the level of extracellular vesicles suggested a specific role for cardiac Cx26 in inter-cellular communication in an independent gap junction manner.

Angiogenesis is involved in regeneration of cardiac tissue following acute myocardial infarction (MI), a disease often investigated in rat models. Therefore, the ability to thoroughly evaluate the angiogenic response following experimentally induced MI in rats, and distinguish it from inflammation, is desired. This would enable evaluation of the angiogenic potential of new therapeutics and improve knowledge on MI pathophysiology. Due to the complex response to MI involving multiple cell types and the limited selection of rat-specific antibodies, careful optimization is crucial to capture this complexity. Here, we present an 8-color flow cytometry-based multicolor panel that will enable quantification of the ongoing angiogenic response as well as characterize the cells involved. A detailed description of tissue preparation, immunostaining, and gating strategy is provided. © 2021 Wiley Periodicals LLC. Basic Protocol: Cardiac tissue preparation and staining to investigate the ongoing angiogenic response in rat cardiac tissue following myocardial infarction Support Protocol: Titration of all antibodies in the presented panel., (© 2021 Wiley Periodicals LLC.)

Considering that sex related differences in cardiac response to flaxseed (FSO) and evening primrose oil (EPO) are insufficiently known present investigation assessed the effect of these two oils, on the cardiac function of isolated rat hearts and the possible role of sex in this. The present study was carried out on 60 adult male Wistar albino rats randomly divided into 6 groups: male rats treated with EPO, dose of 10 mg/kg/day; female rats treated with EPO, dose of 10 mg/kg/day; male rats treated with FSO, dose of 300 mg/kg/day; female rats treated with FSO, dose of 300 mg/kg/day; control group of female rats treated with regular laboratory diet for animals; control group of male rats treated with regular laboratory diet for animals. Using the Langendorff technique, markers of the heart function were evaluated: the maximum and minimum rates of pressure development in the left ventricle (LV; dP/dtmax, dP/dtmin), systolic and diastolic left ventricular pressure (SLVP, DLVP, respectively), heart rate (HR) and coronary flow (CF). Male rats treated with EPO had significantly higher (p = 0.016) mean values of dP/dtmax, dP/dtmin, SLVP and DLVP (average increase for all CPPs 20%, 25%, 30% and 110%, respectively), compared to the group of male rats treated with FSO (p = 0.914). Our study results indicate that both types of PUFA oils only slightly changed the function of the isolated rat heart in male but not in female rats. Nevertheless, the difference between oil treatments was found in male rats who had stronger cardiac response after supplementation with EPO.

Using histological methods of staining with toluidine blue, hematoxylin-eosin and immunohistochemical reactions for the PGP 9,5 protein, tyrosine hydroxylase (TH), Iba-1 protein, cellular changes in different parts of the heart of Wistar rats at the age of 18-23 months were studied. In the connective tissue of the heart base, focal inflammatory infiltrates were found, near which PGP 9.5+ and TH+ plexuses, consisting of parasympathetic and sympathetic nerve fibers, were detected. In the area of the valvular heart apparatus, at the border of the anneau fibreux and the myocardium of the right atrium, pathological changes in nerve structures were found: degeneration of nerve fibers and granular destruction varicose axons of the terminal plexus. A close relationship has been established between axons of the terminal nervous network and cells of inflammatory infiltrates and blood capillaries. The features of the localization of neurocellular inflammatory complexes consisting of nerve fibers, blood capillaries and cells participating in the local inflammatory process (mast cells, histiocytes, monocytes, fibroblasts, plasma cells) in various parts of the myocardium in old animals are described. The chronic nature of neurogenic inflammation in the heart during aging has been established.

The aim of our study was to determine a chemical composition of methanol extract of Galium verum as well as to assess its effects on functional recovery and redox status of isolated rat heart after ischemia. Rats were divided into control and G. verum group, which included animals treated with 500 mg/kg of methanol extract of G. verum for 28 days. Parameters of heart function and oxidative stress markers were estimated. Cell morphology was evaluated by hematoxylin and eosin (HE) staining. Our results demonstrated for the first time that G. verum extract preserved cardiac contractility, systolic, and diastolic function as wells as structural damage of the heart after ischemia. Furthermore, G. verum extract modulated the activity of antioxidant enzymes and alleviated the production of pro-oxidants.

Objective: To detect the effects of shortwave radiation on dose-dependent cardiac structure and function in rats after radiation and to elucidate the mechanism of shortwave radiation induced cardiac injury to identify sensitive indicators and prophylactic treatment., Methods: One hundred Wistar rats were either exposed to 27 MHz continuous shortwave at a power density of 5, 10, and 30 mW/cm 2 for 6 min or undergone sham exposure for the control (the rats had to be placed in the exposure system with the same schedules as the exposed animals, but with an inactive antenna). The Ca 2+ , glutamic oxaloacetic transaminase (AST), creatine kinase (CK) and lactate dehydrogenase (LDH) content in the peripheral serum of the rats were detected by an automatic blood biochemical analyser. The electrocardiogram (ECG) of standard lead II was recorded by a multi-channel physiological recording and analysis system. The cardiac structure of rats was observed by light and electron microscopy., Results: The results showed that the 5, 10, and 30 mW/cm 2 shortwave radiation caused a significant increased in the levels of Ca 2+ , AST, CK, and LDH in the peripheral serum of rats. The cardiac structure was damaged by radiation and showed a disordered arrangement of myocardial fibres, the cavitation and swelling of myocardial mitochondria. These injuries were most significant 7 d after radiation and were not restored until 28 d after radiation., Conclusion: Shortwave radiation of 5, 10, and 30 mW/cm 2 can damage rat cardiac function, including damage to the tissue structure and ultrastructure, especially at the level of the myocardial fibres and mitochondria. Shortwave radiation at 5, 10, and 30 mW/cm 2 induced damage to rat heart function and structure with a dose-effect relationship, i.e., the greater the radiation dose was, the more significant the damage was., (Copyright © 2020 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.)

