Heterotopic Rat Heart Transplantation Research Articles

Abstract 12968: Atorvastatin for the Treatment of Endocardial Fibroelastosis Formation in a Preclinical Animal Model

Background: Endocardial fibroelastosis (EFE) frequently associated with development of HLHS or critical AS, restricting growth of the left ventricle (LV). The mechanism of EFE formation is the transition from endocardial endothelial cells to mesenchymal cells (EndMT). Regulation of EndMT in cardiac fibrosis is mainly through the TGF-β pathway, and upregulated Krüppel-like factor 2 (KLF2) inhibits the TGF-ß pathway by blocking SMAD2/3. Besides their plasma lipid-lowering effect, statins (e.g. Atorvastatin) directly upregulate KLF2 and are thereby atheroprotective (i.e., inhibit EndMT). The aim was to evaluate whether the upregulation of KLF2 with Atorvastatin inhibits EFE formation. Methods: Heterotopic rat heart transplantation was performed using 2-4 day-old donor hearts into adolescent recipients, which induces LV EFE growth. Atorvastatin (n=7) was administered daily at 3 mg/kg intraperitoneally for two weeks and compared to untreated/vehicle (n=5) controls. At postoperative day 14, LV EFE thickness was determined on Masson’s trichrome-stained slides. Gene expression of KLF2 was analyzed by qRT-PCR and protein expression by Westernblot and quantified via densitometry for SMAD2/3 and collagen. Results: Mean LV EFE thickness was significantly decreased in the Atorvastatin group (22.3±2.5μm) compared to 176.8±22.6μm in the untreated/vehicle-treated group (p<0.0001, A ). KLF2 was significantly upregulated in Atorvastatin treated hearts (p=0.0173, B ). SMAD2/3 and collagen were significantly elevated in the control group, p=0.0014 and p=0.048, respectively ( C, D ). Conclusion: LV growth restriction through EFE formation precludes patients from biventricular repair. Until now, surgical resection has been the only available therapeutic option. With this study, we show that Atorvastatin impedes EFE formation within the LV cavity in neonatal rat hearts through the upregulation of KLF2. Atorvastatin is, therefore a promising therapeutic agent.

Modified Heterotopic Abdominal Heart Transplantation and a Novel Aortic Regurgitation Model in Rats.

Over the past 50 years, many researchers have reported heterotopic abdominal heart transplantation in mice and rats, with some variations in the surgical technique. Modifying the transplantation procedure to strengthen the myocardial protection could prolong the ischemia time while preserving the donor's cardiac function. This technique's key points are as follows: transecting the donor's abdominal aorta before harvesting to unload the donor's heart; perfusing the donor's coronary arteries with a cold cardioplegic solution; and topical cooling of the donor's heart during the anastomosis procedure. Consequently, since this procedure prolongs the acceptable ischemia time, beginners can easily perform it and achieve a high success rate. Moreover, a new aortic regurgitation (AR) model was established in this work using a technique different from the existing one,which is created by inserting a catheter from the right carotid artery and puncturing the native aortic valve under continuous echocardiographic guidance. A heterotopic abdominal heart transplantation was performed using the novel AR model. In the protocol, after the donor's heart is harvested, a stiff guidewire is inserted into the donor's brachiocephalic artery and advanced toward the aortic root. The aortic valve is punctured by pushing the guidewire further even after the resistance is felt, thus inducing AR. It is easier to damage the aortic valve using this method than with the procedure described in the conventional AR model. Additionally, this novel AR model does not contribute to the recipient's circulation; therefore, this method is expected to produce a more severe AR model than the conventional procedure.

A Heterotopic Rat Heart Transplantation Model using Circulatory Death Donor Hearts.

