EP3691674A1 - Methods and pharmaceutical compositions for providing cardioprotection in subjects who experienced a myocardial infarction - Google Patents

Abstract

Myocardial infarction occurs when the blood supply to one part of the heart is interrupted, causing the death of certain heart cells. The immediate goal is to quickly restore blood flow to the patient. However, a significant delay in restoring blood flow leads to a second condition known as an ischemia-reperfusion injury that may progressively develop after an ischemic event and may cause irreversible tissue damage. Successful cardioprotection is limited by a relatively small number of therapeutic targets. The present invention satisfies this need. The inventors have indeed shown that a peptide derived from selenoprotein T (SelT), SelT43-52 (PSELT), is effective against an injury of ischemia / reperfusion (I / R).

Description

METHODS AND PHARMACEUTICAL COMPOSITIONS FOR PROVIDING CARDIOPROTECTION IN SUBJECTS WHO EXPERIENCED A MYOCARDIAL

INFARCTION

FIELD OF THE INVENTION:

The present invention relates to methods and pharmaceutical compositions for providing cardioprotection in subjects who experienced a myocardial infarction.

BACKGROUND OF THE INVENTION:

Myocardial infarction, commonly known as a heart attack, occurs when the blood supply to part of the heart is interrupted causing some heart cells to die. This is most commonly due to occlusion of a coronary artery following the rupture of a vulnerable atherosclerotic plaque. The resulting ischemia and oxygen shortage, if left untreated for a sufficient period of time, can cause damage and or death of heart muscle tissue. Accordingly, in clinical situations of myocardial infarction, the immediate goal is to restore blood flow to the patient as quickly as possible. If blood flow is restored within a suitable time period, tissue damage can be averted. However, a significant delay in restoring blood flow leads to a second condition known as ischemia-reperfusion injury that can develop gradually after an ischemic event and may cause irreversible damage to tissues. Clinical examples include cardiac contractile dysfunction, arrhythmias and irreversible myocyte damage (heart cell death) following myocardial infarction. Accordingly, several methods for the cardioprotection after myocardial infarction have been investigated. For example, current therapies aimed at improving contractile function often involve the use of inotropic agents (e.g., calcium, dopamine, epinephrine, ephedrine, phenylephrine, dobutamine). However inotropic drugs have been reportedly associated with increases in intracellular calcium concentration and heart rate, which may be potentially harmful, especially in hearts with impaired energy balance. Thus, successful cardioprotection is limited by a relatively small number of therapeutic targets. The present invention fulfils this need by providing a new therapeutic target for cardioprotection after myocardial infarction.

Selenium (Se) is an essential micronutrient, the benefits of which to human health as an antioxidant are widely recognized. Selenium deficiency has been implicated in a number of disorders, including infertility, increased cancer incidence, susceptibility to viral infection, mental development retardation and accelerated aging (Rayman, 2012). Accumulating evidence suggests that Se is also important for optimal functioning of the cardiovascular system (Benstoem et al., 2015). The majority of Se beneficial effects are actually mediated by the biological activity of a particular class of proteins in which it is incorporated, the selenoproteins (Atkins and Gesteland, 2000), where the oligoelement is inserted as a selenocystein (Sec), the 21st natural amino acid (Driscoll and Copeland, 2003). The genes encoding selenoproteins harbor, at their 3 '-untranslated region (UTR), a specific hairpin motif, designated the Sec insertion sequence (SECIS), which is responsible for the recognition and decoding of the in frame UGA stop codon as a signal for the incorporation of the Sec residue (Driscoll and Copeland, 2003). In mammals, 25 selenoprotein-encoding genes have been identified (Kryukov et al., 2003) whose entire invalidation through Sec tRNA gene knockout leads to early embryonic lethality in rodents (Bosl et al., 1997). Likewise, mutations in human Sec tRNA or SECIS binding protein lead to complex disorders (Schweizer and Fradejas-Villar, 2016). Due to the presence of Sec in their catalytic site, selenoproteins exert important oxidoreductase activities and include several major enzymes, such as glutathione peroxidases (GPx), thioredoxin reductases (TrxR) and iodothyronine deiodinases which protect cells from oxidative stress and catalyze key redox reactions (Labunskyy et al., 2014). In addition to their role in detoxification of free radicals, diverse essential functions were attributed to selenoproteins, such as the implication of deiodinases in thyroid hormone metabolism (Beckett and Arthur., 2005), the selenoproteins N, M or K in Ca²⁺ regulation (Pitts and Hoffmann, 2017) or TrxR in signal transduction and transcription regulation (Labunskyy et al., 2014). However, the role of many selenoproteins and their mechanisms of action are still elusive.

Several selenoproteins are highly expressed during development (Petit et al. 2003; Papp et al., 2007; Tanguy et al 2011) and their loss has been associated with major anatomical alterations and functional defects in rodents (Pitts et al., 2014, Lescure et al., 2009). In particular, Selenoprotein T (SelT) is a key thioredoxin-like enzyme present in the endoplasmic reticulum (Grumolato et al., 2008; Hamieh et al, 2017), which is essential at early stage of mouse development (Boukhzar et al, 2016; Castex et al, 2016). SelT is highly expressed in most embryonic organs, but declines gradually as the organs develop, to disappear in most adult tissues except the endocrine glands (Hamieh et al, 2017; Tanguy et al, 2011; Prevost et al, 2013). Targeted inactivation of SelT in pancreatic β-cells resulted in a reduction in pancreatic islet size and impaired glucose tolerance, thus indicating a possible defect in cell commitment to β-cell lineage. Similarly, SelT depletion in other neuroendocrine and endocrine cells provoked Ca²⁺ store depletion, ER stress, misfolded protein accumulation and hormone production and secretion defects (Grumolato et al, 2008; Hamieh et al, 2017).

Although a large body of evidence has now been accumulated showing that SelT affects the survival, differentiation and function of nervous and endocrine cells (Boukhzar et al., 2016; Castex et al. 2016, Hamieh et al., 2017; Grumolato et al., 2008; Prevost et al. 2013) via its redox activity, nothing is known about the expression and function of SelT in other vital tissues such as the heart, a tissue which is highly exposed to oxidative stress in particular after ischemia/reperfusion (I/R).

SUMMARY OF THE INVENTION:

The present invention relates to methods and pharmaceutical compositions for providing cardioprotection in subjects who experienced a myocardial infarction. In particular, the present invention is defined by the claims.

DETAILED DESCRIPTION OF THE INVENTION:

Selenoprotein T (SelT or SELENOT) is a novel thioredoxin-like enzyme whose genetic ablation in mice results in early embryonic lethality. SelT exerts an essential cytoprotective action during development and after injury through its redox active catalytic site. The inventors aim to determine the expression and regulation of SelT in the mammalian heart in normal and pathological conditions, and to evaluate the cardioprotective effect of a SelT-derived peptide, SelT43-52 (PSELT) encompassing the active site, against ischemia/reperfusion (I/R) injury. For this purpose, the inventors used the isolated Langendorff rat heart to perform physio- pharmacological experiments, and they analyzed SelT expression and different associated signaling mechanisms by immunohistochemistry, western blot analysis and ELISA assays. The inventors found that SelT expression is more abundant in embryo than newborn heart and is undetectable in adult. However, SelT expression was tremendously increased after ischemia/reperfusion, suggesting that SelT could participate to heart protection after an insult. To demonstrate this and decipher the mechanisms associated, the inventors used the small peptide PSELT encompassing the redox motif of SelT which is key to its function. The peptide PSELT (5 nM) was indeed able to induce post-conditioning cardioprotection as evidenced by a significant recovery of contractility (dLVP), without changes in cardiac contracture (LVEDP), and by a significant reduction of infarct size (IS). In contrast, PSELT at a higher dose (100 nM) slightly reduced IS without a significant recovery of contractility. An inert peptide lacking the redox site used as a control did not confer cardioprotection. Immunoblot analysis showed that PSELT-dependent cardioprotection is accompanied by a significant increase of the active forms of Akt, Erkl/2 and Gsk3a-P, and a decrement of active p38MAPK. Concurrently, PSELT inhibited the I/R-induced expression of pro-apoptotic factors Bax, caspase 3 and cytochrome c, while it stimulated the anti-apoptotic factor Bcl-2. Furthermore, PSELT significantly reduced several markers of I/R-induced oxidative and nitrosative stress in heart. These results unravel the role of SelT as a cardiac modulator and identify PSELT, a small SelT-derived peptide, as an effective post-conditioning agent able to protect the heart after ischemic injury.

Accordingly, the first object of the present invention relates to a method for providing cardioprotection in a subject who experienced a myocardial infarction comprising administering the subject with a therapeutically effective amount of the PSELT peptide having the formula of:

H-Phe-Gin-IIe-Cys-Val-Ser-Sec-GIy-Tyr-Arg-OH

As used herein, the term "subject", "individual," or "patient" is used interchangeably and refers to any subject for whom diagnosis, treatment, or therapy is desired, particularly humans. Other subjects may include cattle, dogs, cats, guinea pigs, rabbits, rats, mice, horses, and the like. In some preferred embodiments, the subject is a human.

