BNIP3 PEPTIDES FOR THE TREATMENT OF REPERFUSION INJURY

Abstract

The invention provides peptides capable of inhibiting the individual activity and communication between BNIP3, BAX, and mitochondrial pathways. These peptides can be used in methods for treating a disease or condition in a subject where preventing cell damage and cell death is desirable.

Description

BNIP3 PEPTIDES FOR THE TREATMENT OF REPERFUSION INJURY FIELD OF INVENTION The present invention relates to the treatment of reperfusion injury. In particular, the present invention provides peptides derived from BNIP3 that prevent cell damage and cell death by reducing the activity of BNIP3 and BAX in the mitochondria. BACKGROUND OF THE INVENTION Occlusion of a blood vessel causes the cessation of blood flow to part of the tissue, leading, in particular, to insufficient oxygen supply, reduced nutrient availability, and inadequate removal of metabolic waste products, causing cellular chaos and subsequent cell death. While acute occlusion is unpredictable and unavoidable, restoring vessel patency is feasible and essential for patient outcomes.1 A timely reperfusion regimen is the recommended therapy, but rapid restoration of blood flow, and particularly oxygen delivery, necessitates tissue injury without currently available treatment. The early phase of reperfusion is characterized by high oxygen levels leading to hyperoxic conditions, a surge of reactive oxygen species, and elevated calcium levels without acidosis.The pathology of reperfusion injury has been recognized in the heart, brain, liver, and kidney and is associated with serious clinical manifestations, including myocardial hibernation, acute heart failure, cerebral dysfunction, gastrointestinal dysfunction, renal dysfunction, systemic inflammatory response syndrome, and multiple organ dysfunction syndrome. Consequently, reperfusion injury is a critical medical condition that poses a significant therapeutic challenge. Myocardial infarction (MI) is a sudden, temporally unpredictable event in which reperfusion is essential for survival but determines up to 50% of the final infarct size. This fate also applies to transplanted organs.Myocardial infarction (MI) is the most common cause of heart failure; therefore, therapeutic interventions to reduce reperfusion injury present an opportunity to salvage viable myocardium, limit MI size, preserve cardiac function, and impact the incidence of heart failure.3 In reperfusion-induced infarction progression, two forms of cell death—necrosis and apoptosis—play essential roles. A predominance of necrotic cardiomyocyte death was observed in the initial infarct area. Necrosis induces subsequent tissue responses, such as inflammation, matrix remodeling, and fibrosis.4 Apoptosis occurs in the infarct and pen-infarct area and is a major component of early post-infarction remodeling.5 Cardiac damage is also a critical problem for cancer patients. Due to advances in screening and treatment strategies, the cancer survivor population has grown steadily over the past three decades. Overall 5-year survival improved to 50–70% at 10-year follow-up. As a result, there is an increasing prevalence of cancer therapy side effects, especially cardiovascular toxicity. Conventional chemotherapy (e.g., anthracyclines) is a commonly used therapy for many cancers that has been widely recognized as contributing to both asymptomatic and symptomatic decreases in left ventricular ejection fraction (LVEF), cardiomyopathy, and heart failure (HF).Cancer-mediated cardiomyopathy (cQnn / zznz / q / YiAi) is characterized by a dose-dependent decline in left ventricular systolic function mediated by reactive oxygen species (ROS) that is usually irreversible. Currently, there are no effective approaches to prevent or reduce cardiotoxic side effects (e.g., decreased cardiac function, cardiomyopathy, etc.) in patients undergoing anthracillin chemotherapy or other cancer therapies. Several studies have aimed to address this medical need but have revealed only partial results / benefits. The CECCY trial failed to demonstrate a benefit of carvedilol in breast cancer patients; however, it did show protection by reducing troponin levels.The PRADA trial evaluated candesartan and metoprolol and revealed a significant benefit for candesartan in preventing cardiomyopathy, although the study was not sufficiently powered according to the current definition of cardiotoxicity.7 The primary outcome of the MANTICORE trial was a change in left ventricular diameter. Neither the beta-blocker nor the ACE inhibitor had a significant impact on this outcome measure, but they significantly prevented heart failure as a secondary outcome.8 Taken together, while the existing literature points to a potential benefit of heart failure therapy, no study has yet thoroughly evaluated the value of next-generation heart failure therapy, as defined by current guidelines, in preventing cardiotoxicity in cancer patients. Mitochondria are central to both necrotic and apoptotic signaling.9 These include disruption of electron transport, oxidative phosphorylation, and ATP synthesis, DNA fragmentation, protein and lipid damage, and excessive ROS generation. The defining event of mitochondrial necrosis is the opening of a pore in the inner mitochondrial membrane (IMM), the so-called mitochondrial permeability transition pore (mPTP). This creates an energy collapse and a rapid exchange of solutes with an influx of osmolytes into the mitochondria. Subsequent