The objective of this protocol is to set up a rat heterotopic heart transplantation model with donation after circulatory death (DCD) donor hearts. There are two setups for this protocol: heart donor setup and recipient setup. In the heart donor setup, Sprague Dawley rats are anesthetized, endotracheally intubated, and ventilated. The right carotid artery is cannulated to deliver heparin and the paralytic agent vecuronium-bromide. The DCD process is initiated by terminating the ventilation. After 20 min, the heart is exposed and the aorta distal to the brachiocephalic branch is clamped. At 25 min from terminating the ventilator, ice-cold University of Wisconsin (UW) solution is perfused through the carotid catheter to flush the heart. The heart is procured by dividing the aorta, pulmonary artery, venae cavae, and pulmonary veins and stored in UW solution for implantation. In the recipient setup, the Lewis rat is anesthetized with isoflurane. Slow-release buprenorphine is administered subcutaneously to facilitate a smooth postoperative recovery. Through a midline abdominal incision, the infra-renal aorta and the inferior vena cava are isolated and clamped with an atraumatic vascular clamp. The donor heart aorta and pulmonary artery are sutured to the recipient abdominal aorta and vena cava, respectively, with a running 8-0 Prolene. The vascular clamp is removed to reperfuse the heart. The abdominal wall is closed and the rat is recovered. After a set interval (24 h to 2 weeks), the recipient rat is anesthetized, the transplanted heart is exposed, and a balloon-tip-catheter is inserted into the left ventricle via the apex to record developed pressure and dP/dt using a data acquisition system. The heart tissue is collected for histology, immunology, or molecular analysis. A successful DCD donor rat heart transplantation model will allow further studies on the cardioprotective approaches to improve heart transplantation outcomes from DCD donors.

Spatial Transcriptomic Analysis of Acute Heart Rejection Model

<h3>Purpose</h3> Heart transplantation (HTx) is sometimes the only treatment for end-stage cardiac disease, but survival after HTx is limited. Understanding the molecular mechanisms behind allograft injury, dysfunction, and failure, are critical in improving the patients' outcome. Rapidly evolving single cell and spatial transcriptomic technologies have opened a new field of precision to diagnostics and therapeutics of cardiovascular diseases. Here we studied the cellular mechanisms of acute rejection in a rat heart transplant model with spatial transcriptomics methods. <h3>Methods</h3> We investigated the effects of acute rejection in heterotopic rat heart transplantation model with syngeneic (DA-DA) and major MHC-mismatched allogeneic (DA-WF) recipients. The recipient rats were sacrificed 5 days after transplantation, and graft cross-sections were analyzed with the Visium Spatial Gene Expression platform (10xGenomics). Raw sequencing reads were processed using SpaceRanger. All main data analysis steps were performed using Seurat. Clusters were identified based on the expression of several known cell markers. <h3>Results</h3> From all samples together, a total of 17 clusters of spots were identified. Our analysis suggests that two of the clusters presented mainly cardiomyocytes, one cluster of smooth muscle cells, two clusters of fibroblasts, 11 immune cells and one cluster of erythrocytes. Endothelial cells did not form a unique cluster. Syngeneic HTx alone induced marked transcriptomic changes in 5 clusters based on differential gene expression analysis, mainly increasing the proportion of clusters with certain types of macrophages. Few clusters remained unchanged between the allogenic and the syngeneic groups. Allogenic group had four unique clusters representing mainly different immune cell populations: granulocytes, lymphocytes and a type of macrophages that were not present in other samples. One cluster consisted of erythrocytes from hemorrhagic area. <h3>Conclusion</h3> Acute rejection changes the cellular composition of the heart muscle dramatically and induces transcriptomic changes in the structural cells of the heart. Our spatial transcriptomic analysis provides novel transcriptomic data on cellular changes occurring following acute rejection. This method allows us to study the cells and mechanisms involved in rejection and look for potential targets for drug development and therapeutics.

Immutol regulates CD4+Tregs, CD8+Tregs and pDCs via IDO signaling pathway to induce immune tolerance in rat heart allograft transplant

Indoleamine 2,3-dioxygenase (IDO) can promote tryptophan metabolism to kynurenine and modulate regulatory T cells (Tregs), thereby maintains lower efficiency to induce tolerance. Our aim is to investigate the mechanism of tolerance induction by a IDO metabolite named Immutol. MethodsWe established rat heterotopic heart transplantation models and treated them with Immutol, cyclosporine A (CsA) and 1-methyl-DL-tryptophan (1-MT) in vivo. The drugs were administered via gavage to all but the control group one day before surgery. CsA was gavaged continually for 20 days and Immutol for 60 days; after withdrawal of the drugs, the recipients were observed for at least 10 months. Immune cells were analyzed by flow cytometry. The IDO signaling pathway was evaluated by Western blotting, RT-PCR and immunochemical staining. Enzyme-linked immunosorbent assays (ELISAs) were used to detect changes in cytokines. ResultsCsA or Immutol alone prolonged survival but did not induce tolerance after withdrawal. Immutol+CsA inhibited acute rejection, and the grafts survived more than 400 d, with tolerance detected in most rats (13/15). Increased protein IDO and kynurenine could regulate the accumulation of CD4+Tregs, CD8+Tregs and pDC to induce immune tolerance. I-MT specifically blocked IDO, weakened the expression of IDO and kynurenine, and produced grafts rejection. Additionally, Tregs could down-regulate immune responses through production of the immunosuppressive cytokines IL-10 and TGF-beta, thus induce immune tolerance. CD8+ Tregs produce IFN-γ, and tolerance is dependent on both IFN-γ and IDO. ConclusionImmutol combined with CsA can control acute rejection and induce tolerance in rats with cardiac allografts after withdrawal. Immutol may become a novel drug for future clinical use.