As used herein the term "cardioprotection" means protecting against or reducing damage to the myocardium after a myocardial infarction, after, during or prior to ischemic reperfusion. In particular, cardioprotection includes reducing infarct size, reducing ischemia-reperfusion injury, reducing hypoxia induced apoptosis/necrosis and preventing cardiomyocyte cell death. The method of the present invention is thus particularly suitable for the treatment of myocardial infarction injury in a subject in need thereof.

As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of patient at risk of contracting the disease or suspected to have contracted the disease as well as patients who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a patient during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a patient during treatment of an illness, e.g., to keep the patient in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular interval, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., disease manifestation, etc.]).

In some embodiments, the PSELT peptide is administered to a subject having one or more signs or symptoms of acute myocardial infarction injury. In some embodiments, the subject has one or more signs or symptoms of myocardial infarction, such as chest pain described as a pressure sensation, fullness, or squeezing in the mid portion of the thorax; radiation of chest pain into the jaw or teeth, shoulder, arm, and/or back; dyspnea or shortness of breath; epigastric discomfort with or without nausea and vomiting; and diaphoresis or sweating.

In some embodiments, the PSELT peptide is administered simultaneously or sequentially (i.e. before or after) with a revascularization procedure performed on the subject. In some embodiments, the subject is administered with the PSELT peptide before, during, and after a revascularization procedure. In some embodiments, the subject is administered with the PSELT peptide as a bolus dose immediately prior to the revascularization procedure. In some embodiments, the subject is administered with the PSELT peptide continuously during and after the revascularization procedure. In some embodiments, the subject is administered with the PSELT peptide for a time period selected from the group consisting of at least 3 hours after a revascularization procedure; at least 5 hours after a revascularization procedure; at least 8 hours after a revascularization procedure; at least 12 hours after a revascularization procedure; at least 24 hours after a revascularization procedure. In some embodiments, the subject is administered with the PSELT peptide in a time period selected from the group consisting of starting at least 8 hours before a revascularization procedure; starting at least 4 hours before a revascularization procedure; starting at least 2 hours before a revascularization procedure; starting at least 1 hour before a revascularization procedure; starting at least 30 minutes before a revascularization procedure. In some embodiments, the revascularization procedure is selected from the group consisting of percutaneous coronary intervention; balloon angioplasty; insertion of a bypass graft; insertion of a stent; directional coronary atherectomy; treatment with one or more thrombolytic agent(s); and removal of an occlusion.

In some embodiments, the PSELT peptide is administered in combination with an additional active agent. In some embodiments, the additional active agent is a cardiovascular agent selected from the group consisting of hyaluronidase, a corticosteroid, recombinant superoxide dismutase, prostacyclin, fluosol, magnesium, poloxamer 188, trimetazidine, eniporidine, cariporidine, a nitrate, anti-P selectin, an anti-CD 18 antibody, adenosine, and glucose-insulin-potassium. In some embodiments, the cardiovascular agent is selected from the group consisting of an anti-arrhthymia agent, a vasodilator, an anti-anginal agent, a corticosteroid, a cardioglycoside, a diuretic, a sedative, an angiotensin converting enzyme (ACE) inhibitor, an angiotensin II antagonist, a thrombolytic agent, a calcium channel blocker, a throboxane receptor antagonist, a radical scavenger, an anti-platelet drug, a β-adrenaline receptor blocking drug, oreceptor blocking drug, a sympathetic nerve inhibitor, a digitalis formulation, an inotrope, and an antihyperlipidemic drug. In some embodiments, the active agent is an inotrope. Positive inotropic agents increase myocardial contractility, and are used to support cardiac function in conditions such as decompensated congestive heart failure, cardiogenic shock, septic shock, myocardial infarction, cardiomyopathy, etc. Examples of positive inotropic agents include, but are not limited to, Berberine, Bipyridine derivatives, Inamrinone, Milrinone, Calcium, Calcium sensitizers, Levosimendan, Cardiac glycosides, Digoxin, Catecholamines, Dopamine, Dobutamine, Dopexamine, Epinephrine (adrenaline), Isoprenaline (isoproterenol), Norepinephrine (noradrenaline), Eicosanoids, Prostaglandins, Phosphodiesterase inhibitors, Enoximone, Milrinone, Theophylline, and Glucagon. Negative inotropic agents decrease myocardial contractility, and are used to decrease cardiac workload in conditions such as angina. While negative inotropism may precipitate or exacerbate heart failure, certain beta blockers (e.g. carvedilol, bisoprolol and metoprolol) have been shown to reduce morbidity and mortality in congestive heart failure. Examples of negative inotropic agents include, but are not limited to, Beta blockers, Calcium channel blockers, Diltiazem, Verapamil, Clevidipine, Quinidine, Procainamide, disopyramide, and Flecainide. In some embodiments, the cardiovascular agent is cyclosporine. As used herein, the term "cyclosporine" refers to cyclosporine A, cyclosporine G, and functional derivatives or analogues thereof, e.g., NIM81 1. Cyclosporine A refers to the natural Tolypocladium inflation cyclic non-ribosomal peptide. Cyclosporine G differs from cyclosporine A in the amino acid 2 position, where an L- norvaline replaces the a-aniinobutyric acid. (See generally, Wenger, R. M. 1986. Synthesis of Ciclosporin and analogues: structural and conformational requirements for immunosuppressive activity. Progress in Allergy, 38:46-64).

In some embodiments, the PSELT peptide is synthetized by any well-known method in art. For instance, the solid phase peptide synthesis that is a process used to chemically synthesize peptides on solid supports may be used (see e.g. the EXAMPLE). In solid phase peptide synthesis, an amino acid or peptide is bound, usually via the C-terminus, to a solid support. New amino acids are added to the bound amino acid or peptide via coupling reactions. Due to the possibility of unintended reactions, protection groups are typically used. To date, solid phase peptide synthesis has become standard practice for chemical peptide synthesis. Automated solid phase peptide synthesizers are commercially available and could be used for producing the peptide of the present invention.

As used herein, the term "effective amount" refers to a quantity sufficient of PSELT peptide to achieve cardioprotection. In the context of therapeutic or prophylactic applications, the amount of a composition administered to the subject will depend on the type and severity of the disease and on the characteristics of the individual, such as general health, age, sex, body weight and tolerance to drugs. It will also depend on the degree, severity and type of disease. The skilled artisan will be able to determine appropriate dosages depending on these and other factors. In some embodiments, an effective amount of the PSELT peptides for achieving a therapeutic or prophylactic effect, range from about 0.000001 mg per kilogram body weight per day to about 10,000 mg per kilogram body weight per day. Typically, the dosage ranges are from about 0.0001 mg per kilogram body weight per day to about 100 mg per kilogram body weight per day. For example dosages can be 1 mg/kg body weight or 10 mg/kg body weight every day, every two days or every three days or within the range of 1-10 mg/kg every week, every two weeks or every three weeks. In some embodiments, a single dosage of peptide ranges from 0.1-10,000 micrograms per kg body weight. In some embodiments, aromatic- cationic peptide concentrations in a carrier range from 0.2 to 2000 micrograms per delivered milliliter.

Typically, The PSELT peptide is combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions. "Pharmaceutically" or "pharmaceutically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. Typically, the pharmaceutical compositions contain vehicles, which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Sterile injectable solutions are prepared by incorporating the PSELT peptide in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

The invention will be further illustrated by the following figures and examples.

However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

FIGURES:

Figure 1. Systolic and diastolic function and infarct size. (A) dLVP and (B) LVEDP variations. Data are expressed as changes of dLVP and LVEDP values (mmHg) from the stabilization to the end of the 120-min of reperfusion with respect to the baseline values for I/R alone (n=5) or I/R in the presence of PSELT 5 nM (n=5), PSELT 100 nM (n=5), inert peptide (n=4). Grey boxes indicate the ischemic period (Bonferroni Multiple Comparison test, dLVP=50.53% of total variation between groups (p <0.0001); LVEDP=38.75 % of total variation between groups (p <0.0001). Inset graph shows the dLVP and LVEDP at the end of reperfusion (One-way ANOVA/Newman-Keuls Multiple Comparison Test, *=p <0.05; **=p<0.01). (C) Infarct size (n=5 hearts for I/R alone, PSELT 5 nM, PSELT 100 nM, n=4 hearts for inert peptide group). The amount of necrotic tissue measured after 30-min global ischemia and 120-min reperfusion is expressed as percent of the left ventricle mass (LV) (% IS/LV). p<0.05 (*), p<0.01 (**), p<0.001 (***), by One- Way ANOVA/Newman-Keuls Multiple Comparison Test.

