Daniele Linardi

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.

Cardioprotective effect of δ-opioid receptor agonist vs. mild therapeutic hypothermia in a rat model of cardiac arrest with extracorporeal life support.

BACKGROUND To compare the effect of δ-opioid receptor agonist, d-Ala2-d-Leu5 enkephalin (DADLE) with normothermic control and therapeutic hypothermia on post resuscitation myocardial function in a model of extracorporeal life support (ECLS). METHODS Ventricular fibrillation (VF) was induced in male Wistar rats. After 10 min of untreated VF, venoarterial ECLS was instituted for 60 min. At the beginning of ECLS animals were randomized to three groups of ten: normothermia, hypothermia (32 °C) and DADLE intravenous infusion (1 mg/kg/h). Cooling to 32 °C or normothermia or drug infusion lasted for the entire ECLS. Plasma samples and myocardial biopsies were obtained and left-ventricular (LV) function was assessed by a conductance catheter at baseline and after weaning from ECLS. RESULTS DADLE administration resulted in a significantly enhanced recovery of LV systolic function expressed by slope of the LV end-systolic pressure volume relationship (Ees) and preload recruitable stroke work (PRSW) than hypothermia and normothermia. LV stiffness indicated by end-diastolic pressure volume relationship (EDPVR) was significantly lower after DADLE administration (P<0.01). LV relaxation described by Tau was preserved after DADLE treatment but not after normothermia or mild hypothermia (P<0.01). Plasma lactate concentrations were lower in DADLE group (P<0.05). DADLE and not conventional hypothermia significantly increased phosphorylation of the kinases ERK1 and 2 (3.9±0.3 and 3.1±0.5 vs. 0.4±0.1 and 0.3±0.1-fold of baseline levels) (P<0.001). Both DADLE and hypothermia but not normothermia increase phosphorylation of Akt. CONCLUSIONS DADLE was more effective than mild therapeutic hypothermia in recovering myocardial function and activation of the pro-survival kinases Akt and ERK after ECLS.

Ventricular and pulmonary vascular remodeling induced by pulmonary overflow in a chronic model of pretricuspid shunt.

OBJECTIVES Current preclinical models of pulmonary arterial hypertension do not reproduce the clinical characteristics of congenital heart anomalies. Aortocaval shunt is relevant to a variety of clinical conditions. The pathophysiology and possible determination of pulmonary hypertension in this model are still undefined. METHODS A method to create a standardized and reproducible aortocaval shunt was developed in rats. After creation of the shunt, the animals were followed up for 20 weeks and a sham laparotomy was used as a control. The chronic effects of volume overload on the right and left ventricles and pulmonary hemodynamic modifications were evaluated by biventricular catheterization, echocardiography, and magnetic resonance. Pulmonary vascular changes were defined by histology. RESULTS An increased right ventricular end-diastolic area was confirmed by echocardiography. Left ventricular overload and decreased biventricular ejection fraction were demonstrated by magnetic resonance after 20 weeks in the shunt group compared with the controls (left ventricle, 50% ± 5% vs 62% ± 3%, P = .029; right ventricle, 53% ± 2% vs 65% ± 2%, P = .036). Preload recruitable stroke work of left and right ventricles decreased after 20 weeks in shunt rats (left ventricle: 36 ± 7 vs 98 ± 5, P = .004; right ventricle: 19 ± 2 vs 32 ± 9, P = .047). At the same time point, catheterization showed that effective pulmonary arterial elastance was increased only in the shunt group (1.29 ± 0.20 vs 0.14 ± 0.06 mm Hg/μL; P = .004). Histology showed medial hypertrophy, small artery luminal narrowing, and occlusion. CONCLUSIONS The aortocaval shunt model reliably produces right ventricular volume overload and secondary pulmonary hypertension. Due to a combination of left ventricular dysfunction and pulmonary overflow, the pulmonary hypertension produced shows features similar to those found in patients with chronic atrial-level shunt.