Effects of carvedilol and metoprolol on the myocardium during mechanical unloading in a rat heterotopic heart transplantation model.

Left ventricular assist devices enable recovery from severe heart failure and serve as a bridge to heart transplantation. However, chronic mechanical unloading can impair myocardial recovery. We aimed to assess myocyte size, fibrosis, apoptosis, and β-adrenoreceptor levels after rats with left ventricle unloading induced by heterotopic heart transplantation were administered carvedilol and metoprolol. Thirty rats with heart transplants were divided randomly into control, carvedilol treatment, and metoprolol treatment groups. Follow-up was conducted after 2 and 4 weeks of unloading. Carvedilol and metoprolol treatments did not prevent the decrease in myocyte diameter in unloaded left ventricles. Metoprolol significantly decreased the ratio of the fibrotic area in the unloaded heart, measured using Masson's trichrome staining after 2 weeks. However, carvedilol and metoprolol did not reduce apoptosis, based on measurements of terminal deoxynucleotidyl-transferase-mediated dUTP nick end-labelling positive cells and the expression of caspase-3 in unloaded hearts after 2 and 4 weeks. Metoprolol treatment did not significantly decrease the mRNA expression of myocardial SERCA2a in the unloaded heart after 2 weeks. Compared to carvedilol treatment, metoprolol treatment improved myocardial fibrosis and SERCA2a expression to a greater extent; however, neither drug prevented myocardial apoptosis.

Use of peroxiredoxin for preconditioning of heterotopic heart transplantation in a rat

Peroxiredoxin 6 (Prdx6) is an antioxidant enzyme in the human body that performs a number of important functions in the cell. Prdx6 restores a wide range of peroxide substrates, thus playing a leading role in maintaining redox homeostasis in mammalian cells. In addition to peroxidase activity, Prdx6 has an activity of phospholipase A2, thus taking part in membrane phospholipid metabolism. Due to its peroxidase and phospholipase activity, Prdx6 participates in intracellular and intercellular signal transmission, thereby facilitating the initiation of regenerative processes in the cell, suppression of apoptosis and activation of cell proliferation. Given the functions performed, Prdx6 can effectively deal with oxidative stress caused by various factors, including ischemia-reperfusion injury. On an animal model of rat heterotopic heart transplantation, we showed the cardioprotective potential of exogenous recombinant Prdx6, introduced before transplantation and subsequent reperfusion injury of the heart. It has been demonstrated that exogenous Prdx6 effectively alleviates the severity of ischemia-reperfusion injury of the heart by 2–3 times, providing normalization of its structural and functional state during heterotopic transplantation. The use of recombinant Prdx6 can be an effective approach in preventing/alleviating ischemia-reperfusion injury of the heart, as well as in maintaining an isolated heart during transplantation.

Novel delivery of cellular therapy to reduce ischemia reperfusion injury in kidney transplantation.

Ex vivo normothermic machine perfusion (NMP) of donor kidneys prior to transplantation provides a platform for direct delivery of cellular therapeutics to optimize organ quality prior to transplantation. Multipotent Adult Progenitor Cells (MAPC® ) possess potent immunomodulatory properties that could minimize ischemia reperfusion injury. We investigated the potential capability of MAPC cells in kidney NMP. Pairs (5) of human kidneys, from the same donor, were simultaneously perfused for 7 hours. Kidneys were randomly allocated to receive MAPC treatment or control. Serial samples of perfusate, urine, and tissue biopsies were taken for comparison. MAPC-treated kidneys demonstrated improved urine output (P=.009), decreased expression of injury biomarker NGAL (P=.012), improved microvascular perfusion on contrast-enhanced ultrasound (cortex P=.019, medulla P=.001), downregulation of interleukin (IL)-1β (P=.050), and upregulation of IL-10 (P<.047) and Indolamine-2, 3-dioxygenase (P=.050). A chemotaxis model demonstrated decreased neutrophil recruitment when stimulated with perfusate from MAPC-treated kidneys (P<.001). Immunofluorescence revealed prelabeled MAPC cells in the perivascular space of kidneys during NMP. We report the first successful delivery of cellular therapy to a human kidney during NMP. Kidneys treated with MAPC cells demonstrate improvement in clinically relevant parameters and injury biomarkers. This novel method of cell therapy delivery provides an exciting opportunity to recondition organs prior to transplantation.