Figure 2. Systolic and diastolic function and infarct size. (A) dLVP and (B) LVEDP variations. Data are expressed as changes of dLVP and LVEDP values (mmHg) from the stabilization to the end of the 120-min of reperfusion with respect to the baseline values for Normal Diet (ND), or High Fat Diet (HFD 60% kcal from fat), or ND+PSELT (15 μg/kg/day), or HFD+PSELT (15 μg/kg/day). Boxes indicate the ischemic period (Bonferroni Multiple Comparison test) Inset graph shows the dLVP and LVEDP at the end of reperfusion (One-way ANOVA/Newman-Keuls Multiple Comparison Test, *=p <0.05; **=p<0.01). (C) Infarct size. The amount of necrotic tissue measured after 30-min global ischemia and 120-min reperfusion is expressed as percent of the left ventricle mass (LV) (% IS/LV). p<0.05 (*), p<0.01 (**), p<0.001 (***), by One- Way ANOVA/Newman-Keuls Multiple Comparison Test.

EXAMPLE:

Material & Methods

Animals

Male Wistar rats (Harlan Laboratories Sri, Udine, Italy), weighing 250-300 g, were housed (three per cage) in a ventilated cage rack system under standard conditions. The animals had access to food and water ad libitum. The investigation conforms to Italian law (DL. 26/14) and to the Guide for the Care and Use of Laboratory Animals, according to National Institutes of Health publication 85-23 (revised 1996). The project was approved by the Italian Ministry of Health, Rome, and by the Ethics Review Board of the University of Calabria.

Peptides and drugs

The SelT-derived peptide 43-52 (PSELT) corresponding to the sequence FQICVSUGYR (SEQ ID NO: l) in its reduced form, and an inactive peptide without Sec used as a control, were chemically synthesized with the method of solid phase on a Fmoc resin, using an Applied Biosystems model 433A peptide synthesizer (AB Sciex, Courtaboeuf, France). MDL-12,330A (MDL), a specific inhibitor of adenylate cyclase, wortmannin (WT), a potent phosphatidylinositol 3-kinase (PI3K) inhibitor, PD-98059 (PD), a specific inhibitor of Erk 1/2 and 5 -hydroxy decanoate (5HD), a mitoKATP channel blocker, were purchased from Sigma Aldrich (Saint Louis, Missouri, USA). All drug-containing solutions were freshly prepared just before the experiments.

Isolated heart perfusion

Rats were heparinized (2.500 U i.m.) and anesthetized with ethyl carbamate (2 g/kg rat, i.p.) 10 min later. Hearts were rapidly excised and transferred in ice-cold buffered Krebs- Henseleit solution (KHs) and weighed. The performance of the rat heart was evaluated according to the Langendorff technique. The aorta was immediately cannulated with a glass cannula and connected with the Langendorff apparatus to start the retrograde perfusion (Cerra et al, 2006) at a constant flow-rate of 12 ml/min with oxygenated KHs, containing 4.7 mM KC1, 113 mM NaCl, 25 mM NaHCOs, 1.8 mM CaCk, 1.2 mM MgS0₄, 1.2 mM KH₂P0₄, 1.1 mM mannitol, 11 mM glucose, 5 mM Na-pyruvate (Sigma Aldrich, Saint Louis, Missouri, USA) (pH 7.4; 37 °C; 95% 0₂ and 5% CO2). The perfusion pressure was set to 100 mmHg and kept constant throughout the experiments. The hearts were kept in a temperature controlled chamber (37 °C). To avoid fluid accumulation the apex of the left ventricle (LV) was pierced. A water- filled latex balloon, connected to a pressure transducer (BLPR; WRI, Inc., Sarasota, FL, USA), was inserted into the left ventricle through the mitral valve, to allow isovolumic contractions and to continuously record cardiac mechanical parameters. Another pressure transducer was located above the aorta to measure coronary pressure (CP). The developed left ventricular pressure (dLVP, an index of contractile activity) and the left ventricular end- diastolic pressure (LVEDP, an index of contracture) were measured to evaluate inotropism (Pasqua et al., 2013). The endurance of the preparations was stable up to 190 min. The performance variables were measured every 10 min. Parameters were recorded by using the PowerLab data acquisition system (AD Instruments, Oxford-UK) as previously reported (Pasqua et al, 2013).

Experimental protocols

Ischemia/Reperfusion (I/R) studies: each heart was stabilized for 40 min during which the baseline parameters were recorded. After stabilization, hearts were randomly assigned to one of the groups described below and then subjected to 30 min of global, no-flow ischemia followed by 120 min of reperfusion (I/R). The concentration of PSELT (5 nM), corresponding to the EC50 dose, was chosen on the basis of preliminary dose-response curves (data not shown).

Experimental groups

In the first group (SHAM group), hearts were stabilized and perfused for 190 min. In the second group (I/R alone group), hearts were stabilized and subjected to I/R protocol.

In the third group (PSELT 5 nM group), PSELT at the EC50 dose (5 nM) was infused for 20 min-at the beginning of 120 min of reperfusion (post-conditioning protocol: Post-C PSELT 5 nM). In the fourth group (PSELT 100 nM group), PSELT at a high dose (100 nM) was infused for 20 min at the beginning of 120 min of reperfusion (post-conditioning protocol: Post-C PSELT 100 nM).

In the fifth group (inert PSELT group), inert PSELT, at the same concentration of PSELT EC50 (5 nM) was infused for 20 min at the beginning of 120 min of reperfusion (post- conditioning protocol: Post-C inert PSELT 5 nM).

In the groups 6-9 (PSELT + inhibitors groups) hearts were perfused with PSELT 5 nM plus one of the following inhibitors: MDL (100 nM), WT (100 nM), PD (10 nM) or 5HD (10 μΜ); perfusion with inhibitors was started 5 min before ischemia and continued during the early 20 min of reperfusion.

In all experiments, the inhibitor concentration was selected on the basis of previous reports (Penna et al., 2012). Previous data showed that in the hearts perfused with inhibitors alone, the dLVP recovery, the LVEDP and the infarct size were similar to I/R alone group (data not shown).

Assessment of myocardial injury

To measure the infarct area, hearts were rapidly removed from the perfusion apparatus at the end of reperfusion. The left ventricles were dissected transversely into 2-3 mm slices. After 20 min of incubation at 37°C in 0.1% nitro blue tetrazolium in phosphate buffer (59.8 mM NaH₂P0₄, 484.9 mM Na₂HP0₄, pH: 7.4), unstained necrotic tissues were carefully separated from stained viable tissues by an independent observer who was not aware of the nature of the intervention. The weights of the necrotic and non-necrotic tissues were then determined, and the necrotic mass was expressed as a percentage of total left ventricular mass (% IS/LV), including septum (Pasqua et al, 2015).

Western blotting

Apex of cardiac ventricles were homogenized in ice-cold RIPA buffer (Sigma- Aldrich,

Saint Louis, Missouri, USA) containing a mixture of protease inhibitors (1 mmol/L aprotinin, 20 mmol/L phenylmethylsulfonyl fluoride, and 200 mmol/L sodium ortho vanadate). Then homogenates were centrifuged at 15 000 x g for 20 min at 4 °C for debris removal. Protein concentration was determined using a Bradford reagent according to the manufacturer (Sigma- Aldrich, Saint Louis, Missouri, USA). Equal amounts of proteins were separated on 12% SDS- PAGE gels (30 μg for SelT, β-tubulin, Bax, Bcl-2, Active Caspase 3, cytochrome c, Cyt c, and cytochrome oxidase subunit IV, COX-4) or on 10%> SDS-PAGE gels (30 μg for p-Akt, Akt, p- Erk 1/2, Erkl/2, p-GSIQa/β, GSIQa/β, p-p38MAPK, and p38MAPK), or on 8% SDS-PAGE gels (30 μg for xanthine oxidase, XO and aldehyde oxidase-1, AOX-1), subjected to electrophoresis and transferred to polyvinyl difluoride membranes. The membranes were blocked with non-fat dried milk, and incubated overnight at 4 °C with different antibodies including polyclonal rabbit antibodies against SelT (Acris antibodies, San Diego, California, USA), p-Akt, Akt, GSIQa/β, Erk 1/2, monoclonal mouse antibodies against p-Erkl/2, AOX-1, Bax, Bcl-2, Cyt c, COX-IV, polyclonal goat antibody against XO and β-tubulin (Santa Cruz Biotechnology, Santa Cruz, California, USA), p-GSIQa/β (Sigma Aldrich Saint Louis, Missouri, USA), p-p38MAPK, p38MAPK (Cell Signaling Technology, Danvers, Massachusetts, USA), diluted 1 : 1000 in Tris-buffered saline and 0.2% Tween 20 containing 5% non-fat dry milk (TBSTM). Polyclonal rabbit antibodies against Akt, Erk 1/2, p38MAPK, GSK3a/p and β-tubulin, a monoclonal rabbit antibody against GAPDH and a monoclonal mouse antibody against COX-IV were used as loading controls (Acris antibodies, San Diego, California, USA, Santa Cruz Biotechnology, Santa Cruz, California, Sigma Aldrich, Saint Louis, Missouri, USA, Cell Signaling Technology, Danvers, Massachusetts, USA, Thermofisher Scientific, Waltham, Massachusetts, USA) (n=3 for each group). Anti-rabbit and anti-mouse peroxidase-linked secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, California, USA) were diluted 1 :2000 in TBSTM. Immunodetection was performed using the ECL PLUS enhanced chemiluminescence kit (Amersham, Little Chalfont, UK). Autoradiographs were obtained by membrane exposure to X-ray films (Hyperfilm ECL; Amersham, Little Chalfont, UK). Immunoblots were digitalized; densitometric analyses of the bands were performed evaluating the areas and the pixel intensity represented by 256 Gray values (0 = white; 256 = black) and the background was subtracted. The analyses were carried out using NIH IMAGE 1.6 (National Institutes of Health, Bethesda, Maryland).

