Hearts Of Wistar Rats Research Articles

Evaluation of the repairing effect of collagen type I and MaxGel on the infarcted myocardium in an animal model.

Nowadays and in spite of all the preventive effort made, cardiovascular diseases remain as one of the leading causes of death worldwide. Current treatments decrease the progression of the disease but fail to offer a solution where the contractile function lost due to the injury can be recovered. In response to this, regenerative medicine proposes new approaches and one of this is the use of biomaterials that can mimic the role of the extracellular matrix and provide support for new cells in the affected heart. This study evaluated the repairing effect of collagen type I and MaxGel when injected, alone or combined, into the infarcted heart of Wistar rats. Cardiac function was quantified using the ejection fraction as the main parameter and it was measured in three time points during the study: pre-infarction, post-infarction, and post-treatment. Additionally, histological samples of the hearts were taken for evaluation. The data obtained showed a marked recovery of the cardiac function in the animals were collagen type I was injected with an increase of 8.2% on the mean ejection fraction, suggesting that this biomaterial has the capacity to stop the progressive decline of the cardiac function in an infarcted heart.

NOS UNCOUPLING IS ACCOMPANIED WITH INDUCTION OF THE OXIDATIVE STRESS AND THE CARDIOHEMODYNAMICS DISTURBANCES IN HYPERTENSION

We compared the performance of cardiaohemodynamics and indicators of oxidative and nitrosative stress in the heart and aorta in normotensive Wistar rats (WKR) and spontaneously hypertensive rats (SHR). On the basis of experimentally determined parameters to calculate cNOS uncoupling index and biochemical index of function (BIF) in these organs of the cardiovascular system. In the heart, and especially in the aorta of SHR develop a combined oxidative and nitrosative stress that leads to cNOS uncoupling, BIF lowering that correlate with lowering of systolic and diastolic functions, inhibition of the efficiency Frank-Starling mechanism, oxygen consumption of the heart and increasing arterial stiffness. We made the assumption of the existence of the vicious circle of enhancing oxidative stress in organs of the cardiovascular system due to additional superoxide generation by uncoupling cNOS.

0309 : Cardioprotective effect of a novel snake venom derived natriuretic peptide during myocardial ischemia reperfusion injury

In this study, we aimed at testing the therapeutic potential of a novel Natriuretic Peptide (NPs), identified from Tunisian snake venom, when administered during reperfusion. Langendorff perfused male Wistar rat hearts were subjected to 30min regional coronary artery occlusion followed by 90min reperfusion. Hearts were treated either with brain natriuretic peptide BNP (10nM) or NPs (200nM). In another set of experiments, hearts were pretreated with either isatin (100μM), a natriuretic peptide receptors blocker or 5HD (10μM), a mitochondrial KATP channels blocker. Post ischemic cardiac haemodynamic parameters, using Labt Chart (ADInstruments) and infarct size (IS), using planimetry, were evaluated. Western blotting experiments for studying cGMP and Reperfusion Injury Salvage Kinases pathways were performed. Calcium Retention Capacity (CRC) to evaluate the mitochondrial function was also assessed by oxygraphy. BNP and NPs significantly decreased the IS by 60% and 62% respectively compared to control non treated hearts (p<0.001). Regarding hemodynamic parameters, both BNP and NPs improved the developed pressure (DP) by 135% and 152% respectively (p<0.05). These beneficial effects are abolished after pretreatment by isatin or 5HD. NPs significantly increased the expression of pAKT, pGSK3ß and PKCεwhen BNP increased pERK1/2 compared to control group. Both BNP and NPs increased the CRC by 124% and 86% respectively compared to control group. Our results demonstrate that NPs and BNP have cardioprotective effects during acute myocardial ischemia. These effects are mediated, for both drugs, by natriuretic receptors and by the activation of mitochondrial KATP channels. The cardioprotection involves the two PI3K/Akt/ERK and cGMP-PKC signaling pathways which converge to the mitochondria. Administration of NPs may provide a novel therapeutic strategy in acute myocardial isch

The secretion patterns and roles of cardiac and circulating arginine vasopressin during the development of heart failure