matrix swelling leads to rupture of the outer mitochondrial membrane, cell swelling, and cell disruption.10 Necrotic stimuli, such as Ca2+, are suggested to activate the opening of the mPTP and may be potentiated by ROS.10 Despite extensive research, the components of the mPTP remain unknown, with studies in transgenic animals excluding several putative components, including the adenine nucleotide translocase,11 the voltage-gated anion channel,12 the mitochondrial phosphate carrier13 (SLC25A3), and cyclophilin D.14More recently, it has been suggested that the c subunit of ATP synthase forms the pore in the inner membrane.15,16 Preventing mPTP opening using pharmacological inhibitors such as cyclosporine A has been reported to reduce infarct size in preclinical models of I / R injury.17,18 In larger clinical trials, its effect was neutral.19 The mitochondrial-targeted peptide elamipretide (formerly called Bendavia or MTP-131), as well as the mitochondrial-targeted drug TR040303, have been shown in animal studies to reduce infarct size by attenuating mitochondrial-derived ROS production when administered at the onset of reperfusion.20,22 However, in studies of patients with STEMI, intravenous amipretide20 and TRO40303, both administered before PCI, fail to reduce infarct size23.In particular, more adverse events were reported in patients receiving TR040303 compared to the placebo arm, thus limiting the clinical application of this therapeutic approach. Antioxidants and Na+ / H+ exchange inhibitors also proved ineffective. I bCQnn / 77n7 / q / YIAI such as superoxide dismutase25, and various antineutrophil antibodies26·27. Apoptotic cell death that occurs in the infarct and peri-infarct area is initiated by permeabilization of the outer mitochondrial membrane (OMM) which allows the release of pro-apoptotic proteins, such as cytochrome c, apoptosis induction factor, SMAC / DIABLO (second mitochondrial-derived activator of caspases / direct IAP-binding protein with low Pl), and endonuclease G from the intermembrane space into the cytosol which leads to the initiation of cell death cascades through caspases and DNA fragmentation28-30. The BCL-2 pro-death proteins BNIP3 (BCL-2-interacting protein 3 and 19 kDa adenovirus E1B protein) and BAX (BCL-2-associated protein X) induce MOM permeabilization and represent the mediator and 3' effector of mitochondrial apoptosis by translocation in the MOM and heterodimer formation3135. In addition, BNIP3 and BAX regulate MIM perturbation, thus functioning as crucial activators of necrosis536. The present invention addresses the need for optimal improvement of both acute injury in the central infarct zone and subsequent cell death in immediately surrounding areas by providing an inhibitor of the interaction activity of BNIP3 and BAX, which disrupts intra- and inter-pathway communication between BNIP3, BAX and mitochondria as individuals or triangles to treat reperfusion injury in the heart, brain, liver and kidney and other indications where mitochondrial disturbance leads to cell damage and cell death, such as, for example, heart failure, organ transplantation, cardiac arrest or due to surgical and pharmacological intervention, as well as stroke-induced cardiac injury, cancer and cancer therapy. BRIEF DESCRIPTION OF THE INVENTION The present invention provides peptides that bind to BNIP3 and BAX as monomers, as well as to their homo- and hetero-oligomers, exhibiting broad-spectrum activity by bypassing individual and oligomer activities. Efficacy is not restricted to a single organ or species, as evidenced by the protection of heart and brain tissue, as well as human induced pluripotent stem cell-derived ventricular cardiomyocytes, against reperfusion injury. Myocardial infarct size was also markedly reduced in pigs. The peptides are derived from the A-terminal portion of BNIP3 and an 8-amino-acid stretch consisting of the most active amino acids 13 to 20 of BNIP3. It was highly surprising that such a short peptide was able to inhibit the activities of BNIP3 and BAX, block the formation of homo- and hetero-oligomerization of these proteins, and induce conformational changes in these homo- and hetero-oligomers within the cell. Certain mutations in the peptide sequence even enhanced its efficacy. In view of these results, the present invention provides in a first aspect a peptide comprising (i) a cell uptake signal; and (ii) a fragment of BNIP3 comprising positions 13 to 20 of BNIP3 or an amino acid sequence derived therefrom. The peptide has a length of 50, specifically 40, amino acids or less. I bCQnn / 77n7 / q / YIAI In a second aspect, the present invention provides a pharmaceutical composition comprising the peptide according to the first aspect and its use in the treatment of reperfusion-related and / or mitochondrial-related disorders, as well as cancer therapy-induced cardiotoxicity and prevention thereof. In a third aspect, the invention provides a method for preventing cell damage or cell death, comprising bringing the cell into contact with the peptide according to the first aspect. (i) a cell uptake signal; and (ii) a fragment of BNIP3 comprising positions 13 to 20 of BNIP3 or an amino acid sequence derived therefrom. The invention further relates, in a fourth aspect, to a method of examining a compound suitable for the prevention of reperfusion injury and / or mitochondrial-related disorders and / or cancer therapy-induced cardiotoxicity, comprising (i) providing one or more candidate