Enzyme-linked immunosorbent assay (ELISA)

Detection of reactive oxygen species (ROS) in heart samples was performed by ELISA using a commercial kit (ROS, Sunred Biological Technology, Shanghai, China) as follows: the left ventricles (n=3 for each group) of SHAM, I/R alone or with PSELT (5 nM) groups were homogenized using Ultra-Turrax® in Phosphate-Buffered Saline, PBS (137 mM NaCl, 2.7 mM KC1, 10 mM Na₂HP0₄, 1.8 mM KH₂P0₄; pH 7.4) plus a mixture of protease inhibitors (1 mmol/L aprotinin, 20 mmol/L phenylmethylsulfonyl fluoride, and 200 mmol/L sodium orthovanadate) and centrifuged at 15 000 x g for 20 min (4 °C). The supernatants were then assayed with the ELISA kit.

Mitochondria isolation

Mitochondria were isolated from the ventricles as previously described (Mali et al., 2016). At the end of perfusion, ventricle samples (n=3 for each group) of SHAM, I/R alone and PSELT 5 nM groups were harvested and homogenized in mitochondrial isolation buffer (IBc): Tris-MOPS (0.1 M), EGTA/Tris (0.1 M) and sucrose (1 M). The pH was adjusted to 7.4 and the volume was made to 100 ml with distilled water. The homogenates were centrifuged at 2000 x g for 10 min at 4 °C and the supernatants were collected and centrifuged again at 5000 x g for 10 min at 4 °C. The sedimented mitochondrial pellets were washed twice and resuspended in 50 μΐ of IBc buffer. The 5000 x g supernatant represented the cytosolic fraction. All manipulations were carried out at 4 °C. To confirm the presence of mitochondria in the pellets, the monoclonal mouse antibody against COX-IV was used as mitochondrial loading control.

Immunofluorescence

For immunohistochemistry, rats were anesthetized with sodium pentobarbital (120 mg/kg; Ceva Sante Animale, Libourne, France) and heparinized and perfused through an intracardiac cannula with 0.9% NaCl in 0.1 M phosphate buffer (pH 7.4), followed by 4% paraformaldehyde (PFA) in PBS. Hearts were excised and post-fixed in the same fixative at 4 °C, which was changed to PBS azide after 24 h. Tissues were sectioned into 50-μιη slices with a vibratome. The sections were incubated with 1% donkey serum diluted in 1% bovine serum albumin (BSA) and 0.3% Triton X-100 in PBS for 2 h at room temperature, and then exposed overnight at 4 °C to primary antibodies against SelT (Grumolato et al, 2008) diluted 1 :200, anti-nitrotyrosine used as a marker of nitrosative stress (Merck Millipore, Fontenay sous Bois, France) diluted 1 :200 and calsequestrin-2, used as a marker of cardiac sarcoplasmic reticulum staining (Santa Cruz Biotechnology, Santa Cruz, California, USA) diluted 1 :200. Immunostaining was visualized using Alexa Fluor 488 or 594-conjugated secondary antibodies diluted 1 :200 (Invitrogen, Saint Aubin, France). Counterstaining with 1 μg/ml 4,6- diamino-2- phenylindole (DAPI, Sigma-Aldrich) in PBS for 1 min was performed prior to mounting the slides with PBS/Glycerol 50/50. Samples were analyzed with a Leica SP2 confocal laser scanning microscope (DMRAX- UV) equipped with the Acousto-Optical Beam Splitter system (Leica Microsystems, Nanterre, France). The microscopic observations were made on The Cell imaging platform PRIMACEN (www.primacen-crihan.fr).

Statistics

All data were expressed as mean ± SEM. One-way ANOVA, non-parametric Newman- Keuls multiple comparison test (for post-ANOVA comparisons) was used for western blot and ELISA analyses. Differences at *p =<0.05, **p =<0.01, ***p =<0.001 were considered statistically significant. Two-way ANOVA, non-parametric Bonferroni's multiple comparison test (for post-ANOVA comparisons) was used for the time course of hemodynamic analysis. The statistical analyses was carried out using Graphpad Prism5. Results

SelT expression during heart ontogenesis

We analyzed by immunofluorescence and western blot the cardiac expression of SelT at different stages of heart development and in the adult. For this, we used embryonic (E7), newborn (P14) and adult (3-month old) rat cardiac tissue. A strong SelT immunoreactivity was observed in the embryonic tissue, which co-localized with calsequestrin (data not shown), indicating that cardiomyocytes express high levels of SelT during heart development (data not shown). In newborn heart, only some areas of the cardiac tissue, mostly in the periphery was labeled for SelT whereas the remaining tissue exhibited only calsequestrin staining (data not shown), perhaps reflecting still ongoing postnatal differentiation of heart in this region. In the adult, cardiac tissue was devoid of SelT labeling (data not shown), thus indicating that SelT expression could be linked to cardiac tissue development and remodeling, and is turned off in mature adult tissue. This ontogenetic pattern of SelT expression was confirmed by western blot analysis (data not shown).

SelT expression during cardiac ischemia

To determine whether SelT expression could be induced in adult heart after injury, immunofluorescence histochemical analysis of cardiac tissue and western blot analysis of cardiac extracts were carried out in order to evaluate SelT levels after I/R compared to controls (data not shown). Immunofluorescence analysis revealed that I/R triggers a tremendous increase in SelT immunoreactivity in cardiac tissue (data not shown). In contrast, no change was observed for calsequestrin in this condition (data not shown). In accordance, western blot analysis showed that SelT is not expressed in control hearts as seen by immunohistochemistry. However, I/R induced a large increase in SelT expression compared to control (data not shown).

PSELT effect on post-ischemic cardiac function

In basal condition after 40 min of equilibration, the cardiac parameters for all groups were: dLVP = 72 ± 2 mmHg; LVEDP= 5 - 8 mmHg; CP = 68 ± 7 mmHg; HR = 220 ± 11 mrnHg. Endurance and stability of the preparations were assessed by measuring the performance variables every 10 min. These parameters were stable up to 190 min.

The possibility that PSELT elicits cardioprotection was investigated by comparing the effects induced by the I/R maneuver with those elicited by the peptide administered after I/R for post-conditioning (PostC). The effects of PSELT on both systolic and diastolic functions were analyzed at ECso dose of 5 nM obtained by preliminary dose-response curves (data not shown) and at a higher dose (100 nM). Systolic function was evaluated by measuring the developed left ventricular pressure (dLVP), an index of inotropic activity. A limited contractility recovery was observed in the I/R group (LVP at the end of reperfusion: 27 ± 4 mmHg; baseline values: 75 ± 2 mmHg) (Fig. 1A). PSELT (5 nM) induced an important improvement of LVP recovery during reperfusion (LVP at the end of reperfusion: 56± 6 mmHg; baseline values: 74 ± 4 mmHg) (Fig. 1A). On the contrary, PSELT at a higher dose (100 nM) did not elicit any contractility recovery (LVP at the end of reperfusion: 25 ± 9 mmHg; baseline values: 67 ± 7 mmHg). The "inert" peptide without Sec had no effect, indicating that PSELT exerts a selective action via an intact CXXSec site. It is known that the contracture, evaluated as LVEDP is an index of diastolic function. It has been reported in the rat heart that 4 mmHg or more above the baseline level of LVEDP indicates an important cardiac damage (Pagliaro et al, 2003). Our results showed that in I/R alone group, LVEDP markedly increased compared to baseline (4 ± 4 mmHg at baseline; 24 ± 4 mmHg at the end of reperfusion). A similar effect was observed in the presence of the inert PSELT (5 ± 4 mmHg at baseline; 20 ± 2 mmHg at the end of reperfusion). In contrast, PSELT (5 nM) added during reperfusion abolished contracture, LVEDP being 9 ± 2 mmHg at the end of reperfusion (Fig. IB), indicating that this peptide significantly reduces heart damage after I/R. The higher dose (100 nM) of the intact peptide was less efficient (LVEDP = 14 ± 6 mmHg at the end of reperfusion) (100 nM). The IS was also evaluated and was expressed as a percentage of LV mass. The IS was 75 ± 3% after I/R, which was strongly reduced (38 ± 1%) in the presence of PSELT at 5 nM and less (52 ± 5%) at 100 nM (Fig. 1C). The inert PSELT yielded an IS of 59 ± 9% (Fig. 1C).