Oxygenator Is the Main Responsible for Leukocyte Activation in Experimental Model of Extracorporeal Circulation: A Cautionary Tale.

In order to assess mechanisms underlying inflammatory activation during extracorporeal circulation (ECC), several small animal models of ECC have been proposed recently. The majority of them are based on home-made, nonstandardized, and hardly reproducible oxygenators. The present study has generated fundamental information on the role of oxygenator of ECC in activating inflammatory signaling pathways on leukocytes, leading to systemic inflammatory response, and organ dysfunction. The present results suggest that experimental animal models of ECC used in translational research on inflammatory response should be based on standardized, reproducible oxygenators with clinical characteristics.

Characterization and Expression of Sphingosine 1-Phosphate Receptors in Human and Rat Heart

Aim: Sphingosine 1-phosphate (S1P), sphingolipid derivatives are known anti-inflammatory, anti-apoptotic, and anti-oxidant agent. S1P have been demonstrated to have a role in the cardiovascular system. The purpose of this study was to understand the precise expression and distribution of S1P receptors (S1PRs) in human and rat cardiovascular tissues to know the significance and possible implementation of our experimental studies in rat models. Methods and Results: In this study, we investigated the localization of S1PRs in human heart samples from cardiac surgery department, University of Verona Hospital and rat samples. Immunohistochemical investigation of paraffin-embedded sections illustrated diffused staining of the myocardial samples from human and rat. The signals of the human heart were similar to those of the rat heart in all chambers of the heart. The immunohistochemical expression levels correlated well with the results of RT-PCR-based analysis and western blotting. We confirmed by all techniques that S1PR1 expressed strongly as compared to S1PR3, and are uniformly distributed in all chambers of the heart with no significant difference in human and rat myocardial tissue. S1PR2 expression was significantly weak while S1PR4 and S1PR5 were not detectable in RT-PCR results in both human and rat heart. Conclusion: These results indicate that experimental studies using S1PR agonists on rat models are more likely to have a potential for translation into clinical studies, and second important information revealed by this study is, S1P receptor agonist can be used for cardioprotection in global ischemia-reperfusion injury.

Chronic overcirculation-induced pulmonary arterial hypertension in aorto-caval shunt

Pulmonary arterial hypertension is a common complication of congenital heart defects with left-to-right shunts. Current preclinical models do not reproduce clinical characteristics of shunt-related pulmonary hypertension. Aorto-caval shunt was firstly described as a model of right ventricle volume overload. The pathophysiology and the possible determination of pulmonary arterial hypertension of different periods of shunt exposure are still undefined. A method to create standardized, reproducible aorto-caval shunt was developed in growing rats (260±40 g). Three groups of animals were considered: shunt exposure for 10 weeks, shunt exposure for 20 weeks and control (sham laparotomy). Echocardiography and magnetic resonance revealed increased right ventricular end diastolic area in shunt at 10 weeks compared to control. Hemodynamic analysis demonstrated increased right ventricular afterload and increased effective pulmonary arterial elastance (Ea) in shunt at 20 weeks compared to control (1.29±0.20 vs. 0.14±0.06 mmHg/μl, p=0.004). At the same time point, the maximal slope of end-systolic pressure-volume relationship (Ees) decreased (0.5±0.2 mmHg/ml vs. 1.2±0.3, p<0.001). Consequently, right ventricular-arterial coupling was markedly deteriorated with a ≈50% decrease in the ratio of end-systolic to pulmonary artery elastance (Ees/Ea). Finally, left ventricular preload diminished (≈30% decrease in left ventricular end-diastolic volume). Histology demonstrated medial hypertrophy and small artery luminal narrowing. Chronic exposure to aorto-caval shunt is a reliable model to produce right ventricular volume overload and secondary pulmonary arterial hypertension. This model could be an alternative with low mortality and high reproducibility for investigators on the underlying mechanisms of shunt-related pulmonary hypertension.

Right ventricular laceration caused by an intact sternal wire: a rare complication of median sternotomy.