ObjectiveThe aim of this study is to investigate local cardiac and circulating AVP secretion during heart failure and to determine whether AVP mediates ventricular remodeling. MethodsWe assessed cardiac function and AVP levels of post-myocardial infarction (MI) heart-failure rats 3 weeks (n = 10), 4 weeks (n = 10), 6 weeks (n = 10), 9 weeks (n = 15) after the proximal left anterior descending coronary artery (LAD) ligation. Ten sham-operated rats were used as the control group. In vitro, cardiac microvascular endothelial cells (CMECs) were initiated from isolated Wistar rat hearts and subjected to Ang II to induce AVP expression and secretion. Besides, the effects of AVP stimulation on CMECs and cardiac fibroblasts (CFs) were studied using methylthiazol tetrazolium assay, Western blotting and real-time PCR. ResultsWith cardiac dysfunction, plasma and local cardiac AVP, aldosterone levels increased over time, peaking at 9 weeks post-MI. AVP levels were negatively correlated with serum Na+ and LVEF but positively correlated with LVEDD and myocardial hydroxyproline. In CMECs treated with Ang II, AVP mRNA and protein expression increased. In addition, AVP promoted CFs proliferation and up-regulated the expression of endothelin-1 and connective tissue growth factor. ConclusionCMECs are the cellular sources of elevated local heart AVP stimulated with Ang II/AT1. An intrinsic cardiac AVP system exists. Local cardiac and circulating AVP secretion were enhanced by deteriorating cardiac function. AVP may promote ventricular remodeling. Thus, AVP could be an important mediator of myocardial fibrosis in late-stage heart failure.

Efficacy of mechanical postconditioning following warm, global ischaemia depends on circulating fatty acid levels in an isolated, working rat heart model†.

The number of heart transplantations is limited by donor organ availability. Donation after circulatory determination of death (DCDD) could significantly improve graft availability; however, organs undergo warm ischaemia followed by reperfusion, leading to tissue damage. Laboratory studies suggest that mechanical postconditioning [(MPC); brief, intermittent periods of ischaemia at the onset of reperfusion] can limit reperfusion injury; however, clinical translation has been disappointing. We hypothesized that MPC-induced cardioprotection depends on fatty acid levels at reperfusion. Experiments were performed with an isolated rat heart model of DCDD. Hearts of male Wistar rats (n = 42) underwent working-mode perfusion for 20 min (baseline), 27 min of global ischaemia and 60 min reperfusion with or without MPC (two cycles of 30 s reperfusion/30 s ischaemia) in the presence or absence of high fat [(HF); 1.2 mM palmitate]. Haemodynamic parameters, necrosis factors and oxygen consumption (O2C) were assessed. Recovery rate was calculated as the value at 60 min reperfusion expressed as a percentage of the mean baseline value. The Kruskal-Wallis test was used to provide an overview of differences between experimental groups, and pairwise comparisons were performed to compare specific time points of interest for parameters with significant overall results. Percent recovery of left ventricular (LV) work [developed pressure (DP)-heart rate product] at 60 min reperfusion was higher in hearts reperfused without fat versus with fat (58 ± 8 vs 23 ± 26%, P < 0.01) in the absence of MPC. In the absence of fat, MPC did not affect post-ischaemic haemodynamic recovery. Among the hearts reperfused with HF, two significantly different subgroups emerged according to recovery of LV work: low recovery (LoR) and high recovery (HiR) subgroups. At 60 min reperfusion, recovery was increased with MPC versus no MPC for LV work (79 ± 6 vs 55 ± 7, respectively; P < 0.05) in HiR subgroups and for DP (40 ± 27 vs 4 ± 2%), dP/dtmax (37 ± 24 vs 5 ± 3%) and dP/dtmin (33 ± 21 vs 5 ± 4%; P < 0.01 for all) in LoR subgroups. Effects of MPC depend on energy substrate availability; MPC increased recovery of LV work in the presence, but not in the absence, of HF. Controlled reperfusion may be useful for therapeutic strategies aimed at improving post-ischaemic recovery of cardiac DCDD grafts, and ultimately in increasing donor heart availability.