PSELT affects cardioprotective pathways

The mechanism of action underlying the cardioprotective effect of PSELT (5 nM) was studied by using selective inhibitors of intracellular pathways involved in cardioprotection, such as MDL12,330A, a specific inhibitor of adenylate cyclase, wortmannin, a specific inhibitor of PI3K, PD098059, a selective inhibitor of Erk 1/2, and 5 hydroxy decanoate, a specific inhibitor of mitochondrial KATP channels. The results indicated that PSELT- dependent reduction of IS was abolished in hearts co-treated with the above inhibitors since the IS was 68 ± 2% in the presence of MDL12,330A; in the presence of wortmannin 83 ± 3%; 73 ± 3% in the presence of PD098059; and 67 ± 10% in the presence of 5 hydroxy decanoate (data not shown). A similar trend was observed in the presence of the inhibitors for PSELT - dependent improved recovery of dLVP and contracture (LVEDP) (data not shown).

The involvement of the kinases Akt (protein kinase B), Erkl/2 (extracellular signal- regulated kinases 1/2), p38MAPK (mito gen-activated protein kinase) and GSK3P (glycogen synthase kinase 3-beta) in the PSELT-induced cardioprotection was evaluated by western blot analysis of the phosphorylated and non phosphorylated forms of these enzymes. In homogenates from hearts exposed to 5 nM PSELT at the reperfusion, the levels of phosphorylated Akt (data not shown), Erkl/2 (data not shown) and GSIQa-β (data not shown) were higher, while those of phosphorylated p-38MAPK (data not shown) were lower as compared to I/R alone.

PSELT influence on apoptotic indexes

The effect of PSELT (5 nM) administration at the reperfusion on the expression of the pro- and anti-apoptotic proteins, Bax, Bcl-2, active caspase3 and cyt c was studied. The peptide PSELT induced a significant reduction of the expression of the pro-apoptotic factors Bax (data not shown), active caspase 3 (data not shown) and cyt c (data not shown), and a significant increase of expression of the anti-apoptotic factor Bcl-2 (data not shown) compared to I/R alone.

Mitochondria exhibited a significant decrease of cyt c expression in I/R alone group compared to the SHAM group, while a significant recovery was observed in the mitochondria of PSELT group. In accordance, PSELT treatment caused a significant reduction of cyt c in the cytosol, compared to I/R alone group.

PSELT influence on cellular redox balance control

The control of the redox balance by PSELT was evaluated after I/R by analyzing 3- nitrotyrosine by immunohistohemistry, by measuring intracardiac ROS production by ELISA assay and by assessing the expression of specific markers involved in the production of free radicals, such as xanthine oxidase (XO) and aldehyde oxidase 1 (AOX-1). I/R induced a burst of 3-nitrotyrosine, an indicator of nitrosative stress, which was reversed in the presence of 5 nM PSELT. Similary, I/R provoked an increase of intracardiac ROS production, which was significantly reduced in the presence of 5 nM PSELT (data not shown). In addition, XO and AOX-1 expression was significantly increased under I/R as compared to SHAM, while post- conditioning with 5 nM PSELT abolished the expression of these oxidative stress markers (data not shown).

Cardioprotection induced by PSELT in obese rat

The endoplasmic reticulum, a cellular compartment where Selenoprotein T (SelT) is mainly localized, represents an important target of stress induced by lipid peroxidation. This process plays an essential role in the pathogenesis of obesity dependent-heart failure (Tripathi & Pandey, Front Immunol. 2012; 3: 240). Thus, we aimed to elucidate the cardioprotective effect of the SelT-derived peptide SelT43-52 (PSELT) in a rat model of obesity. Our preliminary results showed a significant increase of SelT expression in the heart of obese mice and rats, suggesting its involvement in the obese heart (data not shown). In order to study the hemodynamic aspect and to evaluate the infarct area, the effect of chronic administration of physiological doses of PSELT (15 μg/kg/day, a concentration similar to that able to induce ex vivo cardioprotection in control rats) have been tested in a rat model fed with high fat diet (HFD) that induces a metabolic syndrome. To this aim, rats were divided in 4 groups and fed with different diets for 12 weeks: i) normal diet (ND) (6.2% kcal from fat) treated with saline solution (NaCl 0.9%); ii) rats fed with HFD (60% kcal from fat) treated with saline solution; iii) rats fed with ND treated with the peptide in the last 4 weeks of the diet; iv) rats fed with HFD treated with the peptide in the last 4 weeks of the diet. After exposing the hearts to ischemia/reperfusion (I/R) ex vivo protocols, we assessed the systolic (i.e. developed left ventricular pressure, dLVP) and diastolic (i.e. left ventricular endo-diastolic pressure, LVEDP) post-ischemic recovery according to the Langendorff method, and we evaluated the infarct size (IS). The hemodynamic analyses indicate that PSELT was able to induce cardioprotection in both control and obese hearts. This is evidenced by a significant recovery of contractility (dLVP) (Fig. 1A) and reduction of IS (Fig. 1C), without changes in cardiac contracture (LVEDP) (Fig. IB) in the ND+PSELT and HFD+PSELT groups compared with ND and HFD groups, respectively.

These results unravel that chronic administration of physiological level of PSELT is able to protect the heart after ischemic injury in both normal and obesity-compromised conditions. This suggests that this peptide is an effective pharmacological agent able to protect the heart under metabolic syndrome condition.

Discussion:

In the present work, we described for the first time the ontogenetic expression of SelT in the rat heart. We observed that the protein is highly expressed in the embryonic heart, but decreases in newborn heart, and is absent in adult heart. Interestingly, the adult rat heart exposed to I/R injury re-expressed SelT, thus arguing for a role of SelT in adult heart under pathological conditions. We showed that administration of a SelT-derived peptide (PSELT) containing the active CVSU motif (U being a Sec residue) to the isolated and Langendorff-perfused adult heart after ischemia, elicited a strong cardioprotective effect. PSELT-dependent cardioprotection is accompanied by activation of the RISK pathway, inhibition of the apoptotic pathway, counteraction of intracellular ROS increase, and regulation of oxidative and nitrosative stress markers. These observations indicate that SelT is induced in adult heart after I/R likely to protect the cardiac tissue from harmful free radicals, and that a short peptide carrying the active catalytic site of SelT is able to protect the heart against I/R-induced oxidative stress and apoptosis.

Cardioexpression of SelT during ontogenesis and ischemic condition

Selenoproteins are increasingly recognized as essential for the development and function of nervous, endocrine and metabolic tissues (Petit et al. 2003; Papp et al, 2007; Tanguy et al 2011). Alteration of their synthesis is associated with major disorders, including muscular dystrophy, diabetes and thyroid disease (Fairweather-Tait et al, 2011). A first interesting finding of our study is that SelT is very abundant during embryonic ontogenesis of rat heart, while its expression was reduced in newborn and was undetectable in the adult heart. It has been shown that proliferative growth of the myocytes for cardiac development is observed during the entire embryonic and fetal period until birth and that myocytes are in principle only able to increase their volume after birth (van den Hoff et al., 1997), suggesting that SelT is required during early hyperplastic growth of cardiomyocytes. The ontogenetic expression pattern of SelT in heart is similar to that observed in the brain and other organs, such as kidney, liver, lung, skeletal muscle and adrenal gland. In fact, SelT expression is drastically reduced after cell differentiation in most tissues, as observed in chromaffin cells and Purkinje cells (Tanguy et al, 2011; Boukhzar et al, 2016; Castex et al, 2016). In contrast, its expression is maintained in several adult endocrine tissues, such as pituitary, thyroid or testis and is highly induced in metabolically active proliferating cells and in cells that are endowed with some plasticity and regenerative capacity (Tanguy et al, 2011; Prevost et al, 2013). Together, these observations suggest that SelT expression is associated with cell plasticity, a process which requires increased metabolism and energy but which generates free radicals. Our recent studies performed in neuronal cells showed that SelT is required for oxidative stress tolerance and that its biosynthesis is coupled to mitochondrial function during differentiation (Castex et al., 2016). Therefore, SelT expression during cardiac tissue ontogenesis is likely involved in cell protection and differentiation. However, in the maturing and adult heart, SelT expression becomes dispensable unless it is submitted to a noxious condition like I/R. This finding is reminiscent of the elevated expression of several selenoproteins in the nervous system (i.e. SelN, SelW, SelT and SelP and GPx) under stressful conditions, which is associated with protection against oxidative stress (Petit et al, 2003, Chung et al, 2009, Lee et al, 2008, Baek et al, 2005; Boukhzar et al, 2016).