compounds; (i) determine the ability of the candidate compounds to interfere with the binding of BNIP3 and BAX; (iii) select those candidate compounds that interfere with the binding of BNIP3 and BAX. Other objects, features, advantages, and aspects of the present invention will become apparent to those skilled in the art from the following description and appended claims. However, it should be understood that the following description, the appended claims, and the specific examples indicating preferred embodiments of the application are provided for illustrative purposes only. Several changes and modifications within the spirit and scope of the described invention will become readily apparent to those skilled in the art from reading the following. BRIEF DESCRIPTION OF THE FIGURES Figures 1A to 1C. BNIP3 deletion in mice reduces myocardial infarct size in vivo. Figure 1A Schematic of the in vivo ischemia / reperfusion model. Figure 1B Schematic of a cardiac section representing a non-ischemic area (remote), an ischemic area (area at risk, AAR), and an infarct area (white, embedded in AAR). Figure 1C Infarct sizes after 24 h of reperfusion in wild-type, BNIP3-deficient (Bnip3'), and Bnip3-treated mice with the indicated doses of TAT-BNIP3 (n = 3-7 mice). AAR - area at risk; Inf - infarct. Data are mean ± week. Statistical analyses are two-way analysis of variance (ANOVA) with Bonferroni correction. Figures 2A to 2D. BNIP3 is a mediator of BAX activity in myocardial reperfusion injury. Mice were exposed to the indicated reperfusion times in vivo after vessel occlusion. Increased mitochondrial BNIP3 levels (Figure 2A) and mitochondrial BAX concentrations (Figure 2B) were observed in the at-risk area at baseline and after 10 minutes of reperfusion (n = 5–7 mice). Figure 2C. Western blot monitoring revealed that BNIP3 co-immunoprecipitates with BAX at baseline and after 10 and 30 minutes of reperfusion. Figure 2D. Quantification of mitochondrial BAX levels in the at-risk area of ​​BNIP3-deficient (Bnip3) mice at baseline and after 10 minutes of reperfusion in untreated and BNIP3-treated mice (n = 3 mice). BAX translocation is dependent on the presence of BNIP3. I bCQnn / 77n7 / q / YIAI data are means ± sem Statistical analyses are two-way analysis of variance (ANOVA) with Bonferroni correction. Figures 3A to 3K. Interaction sites, secondary structure, and in silico docking. Figure 3A Schematic of the experimental setup (JPT, Berlin, Germany). Figure 3B Heat map representation of BNIP3 incubation with the BAX peptide library showing the α5, α6 and α7+α8 helices as interaction sites. Color coding ranges from white (0 or low intensity) to light gray (medium intensity) to dark gray (high intensity). Figure 3C The three-dimensional structure model of BNIP3 obtained by homology modeling using Modeller 9.15. Figure 3D Circular dichroism (CD) spectroscopic analysis of BNIP3. Figures 3E and 3F Vignette representations of BAX / BNIP3 interactions with indicated binding sites resulting from in silico docking experiments using HADDOCK. Figure 3G TAT sequence structure. Figure 3H Structure of the BNIP320A structure. Figure 3I Structure of the BNIP3-20C structure.Figure 3J Representative images of the transmural distribution of TAT-BNIP3-20A (green) after 10 min of reperfusion in vivo. Scale bars, 1 mm. Figure 3K Western blot monitoring of TAT-BNIP3-20A co-immunoprecipitated with BNIP3 after 5 min of reperfusion following vessel occlusion in vivo. Figures 4A to 4D. TAT-BNIP3-20A reduces myocardial reperfusion injury in vivo. Figure 4A Schematic of the in vivo myocardial infarction model. Mice were exposed to the indicated reperfusion time durations following in vivo vessel occlusion. The peptide was administered into the left ventricle 5 min before reperfusion. Figure 4B Infarct sizes after 24 h of reperfusion in wild-type mice treated with vehicle, the control peptide TAT-BNIP3-20C, and TAT-BNIP3-20a (n = 7–10 mice). TAT-BNIP3-20A significantly reduces infarct size. BNIP3-20C and the vehicle are not effective in attenuating infarct size. TAT-BNIP3-20A inhibits the interaction of BNIP3 with mitochondria after 10 minutes of reperfusion (Figure 4C) and caspase-3 activity after 4 h of reperfusion (Figure 4D), whereas the control peptide TAT-BNIP3-20C does not (n = 6 mice). Data are mean ± weekStatistical analyses are two-way analysis of variance (ANOVA) with Bonferroni correction. Figures 5A to 5D. Figure 5A Schematic of in vitro reoxygenation (study design in human ventricular cardiomyocytes derived from human induced pluripotent stem cells (humanCMs). Human induced pluripotent stem cells were exposed to normoxia and 2 h of reoxygenation after hypoxia and treated with TAT-BNIP3-20A and the control peptide BNIP3-20C. Figure 5B TAT-BNIP3-20A markedly inhibits the interaction of BNIP3 with mitochondria. Figure 5C Representative images of apoptotic, necrotic, and healthy human induced pluripotent stem cells. Scale bars, 1 mm (left), 200 pm (right). TAT-BNIP3-20A potently prevents human induced pluripotent stem cell death during reoxygenation. Figure 5D Representative images of depolarized mitochondria. Healthy nucleus. TAT-BNIP3-20A decreases the reoxygenation-induced loss of mitochondrial inner membrane potential.Scale bars, 400 pm (left), 100 pm (right). The control peptide TAT-BNIP3-20C was not effective in inhibiting the interaction of BNIP3 with mitochondria, loss of mitochondrial inner membrane potential, and cell death. Data are mean ± SEM. Statistical analyses are two-way analysis of variance (ANOVA) with Bonferroni correction. I bCQnn / 77n7 / q / YIAI Figures 6A to 6C. Figure 6A Structure of the BNIP3-8B sequence. Figure 6B Structure of the BNIP3-8C sequence. Figure 6C Spectroscopic analysis of circular dichroism (CD) of BNIP3-8B. Figures 7A to 7B. Figure 7A Uptake of fluorescently labeled TAT-BNIP3-8B in different organs 5 min after reperfusion following vessel occlusion. TAT-BNIP3-8B was administered 5 min before the start of reperfusion. Figure 7B Survival of isolated adult cardiomyocytes treated with TAT-BNIP3-8B and TAT-BNIP3-8C control peptides after 24 h. Figures 8A to 8C. Pharmacokinetics of TAT-BNIP3-8B. Fluorescently labeled BNIP3-8B was incubated in human serum (Figure 8A), plasma (Figure 8B), and whole blood (Figure 8C) at 37 °C for the indicated time durations and monitored by Western blot. Treatment with proteinase K served as a control. Figures 9A to 9E. TAT-BNIP3-20A reduces myocardial infarct size in vivo. Figure 9A Schematic of the in vivo myocardial infarction model. Mice were exposed to reperfusion 5 min and 24 h after vessel occlusion, respectively. The peptide was administered into the left ventricle 5 min before the start of reperfusion. Figure 9B Western blot monitoring of TAT-BNIP3-8B co-immunoprecipitated with BNIP3 and BAX 5 min after reperfusion. Figure 9C Immunoblotting for cytosolic BNIP3 and BAX in the at-risk area after native blue PAGE at baseline and 10 min reperfusion. Mice were treated with vehicle (NaCl) and TAT-BNIP3-8B. Sham-operated mice served as controls. Figures 9D and 9E Infarct sizes after 24 h of reperfusion in wild-type mice treated with vehicle, TAT-B-Gal, control peptide TAT-BNIP3-8C and indicated doses of TAT-BNIP3-8B (n = 7-10 mice).TAT-BNIP3-8B significantly reduces infarct size in a dose-dependent manner. The vehicle, TAT-B-Gal, and TAT-BNIP3-8C were not effective in attenuating infarct size. Figures 10A to 10F. BNIP3-8B reduces myocardial reperfusion injury in vivo. Mice were exposed to the indicated reperfusion time durations following vessel occlusion in vivo. The peptide was administered into the left ventricle 5 min before initiating reperfusion. TAT-BNIP3-8B inhibited the interaction of BNIP3 (Figure 10A) and BAX (Figure 10B) with mitochondria after 10 minutes of reperfusion, mitochondrial swelling after 10 minutes of reperfusion (Figure 10C), BAX activation after 30 minutes of reperfusion (Figure 10D), cytochrome c release after 30 minutes of reperfusion (Figure 10E), and caspase-3 activity after 4 h of reperfusion (Figure 10F), whereas the control peptide TAT-BNIP3-8C did not (n = 5–12 mice). Sham-operated mice served as controls (n = 5–8 mice). Data are mean ± week. Statistical analyses were two-way analysis of variance (ANOVA) with Bonferroni correction. Figures 11A to 11C. Figure 11A Schematic of in vitro reoxygenation (study design in human ventricular cardiomyocytes derived from human induced pluripotent stem cells (hIPS)). The hIPS were exposed to normoxia and 2 h of reoxygenation after hypoxia and treated with TAT-BNIP3-8B and the control peptide TAT-BNIP3-8C. Figure 11B TAT-BNIP3-8B potently inhibits hIPS cell death during reoxygenation. Representative images of apoptotic, necrotic, and healthy hIPS. Figure 11C TAT-BNIP3-8B I bCQnn / 77n7 / q / YIAI attenuates reoxygenation-induced loss of mitochondrial inner membrane potential. Representative images of depolarized mitochondria, healthy mitochondria, and nucleus. Scale bars, 200 pm. The control peptide BNIP3-8C was not effective in inhibiting cell death and loss of mitochondrial inner membrane potential. Data are mean ± SEQ. Statistical analyses are two-way analysis of variance (ANOVA) with Bonferroni correction. Figure 12. TAT-BNIP3-8B reduces cerebral infarct size 24 h after reperfusion following transient middle cerebral artery occlusion compared to vehicle treatment. TAT-BNIP3-8B and vehicle were administered immediately before reperfusion. Data are mean ± week. Unpaired Student's t-test was used, and statistical significance was set at P < 0.05. Figure 13. TAT-BNIP3-8B reduces myocardial infarct size after 4 h of reperfusion following left coronary artery occlusion compared to vehicle treatment in pigs. TAT-BNIP3-8B and the vehicle were administered 5 min before reperfusion. Data are mean ± week. The unpaired Student's t-test was used, and statistical significance was set at P < 0.05. Figure 14. TAT-BNIP3-8B protects against doxorubicin-induced mitochondrial injury by preventing mitochondrial swelling. HL-1 cells were treated with 5 μM doxorubicin with or without TAT-BNIP3-8B, and mitochondrial swelling was determined by optical density, where swollen mitochondria have a lower OD. Untreated cells were used as a control. DETAILED DESCRIPTION OF THE INVENTION The invention provides methods, compounds, and compositions for treating a disease or condition in a subject in which it is desirable to inhibit the individual activity and communication between pathways of the BCL-2-interacting protein 3 and 19 kDa protein of adenovirus E1B (BNIP3), the BCL-2-associated protein X (BAX), and the mitochondria to prevent cell damage and cell death. In one aspect, the invention is a peptide that inhibits the BNIP3, BAX, and mitochondrial triangle that activates cascades of cell damage and cell death. The peptide according to the invention comprises a cell uptake signal; and a fragment of BNIP3 comprising positions 13 to 20 of BNIP3 or an amino acid sequence