Effect of Apple (Malus domestica) on Na+/K+-ATPase Activity in Liver, kidney and Heart of Adult Wistar Rats

Effect of Apple (Malus domestica) on Na+/K+-ATPase Activity in Liver, kidney and Heart of Adult Wistar Rats

Zingiber officinale (ginger) extract as a histological dye for muscle fibers and cytoplasm

Background: The rhizome of Zingiber officinale belongs to the family Zingiberaceae, which has a deep yellow color. It is generally called ginger and consumed whole as a medicine or used as a spice in cooking, manufacturing drinks, and tea. Previous studies have shown the importance of this plant in traditional medicine for the treatment of several ailments and even in the dye industry. The later use could be owing to its deep yellowish coloration, which could benefit the histologist and histoscientist when used in an improvised manner.Therefore, this study was carried out to determine the staining potential of crude ethanolic extract of Z.officinale on tissue sections taken from the heart, lungs, spleen, and liver of Wistar rats. Objective: To study Z. officinale (ginger) extract as a histological dye for muscle fibers and cytoplasm. Materials and Methods: Tissue samples were obtained from the heart, spleen, lungs, and liver of adult Wistar rats. The tissues were histologically processed and sectioned with a rotary microtome. Zingiber officinale was obtained from a local store in Owo, Ondo state, Nigeria,and extracted with 90% ethanol, thereby giving rise to a crude extract, which was then used to counterstain the sections earlier stained with alum hematoxylin. The control tissue sections were correspondingly stained with hematoxylin and counterstained with eosin. Result: The ethanolic extract of Z.officinale, when used as a counterstain for alum hematoxylin, stained the muscle fibers and the cytoplasm yellowish, and the nuclei stained deeply green within 4 min. The staining reaction was suspected to be similar to alum hematoxylin and eosin, except for its greenish yellow color. Conclusion: Zingiber officinale is a promising histological natural dye that is not only cheap but also readily available.

Effect of Repeated Dermal Application of Bifenthrin on Oxidative Stress Indices in Heart of Wistar Rats

The present study was aimed to appraise the oxidative stress potential of bifenthrin in heart homogenates in wistar rats after its repeated dermal application for a period of 30 days. The rats were divided into four groups with six rats in each group. Group I and III served as control and were applied with distilled water @ 1.8ml/Kg/day whereas the animals of group II and IV were dermally applied with bifenthrin @ 45mg/Kg/day for 20 and 30 days respectively. Significant increase in MDA level was observed in heart homogenate after 20th and 30th day of dermal treatment. Significant decrease in SOD was observed both after 20th and 30th days of dermal treatment. GSH-Px increased significantly after 20th and decreased significantly after 30th day of treatment. The concentration of GST increased significantly both after 20th and 30th days of dermal application. However, no significant change was observed in CAT activity even after 30th day of dermal treatment.

Isoproterenol-Induced Intramural Cytotoxicity Does Not Correlate with Echocardiographic Functional Abnormalities in Wistar Rat Hearts

Background: Isoproterenol (ISO) is a potent non-selective beta-adrenergic agonist with very low affinity for alpha-adrenergic receptors used for treatment of various cardiac problems including ventricular arrhythmias, asthma and shock. In addition, ISO is also used postoperatively after cardiac surgery. The aim of this study was to analyze the association between histopathological and functional effects of ISO in the rat myocardium. Methods: 24 Wistar rats (healthy, males) were used in these experiments. The animals were randomly divided two groups. Group I (n=12): all animals received ISO subcutaneous administration (0,3 mg/Kg/day for 8 days). Group II (n=12): all animals received Phosphate Buffered Saline (PBS) subcutaneous administration as a control. One day after drug administration has been completed; echocardiographic analysis of cardiac function was performed. Histopathological analysis of the specimens was made by using H&E, Gomori’s Trichrome and picrosirius red staining procedures. The results were analyzed using Student’s t-test (p 0.05), however the heart rate differed significantly between both groups (p=0.0053). Conclusion: Short-term ISO administration (8 days) caused notable intramural cytotoxicity of the heart. In contrast, echocardiographic findings did not show any functional abnormalities in accordance with this cytotoxicity.