Cardiac β-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 β-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-β1 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.

Doxorubicin (DOX) is one of the secondary metabolites of Streptomyces peucetius var. caesius. It is a common and effective chemotherapeutic agent used for the treatment of different diseases, including lymphoma, leukemia, breast cancer, and solid tumors. However, this medicine causes cardiotoxic side effects, which limit its clinical application. The present study examined the cardiomyopathy induced by DOX via echocardiography and transmission electron microscopy (TEM). The main objective was to evaluate the capacity of echocardiography and TEM as diagnostic tools for DOX-induced cardiotoxicity. Moreover, the correlation between intracellular and functional changes due to cardiotoxicity was assessed in a rat model. Cardiomyopathy was induced in rats by two cumulative doses of DOX. Group I received DOX 12 [i.e., 12 mg/kg, intraperitoneal (IP)] and group II received DOX 15 (i.e., 15 mg/kg, IP) in six equal doses over two weeks. Group III as the control (Ctrl) group received normal saline as a vehicle. Mortality during the study was only observed in the DOX 15 group. The echocardiographic assessments revealed significant changes in ejection fraction, fractional shortening, and heart rate in the groups which received DOX. In addition, severe cardiac arrhythmia was evident in DOX-treated groups. Remarkable adverse effects, such as moderately degenerated cells and inflated mitochondria were observed in the TEM analysis of rat hearts in the DOX groups. The present study indicated that rat models are suitable for investigating DOX-induced cardiomyopathy, especially at the dose of 12 mg/kg. Furthermore, echocardiography and TEM examinations were found to be valuable methods for the determination of cardiotoxicity in rats due to DOX., (Copyright © 2020, Archives of Razi Institute. Published by Kowsar.)

Exposure to fine particulate matter (PM 2.5 ) increased the risks of cardiovascular diseases. PM 2.5 -bound 1-nitropyrene (1-NP) and 9-nitroanthracene (9-NA) are released from the incomplete combustion of fossil fuels and derived from polycyclic aromatic hydrocarbons (PAHs). The toxicities of 1-NP and 9-NA are mainly reflected in their carcinogenicity and mutagenicity. However, studies of PM 2.5 -bound 1-NP and 9-NA on the cardiac genotoxicity are limited so far. In this study, histopathology, DNA damage, DNA repair-related gene expression, and oxidative stress were investigated in the hearts of male Wistar rats exposed to PM 2.5 [1.5 mg/kg body weight (b.w.)] or three different dosages of 1-NP (1.0 × 10 - 5 , 4.0 × 10 - 5 , and 1.6 × 10 - 4  mg/kg b.w.) or 9-NA (1.3 × 10 - 5 , 4.0 × 10 - 5 , and 1.2 × 10 - 4  mg/kg b.w.). The results revealed that (1) PM 2.5 , higher dosages of 1-NP (4.0 × 10 - 5 and 1.6 × 10 - 4  mg/kg b.w.) and 9-NA (4.0 × 10 - 5 and 1.2 × 10 - 4  mg/kg b.w.) caused obvious pathological responses and DNA damage (DNA strand breaks, 8-OHdG formation and DNA-protein cross-link), accompanied by increasing OGG1 and GADD153 expression while inhibiting MTH1 and XRCC1 expression in rat hearts. Also, they elevated the hemeoxygenase-1 (HO-1), glutathione S-transferase (GST), and malondialdehyde (MDA) levels and decreased superoxide dismutase (SOD) activity compared with the control. (2) The lowest dosages 1-NP or 9-NA could not cause DNA damage and oxidative stress. (3) At the approximately equivalent dose level, PM 2.5 -induced DNA damage effects were more obvious than 1-NP or 9-NA along with positive correlation. Taken together, heart DNA damage caused by PM 2.5 , 1-NP and 9-NA may be mediated partially through influencing the DNA repair capacity and causing oxidative stress, and such negative effects might be related to the genotoxicity PM 2.5 , 1-NP, and 9-NA.