A 68-year-old man underwent an intervention for acute aortic dissection. After extubation, he was treated with non-invasive ventilation because of respiratory distress. On postoperative day 7, the patient suffered severe hypotension, tachycardia and a raised central venous pressure. The emergent resternotomy revealed that the lower sternal wire had led to a right ventricle laceration (Figs 1 and 2).

168 Cardioprotective role of fingolimod in ischemia-reperfusion injury by activation of akt/erk pathways in rat model

Background and purpose Ischaemic Heart Diseases (IHD) are the most common cause of morbidity and mortality. Incidence and prevalence are continuously growing. There is an escalating risk for revascularisation or resuscitation in patients with IHD. Recently, it has been reported that a sphingosine 1-phosphate receptor agonist play an anti-apoptotic and anti-inflammatory role in the ischemia-reperfusion injury. Objectives The aim of our study is to investigate the cardioprotective effects of sphingosine 1-phosphate receptor agonist fingolimod (FTY720) on global ischemia-reperfusion injury related to the cardiac surgery. Methods In our experimental study, global ischemia-reperfusion was achieved by cardiopulmonary bypass by cardioplegic arrest on ventilated male Sprague-Dawley rats (300–350 g). The global ischaemic period lasted 10 min in the cardioplegic arrest while reperfusion times were maintained for 60 min and 24 hours. ECG monitoring was done using AD instrument and using Millar catheter, heart rate, systolic and diastolic pressures were recorded and mean arterial pressure was calculated. The statistical significance was considered at P 0.05. Results The myocardial protection was observed in the group treated with Fingolimod as compared to control groups. Reduced frequency of apoptotic cells and inflammatory mediators were found in the treated group. The level of adenylates was preserved in the treated group as compared to controls (94%, 61% respectively)(p0.001). Reactive Oxygen Species (ROS) were attenuated in the fingolimod-treated group. Fingolimod treatment improved systolic and diastolic ventricular pressures and contractility strength (p0.005). Conclusions The intravenous administration of fingolimod in global ischemia-reperfusion was cardioprotective. Fingolimod cardioprotection appears to be mediated through preservation of high energy phosphates, reduction in oxidative stress, inhibition of apoptosis and inflammation leading to improved cardiac functions.

233 Anti-arrhythmic role of sphingosine 1-phospahte in post-operative atrial fibrillation by pak1 activation

Background Atrial Fibrillation (AF) is the most common arrhythmia occurring post cardiac surgery. The incidence varies and depends on the type of surgery. Postoperative AF may cause hemodynamically unstable, that increase the risk of stroke and increase mortality. Current management for prophylaxis of postoperative AF is not satisfactory. Objectives The purpose of this study is to investigate the anti-arrhythmic role of sphingosine 1-phosphate to prevent post-cardiac surgery AF. Methods Sprague-Dawley Rats (300-350grams) Obtained from Harlan Laboratories (Udine, Italy). They were fed standard rat chow, which they had access to ad libitum. Randomly categorised into two groups n=10 each group. One group was treated with sphingosine 1-phospahte receptor agonist fingolimod (1 mg/kg, i.v) and the control group was treated with saline. Following 15 min of treatment, cardioplegic arrest with the support of cardiopulmonary bypass. ECG electrodes were attached to limbs and using power lab charts ECG monitored and analysed. Following monitoring of rhythm for 24 hours, all animals were sacrificed and myocardial tissue was collected to analyse Pak1 protein. Results The administration of fingolimod led to significantly better and fast recovery as compared to the control group. The episodes of AF recorded during 24 hours in the treated group was low versus the control group (p0.001). We also investigated Pak1 protein by western blot and immunohistochemistry. Expression of Pak1 observed higher in the fingolimod-treated group(p0.05). Conclusion Sphingosine 1-phosphate receptor agonist fingolimod plays important role in prevention of AF-related to cardiac surgery by activating the Pak1 pathway.Fingolimod is FDA-approved sphingolipid that can be the potential therapeutic drug for post-operative AF prophylaxis.