The effects of glycine, glutamate and their combination on cardiodynamics, coronary flow and oxidative stress in isolated rat hearts

Objectives: To examine the effects of glycine, glutamate and their combination on cardiac function, coronary flow and oxidative stress in isolated rat hearts, and to examine the effects of potential activation of N-methyl-Daspartate (NMDA) receptors in isolated rat hearts. Methods: The hearts of male Wistar albino rats were excised and perfused according to the Langendorff technique, and cardiodynamic parameters (maximum rate of pressure development in the left ventricle [dp/dt max]; minimum rate of pressure development in the left ventricle [dp/dt min], systolic left ventricular pressure, diastolic left ventricular pressure, heart rate) and coronary flow were determined during the subsequent administration of glycine, glutamate and their combination. Oxidative stress biomarkers, including thiobarbituric acid-reactive substances, nitrites (NO2 −), superoxide anion radical (O2 −) and hydrogen peroxide (H2O2), were each determined spectrophotometrically in coronary venous effluent. Results: Treatment with glycine and glutamate alone did not cause a statistically significant change in any of the observed parameters; however, their combined administration induced significant decreases in dp/dt max, dp/dt min, heart rate and coronary flow compared with the control conditions. Treatment with glycine and glutamate together induced significant increases in NO2 −, O2 − and H2O2 levels. After the washout period, all parameters that had changed (cardiodynamic parameters, coronary flow and levels oxidative stress biomarkers) returned to values that were not significantly different from controls. Conclusions: The results of the present study indicate that NMDA receptors likely have important roles in the function of the heart and coronary circulation. In addition, these results are suggestive of a damaging effect of NMDA receptor overstimulation in heart functioning, potentially mediated by oxidative stress.

Effect of Caffeine Chronically Consumed During Pregnancy on Adenosine A1 and A2A Receptors Signaling in Both Maternal and Fetal Heart from Wistar Rats.

Background: Caffeine is the most widely consumed psychoactive substance in the world, even during pregnancy. Its stimulatory effects are mainly due to antagonism of adenosine actions by blocking adenosine A1 and A2A receptors. Previous studies have shown that caffeine can cross the placenta and therefore modulate these receptors not only in the fetal brain but also in the heart. Methods: In the present work, the effect of caffeine chronically consumed during pregnancy on A1 and A2A receptors in Wistar rat heart, from both mothers and their fetuses, were studied using radioligand binding, Western-blotting, and adenylyl cyclase activity assays, as well as reverse transcription polymerase chain reaction. Results: Caffeine did not significantly alter A1R neither at protein nor at gene expression level in both the maternal and fetal heart. On the contrary, A2AR significantly decreased in the maternal heart, although mRNA was not affected. Gi and Gs proteins were also preserved. Finally, A1R-mediated inhibition of adenylyl cyclase activity did not change in the maternal heart, but A2AR mediated stimulation of this enzymatic activity significantly decreased according to the detected loss of this receptor. Conclusions: Opposite to the downregulation and desensitization of the A1R/AC pathway previously reported in the brain, these results show that this pathway is not affected in rat heart after caffeine exposure during pregnancy. In addition, A2AR is downregulated and desensitized in the maternal heart, suggesting a differential modulation of these receptor-mediated pathways by caffeine.

The anabolic androgenic steroid nandrolone decanoate disrupts redox homeostasis in liver, heart and kidney of male Wistar rats.

The abuse of anabolic androgenic steroids (AAS) may cause side effects in several tissues. Oxidative stress is linked to the pathophysiology of most of these alterations, being involved in fibrosis, cellular proliferation, tumorigenesis, amongst others. Thus, the aim of this study was to determine the impact of supraphysiological doses of nandrolone decanoate (DECA) on the redox balance of liver, heart and kidney. Wistar male rats were treated with intramuscular injections of vehicle or DECA (1 mg.100 g−1 body weight) once a week for 8 weeks. The activity and mRNA levels of NADPH Oxidase (NOX), and the activity of catalase, glutathione peroxidase (GPx) and total superoxide dismutase (SOD), as well as the reduced thiol and carbonyl residue proteins, were measured in liver, heart and kidney. DECA treatment increased NOX activity in heart and liver, but NOX2 mRNA levels were only increased in heart. Liver catalase and SOD activities were decreased in the DECA-treated group, but only catalase activity was decreased in the kidney. No differences were detected in GPx activity. Thiol residues were decreased in the liver and kidney of treated animals in comparison to the control group, while carbonyl residues were increased in the kidney after the treatment. Taken together, our results show that chronically administered DECA is able to disrupt the cellular redox balance, leading to an oxidative stress state.