Adeno-associated viruses (AAVs) provide advantages in long-term, cardiac-specific gene expression. However, AAV serotype specificity data is lacking in experimental models relevant to cardiac electrophysiology and cardiac optogenetics. We aimed to identify the optimal AAV serotype (1, 6, or 9) in pursuit of scalable rodent and human models using genetic modifications in cardiac electrophysiology and optogenetics, in particular, as well as to elucidate the mechanism of virus uptake. In vitro syncytia of primary neonatal rat ventricular cardiomyocytes (NRVMs) and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were infected with AAVs 1, 6, and 9 containing the transgene for eGFP or channelrhodopsin-2 (ChR2) fused to mCherry. In vivo adult rats were intravenously injected with AAV1 and 9 containing ChR2-mCherry. Transgene expression profiles of rat and human cells in vitro revealed that AAV1 and 6 significantly outperformed AAV9. In contrast, systemic delivery of AAV9 in adult rat hearts yielded significantly higher levels of ChR2-mCherry expression and optogenetic responsiveness. We tracked the mechanism of virus uptake to purported receptor-mediators for AAV1/6 (cell surface sialic acid) and AAV9 (37/67 kDa laminin receptor, LamR). In vitro desialylation of NRVMs and hiPSC-CMs with neuraminidase (NM) significantly decreased AAV1,6-mediated gene expression, but interestingly, desialylation of hiPSC-CMs increased AAV9-mediated expression. In fact, only very high viral doses of AAV9-ChR2-mCherry, combined with NM treatment, yielded consistent optogenetic responsiveness in hiPSC-CMs. Differences between the in vitro and in vivo performance of AAV9 could be correlated to robust LamR expression in the intact heart (neonatal rat hearts as well as adult human and rat hearts), but no expression in vitro in cultured cells (primary rat cells and hiPS-CMs). The dynamic nature of LamR expression and its dependence on environmental factors was further corroborated in intact adult human ventricular tissue. The combined transgene expression and cell surface receptor data may explain the preferential efficiency of AAV1/6 in vitro and AAV9 in vivo for cardiac delivery and mechanistic knowledge of their action can help guide cardiac optogenetic efforts. More broadly, these findings are relevant to future efforts in gene therapy for cardiac electrophysiology abnormalities in vivo as well as for genetic modifications of cardiomyocytes by viral means in vitro applications such as disease modeling or high-throughput drug testing.

Oxytocin (OT) has a postconditioning effect against the ischemia-reperfusion (I/R) injury. However, its precise cardioprotection mechanism at the early reperfusion phase remains under debate. Our previous study revealed that OT postconditioning (OTpost) is cardioprotective by activating the Reperfusion Injury Salvage Kinase (RISK) pathway. Therefore, the present study is aimed to determine the biological effects of OTpost via the OT receptor and the activation of the JAK/STAT3 signaling pathway, mitochondrial adenosine triphosphate-dependent potassium channel (mitoKATP), nitric oxide (NO) release, and its anti-apoptotic effects against I/R injury in an isolated rat heart model. Sixty-three rats were randomly allocated to one of nine groups. OT was perfused 40 min prior to the regional ischemia or 15 min at the early reperfusion phase. AG490 (a JAK/STAT3 inhibitor), 5HD (a mitoKATP blocker), atosiban (an OT receptor antagonist), L-NAME (a nonspecific nitric oxide synthase inhibitor) were applied either alone or in combination with OT during the pre-ischemia phase and/or in the early reperfusion phase. Myocardial infarct size, hemodynamic factor, ventricular arrhythmia, coronary flow, cardiac biochemical marker, and the apoptosis index were determined at the end of reperfusion. Oxytocin postconditioning reduced infarct size, lactate dehydrogenase activity, arrhythmia score, ventricular fibrillation, and apoptosis. Moreover, AG490, 5HD, atosiban, and L-NAME abrogated the cardioprotective effects of OT. Our results demonstrated that the cardioprotective effects of OT are mediated by NO release, and the activation of mitoKATP and the SAFE pathway through the JAK/STAT3 signaling cascade that finally lead to decrease in the apoptosis index during the early reperfusion phase., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Diabetes mellitus is a major global health disorder and, currently, over 450 million people have diabetes with 90% suffering from type 2 diabetes. Left untreated, diabetes may lead to cardiovascular diseases which are a leading cause of death in diabetic patients. Calcium is the trigger and regulator of cardiac muscle contraction and derangement in cellular Ca 2+ homeostasis, which can result in heart failure and sudden cardiac death. It is of paramount importance to investigate the regional involvement of Ca 2+ in diabetes-induced cardiomyopathy. Therefore, the aim of this study was to investigate the voltage dependence of the Ca 2+ transients in endocardial (ENDO) and epicardial (EPI) myocytes from the left ventricle of the Goto-Kakizaki (GK) rats, an experimental model of type 2 diabetes mellitus. Simultaneous measurement of L-type Ca 2+ currents and Ca 2+ transients was performed by whole-cell patch clamp techniques. GK rats displayed significantly increased heart weight, heart weight/body weight ratio, and non-fasting and fasting blood glucose compared to controls (CON). Although the voltage dependence of L-type Ca 2+ current was unaltered, the voltage dependence of the Ca 2+ transients was reduced to similar extents in EPI-GK and ENDO-GK compared to EPI-CON and ENDO-CON myocytes. TPK L-type Ca 2+ current and Ca 2+ transient were unaltered. THALF decay of L-type Ca 2+ current was unaltered; however, THALF decay of the Ca 2+ transient was shortened in ENDO and EPI myocytes from GK compared to CON rat hearts. In conclusion, the amplitude of L-type Ca 2+ current was unaltered; however, the voltage dependence of the Ca 2+ transient was reduced to similar extents in EPI and ENDO myocytes from GK rats compared to their respective controls, suggesting the possibility of dysfunctional sarcoplasmic reticulum Ca 2+ transport in the GK diabetic rat hearts.