Cardioprotective effect of PSELT

It has been reported that enzymes belonging to the selenoprotein family, e.g. GPx and TrxR in conjunction with Trx play a cardioprotective role after an ischemic injury (Rose and Hoffmann, 2015). For instance, GPxl inhibits I/R-induced apoptosis of cardiac myocytes in mice (Maulik et al, 1999), and its deletion causes heart and vascular dysfunction (Forgione et al., 2002). The TrxR/Trx system exerts a protective effect against I/R injury by reducing infarct size and improving ventricular function recovery (Yoshioka and Lee, 2014). Furthermore, Nakamura et al. (1998) showed that, in patients subjected to bypass surgery, Trx inactivation was deleterious in I/R injury. To date, there are no indications about the role exerted by SelT in cardioprotection. In the present study, we found that post-conditioning with PSELT protects the Langendorff perfused rat hearts exposed to I/R injury. This perfusion method of the heart represents the most widely used technique to probe the impact of ischemia/reperfusion and the effect of pharmacological tools on the heart pathophysiology, including the effect on intracellular signalling and adaption to clinically relevant stressful stimuli (Bell et al., 2011). We showed that, compared to the IS detected in hearts exposed to I/R alone (-75%), hearts post-conditioned with PSELT showed a remarkably reduced IS (~40%)τ IS reduction correlates with systolic function recovery and with the absence of contracture development.

Cardioprotection was more evident in ischemic hearts exposed to PSELT at the ECso concentration (5 nM) than in hearts exposed to the highest peptide concentration (100 nM). This is shown by the better post-ischemic recovery of dLVP observed in hearts exposed to 5 nM of SelT. In both cases, PSELT reduced contracture. The striking protection elicited by the peptide is indicated by the significant IS reduction, observed with both PSELT concentrations tested (5 and 100 nM). This concentration-dependent pattern resembles that described for major antioxidant agents that induce protection at lower doses and are less efficient or deleterious at higher doses (Rukkumani et al., 2004). Of note, PSELT cardioprotection is abolished when a control peptide where the Sec residue was replaced by an Ala, was administrated in post- ischemic condition, suggesting that Sec is important for cardioprotection. This is in agreement with the notion that the unique chemical features of Sec and selenium present in selenoproteins, characterized by a strong nucleophilicity at physiological pH, allow a more efficient redox exchange reactions.

PSELT triggers various intracellular signaling mechanisms to provide cardioprotection

It is known that post-conditioning protection involves components of the RISK cascade, such as PI3K-Akt and Erkl/2, and requires MitoKATP channel opening (Hausenloy et al., 2011). We here evaluated whether these intracellular mediators are involved in PSELT protective effects on the ischemic-reperfused heart. We found that PSELT protection was abolished by exposing the hearts to specific inhibitors of adenylate cyclase, PI3K, Erkl/2 and MitoKATP channels, suggesting the involvement of these pathways. Consistent with these results, we observed an increased phosphorylation of Akt, Erkl/2, and GSIQa-β after reperfusion with PSELT. Our data suggest that PSELT-induced protection takes place if the two pathways activated by cAMP via adenylate cyclase through PI3K or through Erkl/2, as well as mitoKATP channels are simultaneously activated during early reperfusion. Accordingly, mitochondria appear as the terminal effector of PSELT-induced post-conditioning protection. In addition, a decrease in p-38MAPK phosphorylation was observed. This kinase is known for its role in myocyte apoptosis (Maldonado et al., 2005). In cardiomyocytes, activation of p-38MAPK results in a rapid onset of lethal cardiomyopathy associated to cardiomyocyte hypertrophy (Streicher et al., 2010). Conversely, p-38MAPK inhibition may reduce cardiac hypertrophy, inhibit apoptosis and prevent the progression of heart failure (Streicher et al., 2010), thus indicating that PSELT could exert such effects as seen in the present study for its anti-apoptotic action. Indeed, we found that PSELT elicits cardioprotection by modulating the apoptotic signalling. It is known that changes in anti- and pro-apoptotic protein ratios result in inhibition or promotion of cell death (Ola et al., 2011). For instance, the Bcl-2 family, which includes anti- and pro-apoptotic mediators of proteins is a key regulator of apoptosis. Blc-2 prevents cyt c release and caspase activation, while Bax promotes these processes (Shi et al, 2010). Activated caspase-3 is one of the main apoptosis mediators that acts by cleaving other caspases and the anti-apoptotic Bcl-2 (Cullen and Martin., 2009). In agreement with the post- conditioning protection induced by PSELT, we observed that exposure of the ischemic heart to the peptide during the early reperfusion increased Bcl-2 and decreased Bax, activated caspase- 3 and cytosolic release of cyt c.

PSELT inhibits oxidative stress

In the ischemic heart, a notable increase in mitochondrial superoxide radical anion and hydrogen peroxide production leads to cell damage during reperfusion (Valdez et al, 2011). The contribution of ROS to cardiomyocyte cell death and apoptosis, typical of I/R injury, is well established (Valdez et al., 2011). To maintain a redox balance, cells engage several reducing enzymes including members of the selenoprotein family, such as GPx or TrxR, which play instrumental roles in cell survival and homeostasis (Bellinger et al., 2009). These enzymes exert antioxidant activities that impact diverse cellular functions including redox equilibrium, protein folding and Ca²⁺ homeostasis (Bellinger et al., 2009). On the basis of these observations, we investigated the influence of PSELT on the myocardial redox balance by analyzing the intracardiac oxidative and nitrosative stress (Penna et al, 2011) and by evaluating enzymes and factors involved in free radical production such as XO (Raghuvanshi et al., 2007) and AOX-1 (Neumeier et al, 2006). Interestingly, compared to I/R condition, hearts perfused with PSELT after ischemia showed a significant reduction of all the oxidative/nitrosative markers used. It is known that XO is important in ischemic conditions. In fact, ATP depletion and the subsequent loss of membrane Ca²⁺ gradient increases Ca²⁺ levels and activates Ca²⁺-dependent proteases which cause selective proteolysis of the dehydrogenase into XO. This in turn acts on both hypoxanthine and xanthine at the expense of molecular oxygen to produce superoxide ion (Dianat et al., 2016). Accordingly, in the ischemic heart, as well as in myocardial infarction, XO may importantly contribute to free radical-mediated damage (Raghuvanshi et al. 2007). AOX-1 is a member of the molybdo-flavoenzyme family of proteins, which catalyzes the oxidation of a variety of aldehydes, leading to the production of hydrogen peroxide. Under certain conditions, AOX1 can catalyze the formation of the superoxide free radical, and this suggests its involvement in the I/R heart damage (Berry and Hare; 2004). It is of interest that exposure of the reperfused heart to PSELT is accompanied by a decrease of XO and AOX-1. Of note, our results agree with those reported by Boukhzar et al. (2016) which showed that SelT silencing affects oxidative/nitrosative stress and survival of dopaminergic neurons. In fact, thanks to its thioredoxin-like motif, SelT was effective in catalyzing the reduction of oxidized substrates (Boukhzar et al, 2016).

Our findings on the effect of PSELT on components of the redox state in the ischemic heart suggests that SelT is a novel essential effector of the intracardiac antioxidant system able to counteract free radical damage responsible for cell death during I/R.

Conclusion:

In conclusion, by decreasing infarct size and improving post-ischemic cardiac function, PSELT counteracted the damages induced by myocardial reperfusion. This protective action required the activation of prosurvival kinases, mitoKATP channel and a control of the apoptotic process and redox balance. Our observations of the beneficial effects of PSELT, here reported for the first time, provide new information on the biological significance of this protein for cardiac function and protection. They also pave the way for future studies aimed to investigate the possible clinical relevance of PSELT, which might represent a new class of drugs to be tested for reducing cardiac I/R injury. This is important in a medical context since it could allow the development of new adjunctive therapies to be coupled with the reperfusion to reduce morbidity and mortality (Schwartz Longacre et al., 2011).

REFERENCES: Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

1. Atkins JF, Gesteland RF. The twenty-first amino acid. Nature. 2000; 407(6803):463, 465.

2. Baek IJ, Yon JM, Lee BJ, Yun YW, Yu WJ, Hong JT, Ahn B, Kim YB, Kim DJ, Kang JK, Nam SY. Expression pattern of cytosolic glutathione peroxidase (cGPx) mRNA during mouse embryogenesis. Anat Embryol (Berl). 2005 Apr;209(4):315-21. Epub 2005 Mar 24.

3. Beckett GJ, Arthur JR. Selenium and endocrine systems. J Endocrinol. 2005

Mar;184(3):455-65.

4. Bell RM, Mocanu MM, Yellon DM. Retrograde heart perfusion: the Langendorff technique of isolated heart perfusion. J Mol Cell Cardiol. 2011;50(6):940-50. doi: 10.1016/j .yjmcc.2011.02.018.

5. Bellinger FP, Raman AV, Reeves MA, Berry MJ. Regulation and function of selenoproteins in human disease. Biochem J. 2009 Jul 29;422(1): 11-22. doi: 10.1042/BJ20090219.