An Immunological Model for Heterotopic Heart and Cardiac Muscle Cell Transplantation in Rats

Heterotopic heart transplantation in rats has been a commonly used model for diverse immunological studies for more than 50 years. Several modifications have been reported since the first description in 1964. After 30 years of performing heterotopic heart transplantation in rats, we have developed a simplified surgical approach, which can be easily taught and performed without further surgical training or background. After dissection of the ascending aorta and the pulmonary artery and ligation of superior and inferior caval and pulmonary veins, the donor heart is harvested and subsequently perfused with ice-cold saline solution supplemented with heparin. After clamping and incising the recipient abdominal vessels, the donor ascending aorta and pulmonary artery are anastomosed to the recipient abdominal aorta and inferior vena cava, respectively, using continuous running sutures. Depending on different donor-recipient combinations, this model allows analyses of either acute or chronic rejection of allografts. The immunological significance of this model is further enhanced by a novel approach of in-ear injection of vital cardiac muscle cells and subsequent analysis of draining cervical lymphatic tissue.

An Immunological Model for Heterotopic Heart and Cardiac Muscle Cell Transplantation in Rats

An Immunological Model for Heterotopic Heart and Cardiac Muscle Cell Transplantation in Rats

Delivery of FK506-loaded PLGA nanoparticles prolongs cardiac allograft survival.

In this study, FK506-loaded poly(lactide-co-glycolide) nanoparticles (PLGA-FK506-NPs) were developed using an O/W emulsion solvent evaporation method. The PLGA-FK506-NPs were observed to be monodispersed and spherical by transmission and scanning electron microscopy. The mean size and zeta potential measured by dynamic light scattering were 110±1.3nm and -20.56±3.65mV, respectively. The FK506 entrapment and loading efficiency were 94.46±1.88% and 5.38±0.24%, respectively. Moreover, a pharmacokinetics study revealed that the PLGA-FK506-NPs behaved significantly different than free FK506 by exhibiting a higher area under curve (1.69-fold), higher mean residence time (1.29-fold), slower clearance and longer elimination half-life. Notably, the concentrations of FK506 in the spleen and mesenteric lymph nodes of the PLGA-FK506-NP group were 3.1-fold and 2.9-fold higher than those of the free FK506 group. Furthermore, the immunosuppressive efficacy was evaluated in a rat heterotopic heart transplantation model, and the results showed that PLGA-FK506-NP treatment could successfully alleviate acute rejection and prolong allograft survival compared with the free FK506 treatment (mean survival time, 17.1±2.0 versus 13.3±1.7days). In conclusion, PLGA-FK506-NPs are a promising formulation for spleen and lymph node delivery and have potential use in the treatment of cardiac allograft acute rejection.

The method of heterotopic rat heart transplantation for investigation the cardioprotective efficacy of cardioplegic solutions

This article describes in detail the modified method of heterotopic heart transplantation in rats. The methods of animal anesthesia, surgical features of the removal of a donor heart and its implantation to a recipient rat are described in detail. The advantages and disadvantages of donor heart preservation methods are described, as well as methods for evaluating a donor heart preparation, the quality of preservation and the effectiveness of cardioprotection. Experimental approaches to the study of the effectiveness of cardioplegic and preservative solutions using this technique are proposed. The data of our own pilot study on a comparative analysis of the effectiveness of cardioprotection using preservative solutions using this experimental model are presented.