The well-known age-related mitochondrial dysfunction deeply affects heart because of the tissue's large dependence on mitochondrial ATP provision. Our study revealed in aged rat heart a significant 25% decrease in mtDNA relative content, a significant 29% increase in the 4.8 Kb mtDNA deletion relative content, and a significant inverse correlation between such contents as well as a significant 38% decrease in TFAM protein amount. The TFAM-binding activity to specific mtDNA regions increased at those encompassing the mtDNA replication origins, D-loop and Ori-L. The same mtDNA regions were screened for different kinds of oxidative damage, namely Single Strand Breaks (SSBs), Double Strand Breaks (DSBs), abasic sites (AP sites) and oxidized bases as 7,8-dihydro-8-oxoguanine (8oxoG). A marked increase in the relative content of mtDNA strand damage (SSBs, DSBs and AP sites) was found in the D-loop and Ori-L regions in the aged animals, unveiling for the first time in vivo an age-related, non-stochastic accumulation of oxidative lesions in these two regions that appear as hot spots of mtDNA damage. The use of Formamidopyrimidine glycosylase (Fpg) demonstrated also a significant age-related accumulation of oxidized purines particularly in the D-loop and Ori-L regions. The detected increased binding of TFAM to the mtDNA damage hot spots in aged heart suggests a link between TFAM binding to mtDNA and loss of mitochondrial genome likely through hindrance of repair processes., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Ischemia/reperfusion (I/R) injury triggers cardiac dysfunctions via creating reactive oxygen species (ROS). Because xanthine oxidase (XO) is one of the major enzymes that generate ROS, inhibition of XO is expected to suppress ROS-induced I/R injury. However, it remains unclear whether XO inhibition really yields cardioprotection during I/R. The protective effects of the XO inhibitors, topiroxostat and allopurinol, on cardiac I/R injury were evaluated.Methods and Results:Using isolated rat hearts, ventricular functions, occurrence of arrhythmias, XO activities and thiobarbituric acid reactive substances (TBARS) productions and myocardial levels of adenine nucleotides before and after I/R, and cardiomyocyte death markers during reperfusion, were evaluated. Topiroxostat prevented left ventricular dysfunctions and facilitated recovery from arrhythmias during I/R. Allopurinol and the antioxidant, N-acetylcysteine (NAC), exhibited similar effects at higher concentrations. Topiroxostat inhibited myocardial XO activities and TBARS productions after I/R. I/R decreased myocardial levels of ATP, ADP and AMP, but increased that of xanthine. While topiroxostat, allopurinol or NAC did not change myocardial levels of ATP, ADP or AMP after I/R, all of the agents decreased the level of xanthine. They also decreased releases of CPK and LDH during reperfusion., Conclusions: Topiroxostat showed protective effects against I/R injury with higher potency than allopurinol or NAC. It dramatically inhibited XO activity and TBARS production, suggesting suppression of ROS generation.

The Langendorff Perfused Heart Model is an experimental procedure developed at the end of the nineteenth century by Oskar Langendorff. In this procedure, an excised heart has a cannula inserted into its aorta so that the heart can be retrogradely perfused via the coronary artery. The procedure has been improved in recent times, and these improvements are used to evaluate the direct effect of medication on the heart as well as the effect of ischemia-reperfusion injury on heart function. In this chapter, we describe protocols for evaluating heart function in Langendorff perfused rat heart.

Arsenic is a widespread metalloid in the environment, which poses a broad spectrum of adverse effects on human health. However, a global view of arsenic-induced heart toxicity is still lacking, and the underlying molecular mechanisms remain unclear. By performing a comparative quantitative proteomic analysis, the present study aims to investigate the alterations of proteome profile in rat heart after long-term exposure to arsenic. As a result, we found that the abundance of 81 proteins were significantly altered by arsenic treatment (35 up-regulated and 46 down-regulated). Among these, 33 proteins were specifically associated with cardiovascular system development and function, including heart development, heart morphology, cardiac contraction and dilation, and other cardiovascular functions. It is further proposed that the aberrant regulation of 14 proteins induced by arsenic would disturb cardiac contraction and relaxation, impair heart morphogenesis and development, and induce thrombosis in rats, which is mediated by the Akt/p38 MAPK signaling pathway. Overall, these findings will augment our knowledge of the involved mechanisms and develop useful biomarkers for cardiotoxicity induced by environmental arsenic exposure., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