derived therefrom and particularly having a length of 50, in particular 40, amino acids or less. The BNIP3 fragment The BNIP3 fragment within the peptide according to the present invention is particularly capable of binding to BAX and / or BNIP3. Specifically, the BNIP3 fragment is capable of binding to the BNIP3-binding region of BAX, such as the amino acid region 108 to 164 of BAX. The BNIP3 fragment is particularly capable of interfering with or inhibiting the interaction between BNIP3 and BAX. In certain forms, the BNIP3 fragment is 20 amino acids or less in length. Specifically, it is 15 amino acids or less, or even 10 amino acids or less. In particular forms, the BNIP3 fragment is 8 amino acids long. In specific modalities, the BNIP3 fragment has an amino acid sequence from the BNIP3 protein. The term BNIP3, as used herein, specifically refers to protein 3 of I bCQnn / 77n7 / q / YIAI interaction with BCL-2 and 19 kDa adenovirus E1B protein having the amino acid sequence of SEQ ID NO: 1. Accordingly, the BNIP3 fragment may have an amino acid sequence that is identical to a consecutive portion of the amino acid sequence of SEQ ID NO: 1 comprising the sequence of amino acid positions 13 to 20. For example, the BNIP3 fragment may have the amino acid sequence of positions 1 to 20 of SEQ ID NO: 1. In certain embodiments, the BNIP3 fragment has an amino acid sequence selected from the group consisting of positions 4 to 20 of SEQ ID NO: 1, positions 11 to 20 of SEQ ID NO: 1, positions 12 to 20 of SEQ ID NO: 1, and positions 13 to 20 of SEQ ID NO: 1. In specific embodiments, the BNIP3 fragment It consists of the amino acid sequence from positions 13 to 20 of SEQ ID NO: 1.In these forms, the particular BNIP3 fragment does not comprise any additional amino acid residues. The amino acid sequence at positions 13 to 20 of mouse BNIP3 is identical to the amino acid sequence at positions 73 to 80 of human BNIP3 (SEQ ID NO: 2). As an alternative to the mouse BNIP3 amino acid sequences referenced herein, the corresponding human BNIP3 amino acid sequences may also be used. In additional embodiments, the BNIP3 fragment has an amino acid sequence derived from BNIP3. A target amino acid sequence is derived from or corresponds to a reference amino acid sequence if the target amino acid sequence shares homology or identity along its entire length with a corresponding portion of the reference amino acid sequence of at least 60%, more preferably at least 70%, at least 80%, at least 90%, or at least 95%. In particular embodiments, a target amino acid sequence derived from or corresponds to a reference amino acid sequence is 100% homologous, or in particular 100% identical, along its entire length with a corresponding portion of the reference amino acid sequence.A homology or identity of an amino acid or nucleotide sequence is preferably determined according to the invention over the entire length of the reference sequence or over the entire length of the corresponding portion of the reference sequence that corresponds to the sequence whose homology or identity is being defined. The BNIP3 fragment in particular may be derived from one of the BNIP3 fragments described above. For example, the BNIP3 fragment may have an amino acid sequence that is at least 60% identical, and especially at least 70% identical, to an amino acid sequence selected from the group consisting of positions 1 to 20 of SEQ ID NO: 1, positions 4 to 20 of SEQ ID NO: 1, positions 11 to 20 of SEQ ID NO: 1, positions 12 to 20 of SEQ ID NO: 1, and positions 13 to 20 of SEQ ID NO: 1.In specific modalities, the BNIP3 fragment comprises an amino acid sequence that is at least 60% identical to positions 13 to 20 of SEQ ID NO: 1. In certain embodiments, the BNIP3 fragment comprises positions 13 to 20 of BNIP3, which optionally includes 1, 2, or 3 amino acid substitutions compared to positions 13 to 20 of BNIP3. BNIP3 specifically has the amino acid sequence SEQ ID NO: 1. In these embodiments, the 1, 2, or 3 amino acid substitutions are preferably present at one or more of the positions corresponding to positions 13, 15, 17, 18, 19, and 20 of BNIP3, particularly positions 15, 17, and 19 of BNIP3. In particular, the amino acid substitutions are selected from the group consisting of (i) substitution of glutamic acid at position 15 of BNIP3 by phenylalanine, isoleucine, I bCQnn / 77n7 / q / YIAI leucine, valine, tyrosine, cysteine, histidine, arginine or threonine, (i) substitution of histidine at position 17 of BNIP3 to valine, and (iii) substitution of serine at position 19 of BNIP3 to tyrosine, cysteine, phenylalanine or histidine. In particular embodiments, the BNIP3 fragment comprises positions 13 to 20 of BNIP3 comprising 1 or 2 amino acid substitutions compared to positions 13 to 20 of BNIP3, wherein the amino acid substitutions are selected from the group consisting of (i) substitution of glutamic acid at position 15 of BNIP3 to histidine, isoleucine, leucine, valine or tyrosine, and (ii) substitution of deserine at position 19 of BNIP3 to tyrosine, cysteine ​​or phenylalanine. In certain forms, the serine at position 19 of BNIP3 is replaced by tyrosine, cysteine, or phenylalanine, particularly with phenylalanine. In specific modalities, the BNIP3 fragment comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 3 to 17. In special modalities, the BNIP3 fragment consists of the amino acid sequence of SEQ ID NO: 7 or 8, especially 8. The BNIP3 fragment may optionally comprise additional