P6345Chronic cardiac allograft rejection after heterotopic rat heart transplantation: ed-a but not ed-b domain containing fibronectin as novel serum biomarker of cardiac allograft vasculopathy

Abstract Background and aims Diagnosis of Cardiac Allograft Vasculopathy (CAV) following heart transplantation (HTX) is still challenging but of prognostic relevance. Thus, the identification of biomarkers non-invasively detecting the disease is an hitherto unmet clinical need. Fetal variants of the cell adhesion modulating protein Fibronectin (Fn) could be proven to re-occur in the context of chronic rejection in rat and human cardiac tissue after HTX in spatial association to CAV and fibrosis. This study was aimed to determine serum levels of fetal Fn variants in a heterotopic rat HTX model of chronic rejection. Methods Chronic cardiac allograft rejection was induced by heterotopic rat HTX using inbred male Lewis (LEW) rats as donors and Fisher (F344) rats as recipients (n=24). After microsurgical modifications, the allograft was implanted into the abdomen of the recipient animal. Rats were sacrificed after 70 days. Untreated F344 rats were used as controls (n=6). Cardiac tissue was subjected to histological analysis according to a recently established sum-score system between 0 and 10 (0 = no detectable rejection; 1 to 3 = weak rejection; 4 to 7 = moderate rejection; and 8 to 10 = severe rejection). The score includes all relevant parameters of chronic rejection (CAV, lymphocyte infiltration, cardiomyocyte degeneration, fibrosis). Brain natriuretic peptide (BNP) was determined by a commercially available ELISA. Serum levels of both, ED-A domain containing Fn (ED-A+ Fn) and ED-B domain containing Fn (ED-B+ Fn) were quantified using ELISA protocols established in our group. Results Final experiments were performed on day 70, mortality rate was 0%. Histological analysis revealed a chronic cardiac allograft rejection in all transplanted animals (mean sum-score 6.92±1.94) while controls did not exhibit relevant alterations (mean sum-score 0.1±0.2; p&lt;0.001). There were significantly elevated BNP levels in HTX rats (76.16±23.22 pg/ml) compared to controls (352.23±132.09 pg/ml; p&lt;0.001). BNP showed a strong positive correlation to the histological sum-score (r=0.653; p&lt;0.001). Serum levels of ED-A+ Fn (HTX: 34.42±11.4 μg/ml, controls: 17.3±6.13 μg/ml; p=0.009) but not ED-B+ Fn (HTX: 8.58±4.12 μg/ml, controls: 9.4±2.61 μg/ml; p=0.804) were significantly increased in HTX rats qualifying ED-A+ Fn for further analysis with respect to histological signs of chronic rejection. Interestingly, there is a noticeable association to the occurrence of CAV representing 1 out of 4 parameters included in the histological sum-score (r=0.51; p=0.006). Conclusions Chronic cardiac allograft rejection and, in particular, CAV as major long-term prognosis-limiting factor after HTX are accompanied by both, a re-expression and vessel associated tissue deposition of ED-A+ Fn and also a relevant liberation into blood. These results underline the potential impact of ED-A+ Fn as biomarker or potential therapeutic target patients that underwent HTX.

Recipient natural killer cells alter the course of rejection of allogeneic heart grafts in rats.

Rejection of solid organ grafts is regarded to be dependent on T cell responses. Nonetheless, numerous studies have focused on the contribution of NK cells in this process, resulting in contradictory theories. While some conclude that there is no participation of NK cells, others found an inflammatory or regulative role of NK cells. However, the experimental settings are rarely comparable with regard to challenged species, strain combinations or the nature of the graft. Thus, clear definition of NK cell contribution might be impeded by these circumstances. In this study we performed heterotopic heart transplantation (HTx) in rats, choosing one donor-recipient-combination leading to a fast and a second leading to a prolonged course of graft rejection. We intervened in the rejection process, by depletion of recipient NK cells on the one hand and by injection of activated NK cells syngeneic to the recipients on the other. The fast course of rejection could not be influenced by any of the NK cell manipulative treatments. However, the more prolonged course of rejection was highly susceptible to depletion of NK cells, resulting in significant acceleration of rejection, while injection of NK cells induced acceptance of the grafts. We suggest that, depending on the specific setting, NK cells can attenuate the first trigger of immune response, which allows establishing the regulatory activity leading to tolerance of the graft.