In the heart, the left ventricle pumps blood at higher pressure than the right ventricle. Within the left ventricle, the electromechanical properties of ventricular cardiac myocytes vary transmurally and this may be related to the gradients of stress and strain experienced in vivo across the ventricular wall. Diabetes is also associated with alterations in hemodynamic function. The aim of this study was to investigate shortening and Ca 2+ transport in epicardial (EPI) and endocardial (ENDO) left ventricular myocytes in the streptozotocin (STZ)-induced diabetic rat. Shortening, intracellular Ca 2+ and L-type Ca 2+ current (I Ca,L ) were measured by video detection, fura-2 microfluorimetry, and whole-cell patch clamp techniques, respectively. Time to peak (TPK) shortening was prolonged to similar extents in ENDO and EPI myocytes from STZ-treated rats compared to ENDO and EPI myocytes from controls. Time to half (THALF) relaxation of shortening was prolonged in ENDO myocytes from STZ-treated rats compared to ENDO controls. TPK Ca 2+ transient was prolonged in ENDO myocytes from STZ-treated rats compared to ENDO controls. THALF decay of the Ca 2+ transient was prolonged in ENDO myocytes from STZ-treated rats compared to ENDO controls. Sarcoplasmic reticulum (SR) fractional release of Ca 2+ was reduced in EPI myocytes from STZ-treated rats compared to EPI controls. I C a,L activation, inactivation, and recovery from inactivation were not significantly altered in EPI and ENDO myocytes from STZ-treated rats or controls. Regional differences in Ca 2+ transport may partly underlie differences in ventricular myocyte shortening across the wall of the healthy and the STZ-treated rat left ventricle., (© 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.)

Background: Silver fir trunk extract (SFTE) is a complex mixture of antioxidative polyphenols (lignans and phenolic acids) from the trunks of silver fir trees ( Abies alba , lignum). In our previous study, we have shown that SFTE exerts strong antioxidative and protective effects against atherogenic, diet-induced arterial wall damage., Objective: The aim of the present study was to test the potential protective effects of SFTE and its compounds, two phenolic acids (p-coumaric and protocatechuic acids) in ischemia-reperfusion injury of isolated rat hearts., Design: Isolated hearts of Wistar rats aged 4-8 weeks were exposed to perfusion, ischemia, and reperfusion periods. The experiments were performed using the following five groups: control, SFTE (10 µg/L), SFTE (100 µg/L), protocatechuic acid, and p-coumaric. Aortas were isolated to measure vascular responses in the presence of N ω -Nitro-L-arginine., Results: SFTE dose-dependently reduced ischemic-reperfusion heart damage, which was indicated as the decrease in the lactate dehydrogenase (LDH) release rate and arrhythmias duration by 80% and an increase in coronary flow rate during the reperfusion period. Two tested compounds (p-coumaric and protocatechuic acids) acted less cardioprotective, since they decreased the duration of arrhythmias only by 40 and 45%, respectively, and did not decrease LDH release rates during the reperfusion period. Only p-coumaric acid increased coronary flow rates, whereas protocatechuic acid did not., Conclusions: We conclude that the SFTE exerted the strongest cardioprotective effect, whereas its constituents (the p-coumaric and protocatechuic acids) were less effective in inducing cardioprotection., Competing Interests: and funding The authors have not received any funding or benefits from industry or elsewhere to conduct this study.

Purpose: The contribution of extra virgin olive oil (EVOO) macro- and micro-constituents in heart oxidative and inflammatory status in a hypercholesterolemic rat model was evaluated. Fatty acid profile as well as α-tocopherol, sterol, and squalene content was identified directly in rat hearts to distinguish the effect of individual components or to enlighten the potential synergisms., Methods: Oils and oil-products with discernible lipid and polar phenolic content were used. Wistar rats were fed a high-cholesterol diet solely, or supplemented with one of the following oils, i.e., EVOO, sunflower oil (SO), and high-oleic sunflower oil (HOSO) or oil-products, i.e., phenolics-deprived EVOO [EVOO(-)], SO enriched with the EVOO phenolics [SO(+)], and HOSO enriched with the EVOO phenolics [HOSO(+)]. Dietary treatment lasted 9 weeks; at the end of the intervention blood and heart samples were collected., Results: High-cholesterol-diet-induced dyslipidemia was shown by increase in serum total cholesterol, low-density lipoprotein cholesterol, and triacylglycerols. Dyslipidemia resulted in increased malondialdehyde (MDA) and tumor necrosis factor-α (TNF-α) levels, while glutathione and interleukin 6 levels remained unaffected in all intervention groups. Augmentation observed in MDA and TNF-α was attenuated in EVOO, SO(+), and HOSO(+) groups. Heart squalene and cholesterol content remained unaffected among all groups studied. Heart α-tocopherol was determined by oil α-tocopherol content. Variations were observed for heart β-sitosterol, while heterogeneity was reported with respect to heart fatty acid profile in all intervention groups., Conclusions: Overall, we suggest that the EVOO-polar phenolic compounds decreased MDA and TNF-α in hearts of cholesterol-fed rats.