A rat model of pulmonary infection after cardiac transplant.

Cardiac allograft rejection and infection are leading causes of morbidity and mortality after transplant. The lack of an animal model has hindered related research. We have developed a rat model of pulmonary infection after cardiac transplant to address this issue. Lewis rats received Wistar rat heart allografts, cardiac rejection was induced by cessation of cyclosporine injection, and pulmonary infection was induced by Pseudomonas aeruginosa intrabronchial inoculation. Development of pulmonary infection and/or heart rejection was assessed by histopathology. Histopathologic findings showed that a dose of 2 × 108 colony forming units of Pseudomonas aeruginosa intrabronchial inoculation is sufficient to cause severe pneumonia without being lethal to transplanted animals. Daily administration of 10 mg/kg of cyclosporine reliably suppressed rejection, and withdrawal for 7 days can obtain a consistent International Society for Heart and Lung Transplantation 3R rejection. The current study represents a simple and effective rat model of pulmonary infection along, or in combination, with rejection after heart transplant, which can be used for research of infection-rejection in cardiac transplant settings.

A.actinomycetemcomitans adhesi protein increasing IL-8 titre in heart of wistar rat with aggressive periodontitis (Protein adhesin dari A.actinomycetemcomitans meningkatkantiter IL -8 di dalamjantungtikuswistar denganperiodontitis agresif)

Cardiovascular and periodontal diseases are common inflammatory conditions in the human population. Tlymphocytesparticipateinthepathogenesis and inflammatory events of atherosclerosis. These immune cells enter theinflamed artery wall and join macrophages via a number of interferon-c-inducible chemokines. Chemokines (IL-8),cytokines, and growth factors also participate in this process. The interaction between interleukin- 8 and its receptor,CXCR2, can also contribute to lesion formation in mice. The main causes of aggressive periodontitis isActinobacillusactinomycetemcomitans. Previous studies have proven that adhesin protein with 24 kDa molecular weight from A.actinomycetemcomitans is a specific adhesin, this adhesin proteins play a role in the adhesion process on host. thiskind adhesion in the epithelial attachment would lead to colonization and invasion ofA.actinomycetemcomitans thatwill stimulate the host immune response. This study aimed to analyze the influence of induction 24 kDaA.actinomycetemcomitans adhesin protein to the titre of IL-8 in heart of Wistar rat with aggressive periodontitis usingElisa method to measure and analyze the titre of IL-8. After analyzed with analysis of variance, showed significantdifferences of IL-8 titre in the control group and the group with the induction byA.actinomycetemcomitans,A.actinomycetemcomitans plus 24 kDa A.actinomycetemcomitans adhesin protein, and only with 24 kDaA.actinomycetemcomitans adhesin protein. It can be concluded that A.actinomycetemcomitans adhesin protein with 24kDa molecular weight has a role in increasing of IL-8 titre in heart wistar rat with aggressive periodontitis.

200 Diurnal Variation In Sympathetic Control Of Excitation-contraction Coupling: The Role Of ß3 Adrenoceptors And Nitric Oxide

We have previously shown a time-of-day variation in the response of systolic [Ca2+]i to the non-specific ß-adrenergic (ß-ADR) agonist isoproterenol (ISO), which is linked to a variation in Nitric Oxide...

An Evaluation of Lipid Profile and Antioxidant Activities of &lt;i&gt;Carica Papaya&lt;/i&gt; Seed Oil in the Heart and Liver of Female Wistar Rats.