amino acid residues derived from BNIP3. In particular, the entire BNIP3 fragment is derived from a consecutive amino acid sequence of BNIP3. Specifically, the entire BNIP3 fragment is derived from amino acid positions 1 to 20 of BNIP3 or a portion thereof comprising at least positions 13 to 20 of BNIP3. The BNIP3 fragment may have an amino acid sequence identical to the corresponding portion of BNIP3 or may have from 1 to 8 amino acid substitutions, particularly from 1 to 6 amino acid substitutions.The BNIP3 fragment may, for example, comprise positions 12 to 20 of BNIP3, optionally comprising 1, 2, 3 or 4 amino acid substitutions compared to positions 12 to 20 of BNIP3, or may comprise positions 4 to 20 of BNIP3, optionally comprising 1, 2, 3, 4, 5 or 6 amino acid substitutions compared to positions 4 to 20 of BNIP3, or may comprise positions 1 to 20 of BNIP3, optionally comprising 1, 2, 3, 4, 5 or 6 amino acid substitutions compared to positions 1 to 20 of BNIP3. In particular, 1, 2, or 3 of these amino acid substitutions are in positions 13 to 20, and the remaining amino acid substitutions are in positions 1 to 12. In these embodiments, the amino acid substitutions are preferentially present in one or more of the positions corresponding to positions 4, 11, 12, 13, 15, 17, 18, 19, and 20 of BNIP3, in particular to positions 4, 11, 12, 15, 17, and 19 of BNIP3. In specific modalities, the BNIP3 fragment comprises a selected amino acid sequence from the group consisting of SEQ ID NO: 18 to 30 and 70 to 75. A particular substituted amino acid residue is replaced by another naturally occurring amino acid residue. The term “substitution,” as used herein, also includes the use of a chemically derived residue in place of a non-derived residue, provided that this polypeptide exerts the required activity. The term “derivative,” as used herein, refers to a peptide having one or more residues chemically derived by reaction of a functional side group. These derived molecules include, for example, those molecules in which free amino groups have been derived to form amine hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups, tert-butyloxycarbonyl groups, I bCQnn / 77n7 / q / YIAI chloroacetyl or formyl groups. Free carboxyl groups can be derived to form salts, methyl and ethyl esters, or other types of esters or hydrazides. Free hydroxyl groups can be derived to form O-acyl or O-alkyl derivatives. The imidazole nitrogen of histidine can be derived to form nim-benzylhistidine. Also included as derivatives are those peptides that contain one or more derivatives of naturally occurring amino acids from the twenty standard amino acids. For example: 4-hydroxyproline can be substituted for proline; 5-hydroxylysine can be substituted for lysine; 3-methylhistidine can be substituted for histidine; homoserine can be substituted for serine; and omitine can be substituted for usine. In some embodiments, the term substitution includes a linkage of two or more substituted amino acid residues. In particular, two or more amino acid residues may be substituted with crosslinkable and / or linked portions, each optionally comprising an additional α-carbon substitution selected from substituted alkyl, optionally hetero-lower, in particular optionally substituted, optionally hetero-methyl, ethyl, propyl, and butyl. Furthermore, two substituted amino acid residues may be substituted with homocysteines connected via a disulfide bridge to generate a cyclic ring-and-tail peptide. Alternatively, two or more substituted amino acid residues may be replaced by a linker.Suitable linkers with respect to this include, for example, (CH2)nONHCOx(CH2)m-, where X is CH2, NH or O, and ym and n are integers 1-4, forming a lactam peptide; -CH2OCH2CHCHCH2OCH2-, forming an ether peptide; or -(CH2)nCHCH(CH2)m-, forming a staple peptide. In certain forms, the BNIP3 fragment comprises at least one of the E15Y, S19F, and S19Y substitutions with respect to the BNIP3 sequence. The cell uptake signal The cell uptake signal within the peptide according to the invention is particularly capable of mediating the uptake of the peptide in a target cell. Specifically, the cell uptake signal is capable of mediating uptake in a mammalian cell, particularly a human cell. In certain modalities, the cell uptake signal is a peptide. Specifically, the cell uptake signal is a protein transduction domain or cell penetration peptide. It can be 5 to 30 amino acids long, particularly 8 to 20 amino acids or 10 to 16 amino acids. The cell uptake signal can be, for example, a hydrophilic peptide or an amphiphilic peptide. Examples of cell uptake signals include the protein transduction domain of the HIV TAT protein (specifically, amino acid residues 48-59), penetratin, antennapedia PTD, SynB1, SynB3, PTD4, PTD5, FHV Coat-(35-49), BMV Gag-(7-25), HTLV-II Rex-(4-16), D-Tat, R9-Tat, Transportan, MAP, SBP, FBP, MPG, and MPG. <anls>Pep-1, Pep-2, polyarginines, and polylysines. In certain modalities, the cell uptake signal is a peptide that has an amino acid sequence selected from the group consisting of SEQ ID NO: 31 to 50. The cell uptake signal may consist of naturally occurring amino acid residues and may optionally be a peptide derivative comprising chemically derived amino acid residues as described herein. Furthermore, it may be a peptide mimetic or comprise a D-reverse sequence. In certain embodiments, the cell uptake signal comprises a D-reverse sequence of a cell-penetrating peptide as disclosed in