6. Benstoem C, Goetzenich A, Kraemer S, Borosch S, Manzanares W, Hardy G, Stoppe C. Selenium and its supplementation in cardiovascular disease—what do we know? Nutrients. 2015 Apr 27;7(5):3094-l 18. doi: 10.3390/nu7053094.

7. Berry CE, Hare JM. Xanthine oxidoreductase and cardiovascular disease: molecular mechanisms and pathophysiological implications. J Physiol. 2004 Mar 16;555(Pt 3):589-606. Epub 2003 Dec 23.

8. Bosl M. R., Takaku K., Oshima M., Nishimura S., and Taketo M. M. (1997) Early embryonic lethality caused by targeted disruption of the mouse selenocysteine tRNA gene

(Trsp). Proc. Natl. Acad. Sci. U. S. A. 94, 5531-5534

9. Boukhzar L, Hamieh A, Carrier D, Tanguy Y, Alsharif I, Castex M, Arabo A, El Hajji S, Bonnet JJ, Errami M, Falluel-Morel A, Chagraoui A, Lihrmann I, Anouar Y. Selenoprotein T Exerts an Essential Oxidoreductase Activity That Protects Dopaminergic Neurons in Mouse Models of Parkinson's Disease. Antioxid Redox Signal. 2016 Apr 10;24(l l):557-74. doi: 10.1089/ars.2015.6478. Epub 2016 Mar 16.

10. Castex MT, Arabo A, Benard M, Roy V, Le Joncour V, Prevost Gl, Bonnet JJ, Anouar Y, Falluel-Morel A. Selenoprotein T Deficiency Leads to Neurodevelopmental Abnormalities and Hyperactive Behavior in Mice. Mol Neurobiol. 2016 Nov;53(9):5818-5832. doi: 10.1007/sl2035-015-9505-7. Epub 2015 Oct 26.

11. Cerra MC, De Iuri L, Angelone T, Corti A, Tota B. Recombinant N-terminal fragments of chromogranin-A modulate cardiac function of the Langendorff-perfused rat heart. Basic Res Cardiol. 2006 Jan;101(l):43-52. Epub 2005 Sep 12.

12. Chung YW, Jeong D, Noh OJ, Park YH, Kang SI, Lee MG, Lee TH, Yim MB, Kim IY. Antioxidative role of selenoprotein W in oxidant-induced mouse embryonic neuronal cell death. Mol Cells. 2009 May 31;27(5):609-13. doi: 10.1007/sl0059-009-0074-3. Epub 2009 May 15.

13. Cullen SP, Martin SJ. Caspase activation pathways: some recent progress. Cell

Death Differ. 2009 Jul; 16(7): 935 -8. doi: 10.1038/cdd.2009.59.

14. Dianat M, Radmanesh E, Badavi M, Mard SA, Goudarzi G. Disturbance effects of PM₁₀ on iNOS and eNOS mRNA expression levels and antioxidant activity induced by ischemia-reperfusion injury in isolated rat heart: protective role of vanillic acid. Environ Sci Pollut Res Int. 2016 Mar;23(6):5154-65. doi: 10.1007/sl 1356-015-5759-x. Epub 2015 Nov 10.

15. Driscoll DM, Copeland PR. Mechanism and regulation of selenoprotein synthesis. Annu Rev Nutr. 2003;23: 17-40. Epub 2003 Jan 8.

16. Fairweather-Tait SJ, Bao Y, Broadley MR, Collings R, Ford D, Hesketh JE, Hurst R. Selenium in human health and disease. Antioxid Redox Signal. 2011 Apr l;14(7): 1337-83. doi: 10.1089/ars.2010.3275. Epub 2011 Jan 6.

17. Forgione MA, Cap A, Liao R, Moldovan NI, Eberhardt RT, Lim CC, Jones J, Goldschmidt-Clermont PJ, Loscalzo J. Heterozygous cellular glutathione peroxidase deficiency in the mouse: abnormalities in vascular and cardiac function and structure. Circulation 2002 Aug 27;106(9): 1154-8.

18. Grumolato L, Ghzili H, Montero-Hadjadje M, Gasman S, Lesage J, Tanguy Y,

Galas L, Ait-Ali D, Leprince J, Guerineau NC, Elkahloun AG, Fournier A, Vieau D, Vaudry H, Anouar Y. Selenoprotein T is a PACAP -regulated gene involved in intracellular Ca2+ mobilization and neuroendocrine secretion. FASEB J. 2008 Jun;22(6): 1756-68. doi: 10.1096/fj.06-075820. Epub 2008 Jan 15.

19. Hamieh A., Carrier D., Abid H., Bucharles C, Calas A., Burel C, Jehan C,

Grumolato L., Landry M., Lerouge P., Anouar Y. and Lihrmann I. Selenoprotein T is a novel OST subunit that regulates UPR signaling and hormone secretion. EMBO Rep 2017 (in press).

20. Hatfield DL, Gladyshev VN. How selenium has altered our understanding of the genetic code. Mol Cell Biol. 2002 Jun;22(l l):3565-76. 21. Hausenloy DJ, Lecour S, Yellon DM. Reperfusion injury salvage kinase and survivor activating factor enhancement prosurvival signaling pathways in ischemic postconditioning: two sides of the same coin. Antioxid Redox Signal. 2011 Mar 1;14(5):893- 907. doi: 10.1089/ars.2010.3360. Epub 2010 Oct 26.

22. Kryukov GV, Castellano S, Novoselov SV, Lobanov AV, Zehtab O, Guigo R,

Gladyshev VN. Characterization of mammalian selenoproteomes. Science. 2003 May 30;300(5624): 1439-43.

23. Labunskyy VM, Hatfield DL, Gladyshev VN. Selenoproteins: molecular pathways and physiological roles. Physiol Rev. 2014 Jul;94(3):739-77. doi: 10.1152/physrev.00039.2013.

24. Lee SR, Yon JM, Baek IJ, Kim MR, Park CG, Lee BJ, Yun YW, Nam SY. Spatiotemporal expression of the selenoprotein P gene in postimplantational mouse embryos. Int J Dev Biol. 2008;52(7): 1005-11. doi: 10.1387/ijdb.082656sl.

25. Lescure A, Rederstorff M, Krol A, Guicheney P, Allamand V. Selenoprotein function and muscle disease. Biochim Biophys Acta. 2009 Nov;1790(l 1): 1569-74. doi:

10.1016/j .bbagen.2009.03.002

26. Maldonado C, Cea P, Adasme T, Collao A, Diaz-Araya G, Chiong M, Lavandero S. IGF-1 protects cardiac myocytes from hyperosmotic stress-induced apoptosis via CREB. Biochem Biophys Res Commun. 2005 Nov 4;336(4): 1112-8.

27. Mali VR, Pan G, Deshpande M, Thandavarayan RA, Xu J, Yang XP,

Palaniyandi SS. Cardiac Mitochondrial Respiratory Dysfunction and Tissue Damage in Chronic Hyperglycemia Correlate with Reduced Aldehyde Dehydrogenase-2 Activity. PLoS One. 2016 Oct 13;l l(10):e0163158. doi: 10.1371/journal.pone.0163158. eCollection 2016.

28. Maulik N, Yoshida T, Das DK. Regulation of cardiomyocyte apoptosis in ischemic reperfused mouse heart by glutathione peroxidase. Mol Cell Biochem. 1999

Jun;196(l-2): 13-21.

29. Nakamura H, Vaage J, Valen G, Padilla CA, Bjornstedt M, Holmgren A. Measurements of plasma glutaredoxin and thioredoxin in healthy volunteers and during open- heart surgery. Free Radic Biol Med. 1998 May;24(7-8): 1176-86.

30. Neumeier M, Weigert J, Schaffler A, Weiss TS, Schmidl C, Buttner R,

Bollheimer C, Aslanidis C, Scholmerich J, Buechler C. Aldehyde oxidase 1 is highly abundant in hepatic steatosis and is downregulated by adiponectin and fenofibric acid in hepatocytes in vitro. Biochem Biophys Res Commun. 2006;350(3):731-5. 31. Ola MS, Nawaz M, Ahsan H. Role of Bcl-2 family proteins and caspases in the regulation of apoptosis. Mol Cell Biochem. 2011 May;351(l-2):41-58. doi: 10.1007/sl 1010- 010-0709-x. Epub 2011 Jan 6.

32. Pagliaro P, Mancardi D, Rastaldo R, Penna C, Gattullo D, Miranda KM, Feelisch M, Wink DA, Kass DA, Paolocci N (2003) Nitroxyl affords thiol-sensitive myocardial protective effects akin to early preconditioning. Free Radic Biol Med 34(1):33— 43. doi: 10.1016/S0891 -5849(02)01179-6

33. Papp LV, Lu J, Holmgren A, Khanna KK: From selenium to selenoproteins: synthesis, identity, and their role in human health. Antioxid Redox Signal. 2007, 9: 775-806. 10.1089/ars.2007.1528

34. Pasqua T, Corti A, Gentile S, Pochini L, Bianco M, Metz-Boutigue MH, Cerra MC, Tota B, Angelone T. Full-length human chromogranin-A cardioactivity: myocardial, coronary, and stimulus-induced processing evidence in normotensive and hypertensive male rat hearts. Endocrinology. 2013 Sep;154(9):3353-65. doi: 10.1210/en.2012-2210. Epub 2013 Jun 10.