ASP1126, a Novel Sphingosine-1-Phosphate–Selective Agonist With a Favorable Safety Profile, Prolongs Allograft Survival in Rats and Nonhuman Primates in Combination With Tacrolimus With a Broad Safety Margin for Bradycardia

Sphingosine-1-phosphate (S1P) is a biologically active sphingolipid that acts through the members of a family of 5 G protein–coupled receptors (S1P1 to S1P5). Among these, S1P1 is a major regulator of lymphocyte trafficking. Fingolimod, whose active metabolite, fingolimod phosphate, acts as a nonselective S1P-receptor agonist, exerts its immunomodulatory effect, at least in part, by regulating lymphocyte trafficking via downregulation of S1P1 expression on lymphocytes. Here, we describe the pharmacologic profile of a novel S1P1 agonist, ASP1126. ASP1126 preferentially activated S1P1 compared to S1P3 in rat and human guanosine-5'-(γ-thio)-triphosphate (GTPγS) assays. Oral single administration of ASP1126 decreased the number of peripheral lymphocytes and repeated dosing showed a cumulative effect on lymphopenia in both rats and monkeys. ASP1126 prolonged allograft survival in a rat heterotopic heart transplantation model in combination with a subtherapeutic dose of tacrolimus that was independent of drug-drug interactions. In addition, in nonhuman primate (NHP) renal transplantation, pretreatment with ASP1126 reduced not only the number of naive T cells and central memory T cells but also effector memory T cells in the peripheral blood, all of which could contribute to acute graft rejection and prolonged allograft survival in combination with tacrolimus. Further, we confirmed that ASP1126 has a broad ranging safety margin with respect to its effect on lung weight in rats and bradycardia in NHPs, which were the adverse events found in clinical studies of fingolimod. ASP1126 with improved safety profile has the potential to be an adjunct therapy in combination with tacrolimus in clinical transplantation.

Comparison of hemodynamic and infarct-limiting effects of preservation solution based on Krebs–Henseleit buffer and HTK solution in the rat model of heterotopic heart transplantation

Objective. To compare the cardioprotective effcacy of a preservation solution based on Krebs–Henseleit buffer and HTK solution in the model of heterotopic heart transplantation in rat.Design and methods. A study was conducted on 12 Wistar rats. The animals were divided into groups depending on the preservation solution used: 1) Krebs–Henseleit buffer-based solution (n = 7), 2) HTK (n = 5). Each experiment consisted of collecting donor heart, preserving it with an appropriate cardioplegic solution, heterotopic transplantation into a recipient rat followed by explantation and evaluation of left ventricular contractility using the Langendorff model and histochemical assessment of the irreversible myocardial damage. Coronary blood flow in the donor heart was assessed in vivo using ultrasound doppler flowmetry. After 3 hours, the donor heart was explanted and connected to the Langendorff apparatus to assess left ventricular contractility, and the myocardium was subjected to histochemical staining with 1 % triphenyltetrazolium chloride for the assessment of the irreversible myocardial damage.Results. In the group of Krebs–Henseleit buffer-based cardioplegic solution, 7 experiments were performed. Myocardial infarct size was 3,5 ± 1,2%, the coronary flow rate was 4,5 ± 1,3 ml/min, and the developed left ventricular pressure of the donor heart was 70 ± 6,3 mmHg at diastolic left ventricular pressure of 10 mmHg. In the HTK solution group (n = 5), in all of the experiments after the start of blood flow, the transplanted heart did not begin to contract, and all 5 hearts remained in an asystole state. Therefore, after the end of the 3-hour reperfusion period, the assessment of the contractility of the left ventricle using the Langendorff apparatus was not carried out. Coronary flow rate was only 0,4 ± 0,1 ml/min, probably resulting from inadequate cardioprotection with HTK solution. Due to the lack of adequate reperfusion, the accurate assessment of the irreversible myocardial damage was impossible in the HTK solution group.Conclusions. The technique of heterotopic heart transplantation in rats is adequate and informative for the study of ischemia-reperfusion myocardial damage as well as for the study of the effectiveness of cardioplegic and cardiac preservation solutions. Cardioplegic solution based on Krebs– Henseleit buffer demonstrated greater cardioprotective effectiveness in our model compared to HTK solution.