Increasing attention is been given to Carica papaya seed due to its oil rich nature, and its medicinal value. Four feed diets consisting of 3, 5, 7, and 10 % of the oil were composed and examined for its in-vivo effect on lipid and antioxidants status of female wistar rats compared to those fed with 5 % groundnut oil based diet. The feeds were fed into the rats for 32 days, and examined for their lipid profile and antioxidant status in the liver and heart. Free fatty acids were significantly reduced (P&lt;0.05) by 3-10 % diets in the liver, and by 10 % diet in the heart. HMG-CoA/Mevalonate ratio in the liver was significant increased (P&lt;0.05) by the 7-10 % diets. All diets significantly increased (P&lt;0.05) superoxide dismutase, peroxidase, and reduced glutathione in liver. Lipid peroxidation in the heart was significantly increased (P&lt;0.05) by the 3-7 % diets. Lipid peroxidation, and phospholipid in heart were significantly reduced, and increased (P&lt;0.05) respectively by 10 % diets. The biochemical status of rats fed with the Carica papaya seed oil based diets generally remained comparable to those fed with the groundnut oil diet in the two organs. The Carica papaya seed oil exhibited better antioxidant activities, and health benefits than the commonly consumed groundnut oil.Key words: - Carica papaya; Seeds; Oil, Quality; Antioxidants; Lipids.

Isoflurane induces substrate‐dependent early mitochondrial PTP opening (648.13)

The volatile anesthetic isoflurane (ISO) may increase permeability of mitochondria to protect against mitochondrial injury. Prolonged opening of the mitochondrial permeability transition pore (mPTP) inevitably causes mitochondrial demise, whereas transient mPTP opening may release accumulated Ca2+ from the matrix to avoid subsequent irreversible mPTP opening due to Ca2+ overload. To test if ISO alters mPTP opening, we isolated mitochondria from Wistar rat hearts to assess buffer Ca2+ uptake by Fura 4F. We found that: (a) ISO (1 mM) induced earlier mPTP opening by increasing Ca2+ uptake rate with succinate (SUC). (b) ISO did not alter mPTP opening but reduced Ca2+ uptake with pyruvate/malate. (c) 10 µM ryanodine or 10 nM imperatoxin A with SUC increased Ca2+ uptake and induced earlier mPTP opening; this suggests that ISO’s effects on mPTP may be due to increased conductance via mitochondrial ryanodine receptors (mRyR). We have patch‐clamped single mRyRs in mitoplasts. Our data infer indirectly that SUC substrate ‐dependent transient mPTP opening may be due to enhanced RyR Ca2+ uptake. Transient Ca2+ release could be a mechanism underlying ISO –induced cardiac protection in ischemia‐reperfusion injury.Grant Funding Source: P01‐GM066730, R01‐HL095122, R01‐HL089514

The Effects of Diclofenac and Ibuprofen on Heart Function and Oxidative Stress Markers in the Isolated Rat Heart

ABSTRACT Eicosanoids lead to the promotion of inflammation, cause fever and pain and have many other eff ects. NSAIDs block the action of cyclooxygenase (COX) during the process of converting arachidonic acid into inflammatory mediators, thus reducing the symptoms of inflammation. Investigations focusing on nonselective COX inhibitors, used in high doses, revealed harmful eff ects on myocardial function. Th e aim of our study was to assess the eff ects of two nonselective NSAIDs, diclofenac and ibuprofen, on cardiodynamic parameters, coronary flow and oxidative stress biomarkers in isolated rat hearts. Th e hearts of male Wistar albino rats were excised and retrogradely perfused according to the Langendorff technique at gradually increased coronary perfusion pressures (40-120 cm H2O). Th e experiments were performed under controlled conditions (Krebs-Henseleit physiological solution). Th e hearts were perfused with 10 μmol/l diclofenac and 10 μmol/l ibuprofen. Th e heart function parameters, including the maximum rate of pressure development (dp/dt max), minimum rate of pressure development (dp/dt min), systolic left ventricular pressure (SLVP), diastolic left ventricular pressure (DLVP), mean perfusion pressure (MBP) and heart rate (HR), were continuously registered. Coronary flow (CF) was measured flowmetrically. Oxidative stress markers, including the index of lipid peroxidation measured as TBARS, nitric oxide measured through nitrites (NO2 -), superoxide anion radical (O2 -), and hydrogen peroxide (H2O2) in the coronary venous effluent, were assessed spectrophotometrically. Our results showed that diclofenac aff ected cardiodynamic parameters more significantly than did ibuprofen. Furthermore, the present data indicate that both estimated COX inhibitors do not promote the production of reactive oxygen species.