35. Pasqua T, Filice E, Mazza R, Quintieri AM, Carmela Cerra M, Iannacone R, Melfi D, Indiveri C, Gattuso A, Angelone T. Cardiac and hepatic role of r-AtHSP70: basal effects and protection against ischemic and sepsis conditions. J Cell Mol Med. 2015 Jul;19(7): 1492-503. doi: 10.1111/jcmm. l2491. Epub 2015 Apr 23

36. Penna C, Perrelli MG, Tullio F, Mora F, Parisella ML, Merlino A, Pagliaro P.

Post-ischemic early acidosis in cardiac postconditioning modifies the activity of antioxidant enzymes, reduces nitration, and favors protein S-nitrosylation. Pflugers Arch. 2011 Aug;462(2):219-33. doi: 10.1007/s00424-011-0970-1. Epub 2011 May 5.

37. Penna C, Pasqua T, Perrelli MG, Pagliaro P, Cerra MC, Angelone T. Postconditioning with glucagon like peptide-2 reduces ischemia/reperfusion injury in isolated rat hearts: role of survival kinases and mitochondrial KATP channels. Basic Res Cardiol. 2012 Jul;107(4):272. doi: 10.1007/s00395-012-0272-6. Epub 2012 Jun 15.

38. Petit N, Lescure A, Rederstorff M, Krol A, Moghadaszadeh B, Wewer UM, Guicheney P. Selenoprotein N: an endoplasmic reticulum glycoprotein with an early developmental expression pattern. Hum Mol Genet. 2003 May 1; 12(9): 1045-53.

39. Pitts MW, Byrns CN, Ogawa-Wong AN, Kremer P, Berry MJ. Selenoproteins in nervous system development and function. Biol Trace Elem Res. 2014 Dec; 161(3) :231-45. doi: 10.1007/sl2011-014-0060-2. Epub 2014 Jul 1. 40. Pitts MW, Hoffmann PR. Endoplasmic reticulum-resident selenoproteins as regulators of calcium signaling and homeostasis. Cell Calcium. 2017 May 4. pii: S0143- 4160(17)30047-7. doi: 10.1016/j.ceca.2017.05.001.

41. Prevost G, Arabo A, Jian L, Quelennec E, Carrier D, Hassan S, Falluel-Morel A, Tanguy Y, Gargani S, Lihrmann I, Kerr-Conte J, Lefebvre H, Pattou F, Anouar Y. The PACAP- regulated gene selenoprotein T is abundantly expressed in mouse and human β-cells and its targeted inactivation impairs glucose tolerance. Endocrinology. 2013 Oct;154(10):3796-806. doi: 10.1210/en.2013-1167. Epub 2013 Aug 2.

42. Raghuvanshi R, Kaul A, Bhakuni P, Mishra A, Misra MK. Xanthine oxidase as a marker of myocardial infarction. Indian J Clin Biochem. 2007;22(2):90-2. doi:

10.1007/BF02913321.

43. Rayman MP. Selenium and human health. Lancet. 2012;379(9822): 1256-68. doi: 10.1016/S0140-6736(11)61452-9.

44. Rose AH, Hoffmann PR. Selenoproteins and cardiovascular stress. Thromb Haemost. 2015 Mar;l 13(3):494-504. doi: 10.1160/TH 14-07-0603.

45. Rukkumani R, Aruna K, Varma PS, Menon VP. Influence of ferulic acid on circulatory prooxidant-antioxidant status during alcohol and PUFA induced toxicity. J Physiol Pharmacol. 2004 Sep;55(3):551-61.

46. Schwartz Longacre L, Kloner RA, Arai AE, Baines CP, Bolli R, Braunwald E, Downey J, Gibbons RJ, Gottlieb RA, Heusch G, Jennings RB, Lefer DJ, Mentzer RM, Murphy

E, Ovize M, Ping P, Przyklenk K, Sack MN, Vander Heide RS, Vinten-Johansen J, Yellon DM; National Heart, Lung, and Blood Institute, National Institutes of Health. New horizons in cardioprotection: recommendations from the 2010 National Heart, Lung, and Blood Institute Workshop. Circulation. 2011 Sep 6; 124(10): 1172-9. doi: 10.1161/CIRCULATIONAHA. l 11.032698.

47. Schweizer U, Fradejas-Villar N. Why 21? The significance of selenoproteins for human health revealed by inborn errors of metabolism. FASEB J. 2016 Nov;30(l 1):3669-3681. Epub 2016 Jul 29.

48. Shi L, Chen J, Yang J, Pan T, Zhang S, Wang Z. MiR-21 protected human glioblastoma U87MG cells from chemotherapeutic drug temozolomide induced apoptosis by decreasing Bax/Bcl-2 ratio and caspase-3 activity. Brain Res. 2010 Sep 17; 1352:255-64. doi: 10.1016/j.brainres.2010.07.009. Epub 2010 Jul 13. 49. Streicher JM, Ren S, Herschman H, Wang Y. MAPK-activated protein kinase-2 in cardiac hypertrophy and cyclooxygenase-2 regulation in heart. Circ Res. 2010 Apr 30;106(8): 1434-43. doi: 10.1161/CIRCRESAHA.109.213199. Epub 2010 Mar 25.

50. Tanguy Y, Falluel-Morel A, Arthaud S, Boukhzar L, Manecka DL, Chagraoui A, Prevost G, Elias S, Dorval-Coiffec I, Lesage J, Vieau D, Lihrmann I, Jegou B, Anouar Y.

The PACAP-regulated gene selenoprotein T is highly induced in nervous, endocrine, and metabolic tissues during ontogenetic and regenerative processes. Endocrinology. 2011;152(l l):4322-35. doi: 10.1210/en.2011-1246.

51. Tripathi YB & Pandey V. Obesity and endoplasmic reticulum (ER) stresses. Frontiers in Immunology. 2012; 3: 240. doi: 10.3389/fimmu.2012.00240.

52. Valdez LB, Zaobornyj T, Bombicino S, Iglesias DE, Boveris A, Donato M, DAnnunzio V, Buchholz B, Gelpi RJ. Complex I syndrome in myocardial stunning and the effect of adenosine. Free Radic Biol Med. 2011 Sep 15;51(6): 1203-12. doi: 10.1016/j.freeradbiomed.2011.06.007. Epub 2011 Jun 30.

53. van den Hoff MJ, Deprez RH, Monteiro M, de Boer PA, Charles R, Moorman AF.

Developmental changes in rat cardiac DNA, RNA and protein tissue base: implications for the interpretation of changes in gene expression. J Mol Cell Cardiol. 1997 Feb;29(2):629-39.

54. Yoshioka J, Lee RT. Thioredoxin-interacting protein and myocardial mitochondrial function in ischemia-reperfusion injury. Trends Cardiovasc Med. 2014 Feb;24(2):75-80. doi: 10.1016/j.tcm.2013.06.007. Epub 2013 Jul 26.

Claims

CLAIMS:

1. A method for providing cardioprotection in a subject who experienced a myocardial infarction comprising administering the subject with a therapeutically effective amount of the PSELT peptide having the formula of:

2. The method of claim 1 wherein the PSELT peptide is suitable for reducing infarct size, reducing ischemia-reperfusion injury, reducing hypoxia induced apoptosis/necrosis and/or preventing cardiomyocyte cell death.

3. The method of claim 1 wherein the PSELT peptide is administered to a subject having one or more signs or symptoms of acute myocardial infarction injury such as chest pain described as a pressure sensation, fullness, or squeezing in the mid portion of the thorax; radiation of chest pain into the jaw or teeth, shoulder, arm, and/or back; dyspnea or shortness of breath; epigastric discomfort with or without nausea and vomiting; and diaphoresis or sweating.

4. The method of claim 1 wherein the PSELT peptide is administered simultaneously or sequentially (i.e. before or after) with a revascularization procedure performed on the subject.

5. The method of claim 4 wherein the subject is administered with the PSELT peptide as a bolus dose immediately prior to the revascularization procedure.

6. The method of claim 4 wherein the subject is administered with the PSELT peptide for a time period selected from the group consisting of at least 3 hours after a revascularization procedure; at least 5 hours after a revascularization procedure; at least 8 hours after a revascularization procedure; at least 12 hours after a revascularization procedure; at least 24 hours after a revascularization procedure.

7. The method of claim 4 wherein the revascularization procedure is selected from the group consisting of percutaneous coronary intervention; balloon angioplasty; insertion of a bypass graft; insertion of a stent; directional coronary atherectomy; treatment with one or more thrombolytic agent(s); and removal of an occlusion.