the bCQnn / zznz / q / YiAi present. The peptide The peptide according to the invention comprises the cell uptake signal and the BNIP3 fragment. The term "comprises," as used herein, in addition to its literal meaning, also includes and specifically refers to the expressions "consists essentially of" and "consists of." Accordingly, the term "comprises" refers to embodiments where the subject matter comprising specifically listed elements does not comprise additional elements, as well as embodiments where the subject matter comprising specifically listed elements may and / or does in fact comprise additional elements. Similarly, the term "have" should be understood to mean the expression "comprises," which also includes and specifically refers to the expressions "consists essentially of" and "consists of."The term essentially consists of, where possible, in particular refers to modalities where the present subject matter comprises 20% or less, in particular 15% or less, 10% or less or especially 5% or less additional elements in addition to the specifically listed elements of which the present subject matter essentially consists of. In certain embodiments, the peptide according to the invention consists of the cell uptake signal and the BNIP3 fragment. In further embodiments, the peptide according to the invention comprises the cell uptake signal, the BNIP3 fragment, and a linker between the cell uptake signal and the BNIP3 fragment. Specifically, the peptide according to the invention comprises the cell uptake signal, the BNIP3 fragment, and a linker between the cell uptake signal and the BNIP3 fragment. The linker may be a peptide linker composed of amino acids or a chemical linker. The linker, in particular, is small in size. For example, a peptide linker may have 10 or fewer amino acids, such as 8 or fewer, 6 or fewer, or 5 or fewer amino acids. A chemical linker, for example, may have a molecular weight of 1,500 Da or less, such as 1,000 Da or less, 750 Da or less, or 500 Da or less. In modalities where the cell uptake signal is a peptide portion, the ligand is preferably a peptide ligand. In certain embodiments, the peptide according to the invention has a length of 40 amino acids or less. In specific embodiments, the peptide according to the invention has a length of 35 amino acids or less, especially 30 amino acids or less. The peptide according to the invention may, for example, have a length of 25 amino acids or less, such as approximately 20 amino acids. Shorter peptides are particularly desirable because they are easier to produce, formulate, and handle. Accordingly, the peptide according to the invention preferably has a length of 35 amino acids or less, especially 25 amino acids or less. This particularly applies to embodiments where the cell uptake signal is a peptide portion. In specific embodiments, the peptide according to the invention has a molecular weight of 10,000 Da or less, especially 5,000 Da or less, 4,000 Da or less, or 3,000 Da or less. In certain embodiments, the peptide according to the invention has an amino acid sequence selected from the group consisting of SEQ ID NO: 51 to 67. In general, the peptide according to the invention is composed of naturally occurring L-amino acids. In certain embodiments, the peptide may also include artificial amino acids. For example, The peptide in bCQnn / 77n7 / q / YIAI may comprise one or more D-amino acids, EB-homo amino acids, and / or N-methylated amino acids; or it may be composed of these. In certain modalities, the cell uptake signal and / or the BNIP3 fragment is the D-reverse sequence, especially the D-reverse sequence of an amino acid sequence described herein. For example, the peptide has the D-reverse sequence QPRRRQRRKKRG-NSFHLEVWSGQLNEEGSQSM (SEQ ID NO: 68) or QPRRRQRRKKRGNSFHLEVW (SEQ ID NO: 69). In certain forms, the peptide is acetylated, acylated, formylated, amidated, phosphorylated, sulfated, nitrosated, glycosylated, sumomylated, hydroxylated, alkylated, and / or isomerized. For example, the peptide may comprise an N-terminal formyl, myristol, palmitoyl, carboxyl, 2-furosyl, or acetyl group, and / or a C-terminal amide, ester, thioester, or hydroxyl group. Furthermore, the peptide may be cyclized. In specific embodiments, the peptide according to the invention is a peptide mimetic of any of the peptides described herein. The term peptide mimetic, as used herein, refers to structures that serve as substitutes for peptides in intermolecular interactions. Peptide mimetics include synthetic structures that may or may not contain amino acids and / or peptide linkages but retain the structural and functional characteristics of a BNIP3 peptide. Peptide mimetics also include molecules that incorporate peptides into larger molecules with other functional elements, peptides, oligopeptoids, and peptide libraries containing peptides of a designated length representing all possible amino acid sequences corresponding to a peptide of the invention.All these peptides, as well as molecules that are substantially homologous, complementary, or otherwise functionally or structurally equivalent to these peptides, may be used for the purposes of the present invention. In certain embodiments, the peptide according to the invention is present in a composition that further comprises nanoparticles. Specifically, the peptide is present within a nanoparticle. The nanoparticles can be any nanoparticle suitable for encapsulating the peptide. Example nanoparticles include