Sphingosine-1-Phosphate Receptor Agonist Fingolimod Reduces Myocardial Ischemia-Reperfusion Injury and Apoptosis Increasing Long-Term Left Ventricular Function after Heart Transplantation

Purpose Fingolimod, a sphingosine-1-phosphate receptor agonist, is approved for the treatment of multiple sclerosis and exerts antiapoptotic properties. We hypothesized that sphingosine-1-phosphate receptor activation with fingolimod in the donor hearts could protect against ischemia/reperfusion injury and preserve myocardial function in a heterotopic rat heart transplantation model. Methods Before explantation, donor Wistar rats received finfolimod (1 mg/kg iv, n= 40) or saline (control, n=40). The hearts were excised, stored in cold preservation solution for 1 hour and heterotopically transplanted. Left ventricular function of the graft and myocardial markers of apoptosis, nitro-oxidative stress, inflammation and fibrosis have been assessed after 24 hour (early phase) and 1 month (late phase) from transplantation. Results Fingolimod treatment activated the cardioprotective reperfusion injury salvage kinase Akt and extracellular receptor kinase 1/2 in the early phase after transplantation, leading to decreased cardiomyocyte apoptosis (TUNEL) and reduced myocardial nitro-oxidative stress (malondialdehyde, nitrotyrosine) and inflammation (CCL5/RANTES). In the late phase fingolimod decreased interstitial fibrosis and improved LV systolic and diastolic functions (pressure-volume analysis). Conclusion Sphingosine-1-phosphate receptor activation with fingolimod before heart transplantation activates anti-apoptotic and anti-inflammatory pathway reducing graft ischemia-reperfusion injury. This produces a reduced myocardial fibrosis and improved left ventricular function in a chronic phase. Fingolimod might represent a useful tool for cardioprotection in the clinical setting of heart transplantation surgery soon. Fingolimod, a sphingosine-1-phosphate receptor agonist, is approved for the treatment of multiple sclerosis and exerts antiapoptotic properties. We hypothesized that sphingosine-1-phosphate receptor activation with fingolimod in the donor hearts could protect against ischemia/reperfusion injury and preserve myocardial function in a heterotopic rat heart transplantation model. Before explantation, donor Wistar rats received finfolimod (1 mg/kg iv, n= 40) or saline (control, n=40). The hearts were excised, stored in cold preservation solution for 1 hour and heterotopically transplanted. Left ventricular function of the graft and myocardial markers of apoptosis, nitro-oxidative stress, inflammation and fibrosis have been assessed after 24 hour (early phase) and 1 month (late phase) from transplantation. Fingolimod treatment activated the cardioprotective reperfusion injury salvage kinase Akt and extracellular receptor kinase 1/2 in the early phase after transplantation, leading to decreased cardiomyocyte apoptosis (TUNEL) and reduced myocardial nitro-oxidative stress (malondialdehyde, nitrotyrosine) and inflammation (CCL5/RANTES). In the late phase fingolimod decreased interstitial fibrosis and improved LV systolic and diastolic functions (pressure-volume analysis). Sphingosine-1-phosphate receptor activation with fingolimod before heart transplantation activates anti-apoptotic and anti-inflammatory pathway reducing graft ischemia-reperfusion injury. This produces a reduced myocardial fibrosis and improved left ventricular function in a chronic phase. Fingolimod might represent a useful tool for cardioprotection in the clinical setting of heart transplantation surgery soon.

Sphingosine 1-Phosphate Receptor Modulator Fingolimod (FTY720) Attenuates Myocardial Fibrosis in Post-heterotopic Heart Transplantation.

Background and Objective: Sphingosine 1-phosphate (S1P), and S1P receptor modulator fingolimod have been suggested to play important cardioprotective role in animal models of myocardial ischemia/reperfusion injuries. To understand the cardioprotective function of S1P and its mechanism in vivo, we analyzed apoptotic, inflammatory biomarkers, and myocardial fibrosis in an in vivo heterotopic rat heart transplantation model.Methods: Heterotopic heart transplantation is performed in 60 Sprague–Dawley (SD) rats (350–400 g). The heart transplant recipients (n = 60) are categorized into Group A (control) and Group B (fingolimod treated 1 mg/kg intravenous). At baseline with 24 h after heart transplantation, blood and myocardial tissue are collected for analysis of myocardial biomarkers, apoptosis, inflammatory markers, oxidative stress, and phosphorylation of Akt/Erk/STAT-3 signaling pathways. Myocardial fibrosis was investigated using Masson’s trichrome staining and L-hydroxyline.Results: Fingolimod treatment activates both Reperfusion Injury Salvage Kinase (RISK) and Survivor Activating Factor Enhancement (SAFE) pathways as evident from activation of anti-apoptotic and anti-inflammatory pathways. Fingolimod treatment caused a reduction in myocardial oxidative stress and hence cardiomyocyte apoptosis resulting in a decrease in myocardial reperfusion injury. Moreover, a significant (p < 0.001) reduction in collagen staining and hydroxyproline content was observed in fingolimod treated animals 30 days after transplantation demonstrating a reduction in cardiac fibrosis.Conclusion: S1P receptor activation with fingolimod activates anti-apoptotic and anti-inflammatory pathways, leading to improved myocardial salvage causing a reduction in cardiac fibrosis.