We investigated whether reactive oxygen species (ROS) are involved in heart adaptive responses administering a vitamin E-enriched diet to trained rats. Using the homogenates and/or mitochondria from rat hearts we determined the aerobic capacity, tissue level of mitochondrial proteins, and expression of cytochrome c and factors (PGC-1, NRF-1, and NRF-2) involved in mitochondrial biogenesis. We also determined the oxidative damage, glutathione peroxidase (GPX) and reductase activities, glutathione content, mitochondrial ROS release rate, and susceptibility to in vitro oxidative challenge. Glutathione (GSH) content was not affected by both training and antioxidant supplementation. Conversely, antioxidant supplementation prevented metabolic adaptations to training, such as the increases in oxidative capacity, tissue content of mitochondrial proteins, and cytochrome c expression, attenuated some protective adaptations, such as the increase in antioxidant enzyme activities, and did not modify the decrease in ROS release by succinate supplemented mitochondria. Moreover, vitamin E prevented the training-linked increase in tissue capacity to oppose an oxidative attach. The antioxidant effects were associated with decreased levels of PGC-1, NRF-1, and NRF-2 expression. Our results support the idea that some heart adaptive responses to training depend on ROS produced during the exercise sessions and are mediated by the increase in PGC-1 expression which is involved in both the regulation of respiratory capacity and antioxidant protection. However, vitamin inability to prevent some adaptations suggests that other signaling pathways impinging on PGC-1 can modify the response to the antioxidant integration.

Chronic hypoxia increases the myocardial resistance to acute ischemia-reperfusion injury by affecting the mitochondrial redox balance. Hexokinase (HK) bears a high potential to suppress the excessive formation of reactive oxygen species because of its increased association with mitochondria, thereby inhibiting the membrane permeability transition pore opening and preventing cell death. The purpose of this study was to determine the effect of severe intermittent hypobaric hypoxia (7,000 m, 8 h/day, 5 wk) on the function and colocalization of HK isoforms with mitochondria in the left (LV) and right ventricles of rat myocardium. The real-time RT-PCR, Western blot, enzyme coupled assay, and quantitative immunofluorescence techniques were used. Our results showed significantly elevated expression of HK isoforms (HK1 and HK2) in the hypoxic LV. In addition, intermittent hypoxia increased the total HK activity and the association of HK isoforms with mitochondria in both ventricles. These findings suggest that HK may contribute to the cardioprotective phenotype induced by adaptation to severe intermittent hypobaric hypoxia., (Copyright © 2015 the American Physiological Society.)

Serpinin peptides derive from proteolytic cleavage of Chromogranin-A at C-terminus. Serpinin and the more potent pyroglutaminated-serpinin (pGlu-Serp) are positive cardiac β-adrenergic-like modulators, acting through β1-AR/AC/cAMP/PKA pathway. Because in some conditions this pathway and/or other pro-survival pathways, activated by other Chromogranin-A fragments, may cross-talk and may be protective, here we explored whether pGlu-Serp cardioprotects against ischemia/reperfusion injury under normotensive and hypertensive conditions. In the latter condition, cardioprotection is often blunted because of the limitations on pro-survival Reperfusion Injury Salvage Kinases (RISK) pathway activation. The effects of pGlu-Serp were evaluated on infarct size (IS) and cardiac function by using the isolated and Langendorff perfused heart of normotensive (Wistar Kyoto, WKY) and spontaneously hypertensive (SHR) rats exposed to ischemic pre-conditioning (PreC) and post-conditioning (PostC). In both WKY and SHR rat, pGlu-Serp induced mild cardioprotection in both PreC and PostC. pGlu-Serp administered at the reperfusion (Serp-PostC) significantly reduced IS, being more protective in SHR than in WKY. Conversely, left ventricular developed pressure (LVDevP) post-ischemic recovery was greater in WKY than in SHR. pGlu-Serp-PostC reduced contracture in both strains. Co-infusion with specific RISK inhibitors (PI3K/Akt, MitoKATP channels and PKC) blocked the pGlu-Serp-PostC protective effects. To show direct effect on cardiomyocytes, we pre-treated H9c2 cells with pGlu-Serp, which were thus protected against hypoxia/reoxygenation. These results suggest pGlu-Serp as a potential modulatory agent implicated in the protective processes that can limit infarct size and overcome the hypertension-induced failure of PostC., (© 2015 Society for Endocrinology.)

Mitochondrial biogenesis emerges as a compensatory mechanism involved in the recovery process in endotoxemia and sepsis. The aim of this work was to analyze the time course of the cardiac mitochondrial biogenesis process occurring during endotoxemia, with emphasis on the quantitative analysis of mitochondrial function. Female Sprague-Dawley rats (45 days old) were ip injected with LPS (10 mg/kg). Measurements were performed at 0-24 h after LPS administration. PGC-1α and mtTFA expression for biogenesis and p62 and LC3 expression for autophagy were analyzed by Western blot; mitochondrial DNA levels by qPCR, and mitochondrial morphology by transmission electron microscopy. Mitochondrial function was evaluated as oxygen consumption and respiratory chain complex activity. PGC-1α and mtTFA expression significantly increased in every time point analyzed, and mitochondrial mass was increased by 20% (P<0.05) at 24 h. p62 expression was significantly decreased in a time-dependent manner. LC3-II expression was significantly increased at all time points analyzed. Ultrastructurally, mitochondria displayed several abnormalities (internal vesicles, cristae disruption, and swelling) at 6 and 18 h. Structures compatible with fusion/fission processes were observed at 24 h. A significant decrease in state 3 respiration was observed in every time point analyzed (LPS 6h: 20%, P<0.05). Mitochondrial complex I activity was found decreased by 30% in LPS-treated animals at 6 and 24h. Complex II and complex IV showed decreased activity only at 24 h. The present results show that partial restoration of cardiac mitochondrial architecture is not accompanied by improvement of mitochondrial function in acute endotoxemia. The key implication of our study is that cardiac failure due to bioenergetic dysfunction will be overcome by therapeutic interventions aimed to restore cardiac mitochondrial function., (Copyright © 2014 Elsevier Inc. All rights reserved.)