Cardioprotection by remote ischemic preconditioning of the rat heart is mediated by extracellular vesicles

Remote ischemic preconditioning (RIPC) of the heart is exerted by brief ischemic insults affected on a remote organ or a remote area of the heart before a sustained cardiac ischemia. To date, little is known about the inter-organ transfer mechanisms of cardioprotection by RIPC. Exosomes and microvesicles/microparticles are vesicles of 30–100nm and 100–1000nm in diameter, respectively (collectively termed extracellular vesicles [EVs]). Their content of proteins, mRNAs and microRNAs, renders EV ideal conveyors of inter-organ communication. However, whether EVs are involved in RIPC, is unknown. Therefore, here we investigated whether (1) IPC induces release of EVs from the heart, and (2) EVs are necessary for cardioprotection by RIPC. Hearts of male Wistar rats were isolated and perfused in Langendorff mode. A group of donor hearts was exposed to 3×5-5min global ischemia and reperfusion (IPC) or 30min aerobic perfusion, while coronary perfusates were collected. Coronary perfusates of these hearts were given to another set of recipient isolated hearts. A group of recipient hearts received IPC effluent depleted of EVs by differential ultracentrifugation. Infarct size was determined after 30min global ischemia and 120min reperfusion. The presence or absence of EVs in perfusates was confirmed by dynamic light scattering, the EV marker HSP60 Western blot, and electron microscopy. IPC markedly increased EV release from the heart as assessed by HSP60. Administration of coronary perfusate from IPC donor hearts attenuated infarct size in non-preconditioned recipient hearts (12.9±1.6% vs. 25.0±2.7%), similarly to cardioprotection afforded by IPC (7.3±2.7% vs. 22.1±2.9%) on the donor hearts. Perfusates of IPC hearts depleted of EVs failed to exert cardioprotection in recipient hearts (22.0±2.3%). This is the first demonstration that EVs released from the heart after IPC are necessary for cardioprotection by RIPC, evidencing the importance of vesicular transfer mechanisms in remote cardioprotection.

Electrical Propagation of Three-Dimensional Engineered Hearts using Decellularized Extracellular Matrix

[Background] Construction of three-dimensional cardiac tissues using decellularized extracellular matrix could be a new technique to create an “organ-like” structure of the heart. To engineer functional artificial hearts comparable for orthotopic hearts, however, much remain to be solved including electrical stability for efficient contraction. To elucidate the points, we examined electrophysiological properties of recellularized heart tissues. [Methods] Entire hearts of adult Wistar rats were decellularized using 0.5% SDS and 1% triton-X, and then recellularized with enzymatically-dispersed neonatal rat cardiac cells (1x10⊥8 cells) through antegrade coronary circulation. Three and seven days later after cell seeding, we observed excitation of spontaneous and pacing-induced beatings of recellularized heart tissues expressing Ca2+-indicating protein (GCaMP2) using high resolution cameras. We also conducted immunofluorescence staining to examine morphological aspects of engineered tissues. [Results] Live tissue fluorescence imaging revealed that GFP-labeled-isolated cardiac cells were dispersed into interstitial spaces through extravasation from coronary arteries. Engineered hearts seeded with GCaMP2-expressing cardiac cells started showing spontaneous beating, and were subjected to further electrophysiological experiments using optical imaging system. Both in spontaneous and pacing-induced beating hearts, we observed well-organized conduction of stable excitation in substantial areas of the engineered heart tissues, whereas we also recorded disorganized propagation of asynchronous excitation with multiple origins. Conduction velocity was markedly decreased (∼5 cm/sec) during early stages (up to nine days) after recellularization. Immunofluorescence study revealed randomly-mixed alignment of cardiomyocytes, endothelial cells and smooth muscle cells, stained with alpha-actinin, CD31 and sm-actin, respectively. Recellularized heart tissues also showed disarray of cardiomyocytes and markedly decreased expression of connexin43. [Conclusion] Three-dimensional engineered hearts showed electrical heterogeneity and proarrhythmic propensity during early stages of culture. These findings may be due to, at least in part, disorganized alignment of microstructure and immature formation of gap junction.