Erythroblast transformation-specific 2 correlates with vascular smooth muscle cell apoptosis in rat heterotopic heart transplantation model.

Cardiac allograft vasculopathy (CAV) decreases the long-term survival of heart transplantation recipients. Vascular smooth muscle cell (VSMC) apoptosis is an important pathological feature of CAV. Erythroblast transformation-specific 2 (Ets-2), as a transcription factor, participates in cell apoptosis and plays an important role in organ transplantation. Hearts from Wistar-Furth (WF:RT1u) rats were heterotopically transplanted into Lewis (Lew:RT1(l)) rats without immunosuppression. Additional syngeneic heterotopic cardiac transplantations were performed in Lewis rats. HE staining was used to identify CAV. Ets-2 expression was examined by western blot. Ets-2 tissue location was examined by immunohistochemical assay and double immunostaining. Cleaved caspase 3 expression was detected by western blot. Co-localization of Ets-2 and cleaved caspase 3 was detected by double immunostaining. Ets-2, p53, cleaved caspase 3 and Bcl-xl expression in rat VSMC line A7R5 was examined after Ets-2 siRNA transfection. TUNEL assay was applied to detect A7R5 apoptosis with or without ETS-2 siRNA transfection. Immunoprecipitation was performed to explore the interaction between Ets-2 and p53. Ets-2 expression decreased in the allograft group but had no obvious change in the isograft group. Meanwhile, the phenomenon of CAV was observed in the allograft group and there is neointima formation in the isograft group which is not obvious compared with allograft group. Additionally, Ets-2 expression was opposite to VSMC apoptosis in the allograft group. In vitro, Ets-2 siRNA transfection in A7R5cells resulted in enhanced cell apoptosis. Finally, Ets-2 interacted with p53. Ets-2 might inhibit VSMC apoptosis via p53 pathway. The results further elucidate the molecular mechanism of VSMC apoptosis after heart transplantation during CAV and provide theoretical basis for seeking new specific drug targets for CAV prevention and treatment.

A New Animal Model for Investigation of Mechanical Unloading in Hypertrophic and Failing Hearts: Combination of Transverse Aortic Constriction and Heterotopic Heart Transplantation

Objectives Previous small animal models for simulation of mechanical unloading are solely performed in healthy or infarcted hearts, not representing the pathophysiology of hypertrophic and dilated hearts emerging in heart failure patients. In this article, we present a new and economic small animal model to investigate mechanical unloading in hypertrophic and failing hearts: the combination of transverse aortic constriction (TAC) and heterotopic heart transplantation (hHTx) in rats. Methods To induce cardiac hypertrophy and failure in rat hearts, three-week old rats underwent TAC procedure. Three and six weeks after TAC, hHTx with hypertrophic and failing hearts in Lewis rats was performed to induce mechanical unloading. After 14 days of mechanical unloading animals were euthanatized and grafts were explanted for further investigations. Results 50 TAC procedures were performed with a survival of 92% (46/50). When compared to healthy rats left ventricular surface decreased to 5.8±1.0 mm² (vs. 9.6± 2.4 mm²) (p = 0.001) after three weeks with a fractional shortening (FS) of 23.7± 4.3% vs. 28.2± 1.5% (p = 0.01). Six weeks later, systolic function decreased to 17.1± 3.2% vs. 28.2± 1.5% (p = 0.0001) and left ventricular inner surface increased to 19.9±1.1 mm² (p = 0.0001). Intraoperative graft survival during hHTx was 80% with 46 performed procedures (37/46). All transplanted organs survived two weeks of mechanical unloading. Discussion Combination of TAC and hHTx in rats offers an economic and reproducible small animal model enabling serial examination of mechanical unloading in a truly hypertrophic and failing heart, representing the typical pressure overloaded and dilated LV, occurring in patients with moderate to severe heart failure.