A simple, rapid and sensitive LC-MS/MS method was developed and validated for simultaneous determination of creatine phosphate (CP), creatine (Cr) and 12 nucleotides in rat heart. The analytes, ATP, ADP, AMP, GTP, GDP, GMP, CTP, CDP, CMP, UTP, UDP, UMP, CP, Cr, were extracted from heart tissue with pre-cooled (0°C) methanol/water (1:1, v/v) and separated on a Hypersil Gold AQ C18 column (150mm×4.6mm, 3μm) using an isocratic elution with a mobile phase consisting of 2mmol/L ammonium acetate in water (pH 10.0, adjusted with ammonia). The detection was performed by negative ion electrospray ionization in selective reaction monitoring mode (SRM). In the assay, all the analytes showed good linearity over the investigated concentration range (r>0.99). The accuracy was between 80.7% and 120.6% and the precision expressed in RSD was less than 15.6%. This method was successfully applied to measure the concentrations of the 12 nucleotides, creatine phosphate and creatine in rat heart for the first time., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Unlabelled: The assessment of myocardial radiotracer kinetics, including cardiac extraction fraction and washout, requires the study of isolated perfused hearts to avoid analytic error due to tracer recirculation and systemic metabolites. Analysis of the isolated perfused rat heart by a high-resolution small-animal PET system may offer both reliable evaluation of cardiac tracer kinetics and tomographic images., Methods: An isolated perfused heart system was modified to accommodate the small PET gantry bore size. Isolated rat hearts were perfused via the Langendorff method under a constant flow of Krebs-Henseleit buffer containing (18)F-FDG with a rate of 5 mL/min and placed in the field of view of the commercially available small-animal PET system. Dynamic PET imaging was then performed, with (18)F-FDG uptake in the isolated perfused heart verified by γ counter measurements. Additionally, a rat heart of myocardial infarction was also studied in this system with static PET imaging., Results: Dynamic PET acquisition of the isolated heart under constant (18)F-FDG infusion demonstrated continuous increase of activity in the heart. Correlation between cardiac activity (MBq) measured with the PET system and measurements made with the γ counter were excellent (R(2) = 0.98, P < 0.001, n = 10). Tracer input rate (MBq/min) was also well correlated with cardiac tracer uptake rate (MBq/min) (R(2) = 0.87, P < 0.001, n = 12). PET imaging of the heart with myocardial infarction showed a clear tracer uptake defect corresponding to the location of scar tissue identified by autoradiography and histology., Conclusion: Combining the Langendorff method of isolated rat heart perfusion with high-resolution small-animal PET allows for the reliable quantification of myocardial tracer kinetics. This novel assay is readily adapted to available small-animal PET systems and may be useful for understanding myocardial PET tracer kinetics.

Aims: Oxidative stress emerges after reperfusion of an organ following an ischemic period and results in tissue damage. In the heart, an amplified generation of reactive oxygen species and a significant Ca(2+) accumulation cause ventricular arrhythmias and mitochondrial dysfunction. This occurs in consequence of increased non-specific permeability. A number of works have shown that permeability transition is a common substrate that underlies the reperfusion-induced heart injury. The aim of this work was to explore the possibility that CDP-choline may circumvent heart damage and mitochondrial permeability transition., Main Methods: Rats were injected i.p. with CDP-choline at 20 mg/kg body weight. Heart electric behavior was followed during a closure/opening cycle of the left coronary descendent artery. Heart mitochondria were isolated from rats treated with CDP-choline, and their function was evaluated by analyzing Ca(2+) movements, achievement of a high level of the transmembrane potential, and respiratory control. Oxidative stress was estimated following the activity of the enzymes cis-aconitase and superoxide dismutase, as well as the disruption of mitochondrial DNA., Key Findings: This study shows that CDP-choline avoided ventricular arrhythmias and drop of blood pressure. Results also show that mitochondria, isolated from CDP-choline-treated rats, maintained selective permeability, retained accumulated Ca(2+), an elevated value of transmembrane potential, and a high ratio of respiratory control. Furthermore, activity of cis-aconitase enzyme and mDNA structure were preserved., Significance: This work introduces CDP-choline as a useful tool to preserve heart function from reperfusion damage by inhibiting mitochondrial permeability transition., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Ventricular fibrillation (VF) is a life-threatening arrhythmia, whose occurrence precedes the development of myocardial arrhythmogenic substrate resulting from either chronic or acute pathophysiological conditions. The authors' previous and current studies suggest that downregulated and/or heterogeneously distributed cell-to-cell coupling protein - connexin-43 (Cx43) - facilitates the development of malignant arrhythmias. It was hypothesized that VF itself deteriorates Cx43, and may hamper cardioversion into sinus rhythm. The purpose of the present study was to examine whether myocardial expression and the phosphorylated status of Cx43 is altered due to VF and during sinus rhythm restoration. Experiments were performed using 10-month-old male and female Wistar rats. Isolated Langendorff-mode-perfused rat hearts were subjected to the following events: basal condition, electrically induced VF lasting 2 min, electrically induced VF lasting 10 min, and sustained VF followed by spontaneous sinus rhythm restoration due to transient stop perfusion. The hearts were snap frozen at each event; ventricular tissue was sent for Cx43 immunoblotting using rabbit antiCx43 polyclonal antibody to detect phosphorylated (P-Cx43) as well as unphosphorylated (noP-Cx43) forms of Cx43, and mouse antiCx43 monoclonal antibody to detect noP-Cx43 only. Compared with basal conditions, total Cx43 expression did not change during experiments in either male or female rat hearts. However, P-Cx43 and the ratio of P-Cx43 to total Cx43 decreased significantly due to VF lasting 2 min and 10 min in male rat hearts only. In parallel, there was a significant increase in noP-Cx43 due to VF lasting 2 min and 10 min in male rat hearts only. Surprisingly, an enhancement of noP-Cx43 linked with suppression of P-Cx43 was detected during stop perfusion-induced termination of VF lasting 2 min, followed by sinus rhythm restoration in both male and female rat hearts. Sinus rhythm was not restored after 10 min of VF, which caused pronounced Cx43 dephosphorylation. In conclusion, there is a downregulation of Cx43 due to sustaining of VF, and it occurs earlier in male rat hearts compared with female rat hearts. It appears that transient no-flow-related inhibition of cell-to-cell coupling, as indicated by an increase in nonP-Cx43, can terminate VF followed by sinus rhythm restoration depending on the degree of previous Cx43 downregulation.

Background: The aim of this study was to examine the cardiac function and transcriptional response of the heart to propofol after ischemia-reperfusion., Methods: Rat hearts were Langendorff-perfused using the modified Krebs-Henseleit buffer, and took 20 min stabilizing periods, 40 min ischemia periods, and then 120 min reperfusion period. The hearts were divided into 5 groups;, Control: 180 min perfusion after stabilization, Ischemic: 40 min global ischemia after stabilization, followed by 120 min reperfusion, Pre: 2 microM propofol treatment was preformed only before ischemia, Post: 2 microM propofol treatment was performed only during reperfusion after ischemia, Pre/Post: 2 microM propofol treatment was performed both before and after ischemia. The measurement for cardiac performances, such as left ventricular developed pressure (LVDP), rate of left ventricular pressure generation (dP/dt), heart rate, and coronary flow were obtained. The expression profiles of isolated mRNA were determined by using Agilent microarray and real time-polymerase chain reaction (RT-PCR) was used to confirm the microarray results for a subset of genes., Results: The Post group showed better LVDP and dP/dt than the Ischemic group. But there were no significant differences in heart rate and coronary flow among the groups. On the results of RT-PCR, the expressions of Abcc9, Bard1, and Casp4 were increased, but the expressions of Lyz, Casp8, and Timp1 were decreased in the Post group compared with the Ischemic group., Conclusions: This study suggests that 2 microM propofol may provide cardioprotective effect, and modulate gene expression such as apoptosis, and K(ATP) ion channel related-genes during reperfusion in the isolated rat hearts.

Background: Extract of ligustrum leaves (Ligustrum delavayanum Hariot [Oleaceae]) is well known in traditional Chinese medicine. One of the active components, oleuropein, displays vasodilating and hypotensive effects., Objective: To analyze the effect of 0.008% lyophilized extract of ligustrum dissolved in 0.5% ethanol on heart function., Animals and Methods: Experiments were done on isolated rat hearts perfused by the Langendorff method in control conditions and during ischemic-reperfusion injury., Results: Application of ligustrum induced positive inotropic and vasodilating effects in spontaneously beating hearts. Pretreatment of the hearts with ligustrum reduced left ventricular diastolic pressure measured during reperfusion and improved left ventricular contraction compared with hearts without any pretreatment. Ligustrum significantly suppressed the incidence and duration of cardiac reperfusion arrhythmias, expressed as G-score, from 7.40+/-0.58 in nontreated rats to 1.97+/-0.50., Discussion: Application of ligustrum or ethanol alone induced changes in coordination between atria and ventricles during ischemia-reperfusion injury. The 'g-score', a new parameter summing the incidence and duration of atrioventricular blocks, atrioventricular dissociation and cardiac arrest, is introduced. The g-scores with ligustrum pretreatment were higher during ischemia than during reperfusion. Ethanol significantly depressed myocardial contractility and coronary flow, and nonsignificantly decreased heart rate of isolated rat hearts. Electrical changes observed during coronary reperfusion in the presence of ethanol were accompanied by deterioration of contractile function., Conclusions: Ligustrum had a significant protective effect on rat myocardium against ischemic-reperfusion injury. Ethanol partially attenuated the protective effect of ligustrum.