liposomes, nanoemulsions, solid-liquid nanoparticles, nanostructured lipid carriers, polymeric nanoparticles, and dendrimers. In certain embodiments, the nanoparticle comprises targeting molecules on its external surface, such as peptides, ligands, or antibodies, that enable the peptide of the invention to be delivered to the desired cells or tissues. In specific embodiments, the nanoparticles enable the uptake of the peptide according to the invention into target cells. In these embodiments, the nanoparticle can perform the function of the cell uptake signal and, in particular, be or replace the cell uptake signal. Accordingly, the present invention also provides a nanoparticle comprising a fragment of BNIP3 as defined herein. Therapeutic uses of the peptide The invention provides methods, compounds, and compositions for treating a disease or condition in a subject in which it is desirable to inhibit the individual activity and communication between BNIP3, BAX, and mitochondrial pathways, comprising administering the compound to the subject in an amount effective to treat the disease or condition in a subject. The invention also provides pharmaceutical compositions comprising the peptide of I bCQnn / 77n7 / q / YIAI according to the invention. In particular, the pharmaceutical compositions comprise the peptide according to the invention in an administerable, unit-dose form. The invention further provides methods for inhibiting cell damage and cell death, comprising administering an effective amount of the peptide according to the invention to a person in need thereof. The present invention also provides for the use of the peptide according to the invention or the pharmaceutical composition comprising it in medicine, especially in the treatment of reperfusion-related and / or mitochondrial-related disorders, as well as cancer therapy-induced cardiotoxicity and the prevention thereof, respectively. The invention includes all combinations of the particular modalities mentioned as if each combination had been laboriously cited separately. The invention also provides a method for inhibiting BNIP3 in a subject comprising contacting BNIP3 with one or more of any of the peptides or pharmaceutical compositions disclosed herein in an amount effective to inhibit BNIP3. Preferably, BNIP3 is present in a subject, and the one or more peptides or compositions are administered to the subject. The invention also provides a method for inhibiting BAX in a subject comprising contacting BAX with one or more of any of the peptides or pharmaceutical compositions disclosed herein in an amount effective to inhibit BAX. Preferably, the BAX is present in a subject, and the one or more peptides or compositions are administered to the subject. The invention also provides a method for inhibiting the dimer and / or oligomer activity of BNIP3 / BAX in a subject, comprising contacting the BNIP3 / BAX dimers and / or oligomers with one or more of any of the peptides or pharmaceutical compositions disclosed herein in an amount effective to inhibit the dimer / oligomer activity of BNIP3 / BAX. Preferably, the BNIP3 and BAX are present in a subject, and the one or more peptides or compositions are administered to the subject. The invention also provides a method of treating reperfusion-related and / or mitochondrial-related disorders in a subject, comprising administering the peptide or pharmaceutical composition according to the invention to the subject in a therapeutically effective amount. The invention also provides a method for treating or preventing tissue damage due to apoptosis or mitochondrial-induced necrosis in a subject, comprising administering the peptide or pharmaceutical composition according to the invention to the subject in a therapeutically effective amount. The peptide can be administered to the subject before, during, and / or after the event of cell damage or death. The subject receiving the peptide or pharmaceutical composition, and who is being treated, may have, for example, a selected disease or condition from the group consisting of hypoxic and / or ischemic cells; cardiac, brain, liver, kidney, bowel, limb ischemia, limb vessel occlusion; cardiac, brain, liver, kidney, bowel, limb reperfusion injury; myocardial infarction and reperfusion injury; cytotoxicity induced by chemotherapy, radiotherapy, targeted therapy, and immunotherapy; atherosclerosis; heart failure; heart, liver, or kidney transplantation; aneurysm; chronic lung disease; ischemic heart disease; hypertension; pulmonary hypertension; embolisms; thrombosis; cardiomyopathy; stroke; a neurodegenerative disease or disorder; an immunological disorder; renal hypoxia; hepatitis; a liver disease; a I bCQnn / 77n7 / q / YIAI kidney disease; cerebellar degeneration; organ transplant rejection; and a disease or disorder involving cell death and / or tissue damage. In certain modalities, the subject has a disorder related to ischemia, reperfusion, and / or mitochondria, especially following vessel occlusion, particularly myocardial infarction, ischemic stroke, acute kidney injury, trauma, circulatory arrest, and ischemia during organ transplantation. In additional modalities, the subject suffers from cancer therapy-induced cardiotoxicity, for example, cardiotoxicity induced by chemotherapy, radiotherapy, immunotherapy, and / or targeted therapy. Or the subject may be undergoing any type of cancer therapy, and the therapy may be applied for the prevention of cardiotoxicity. Chemotherapy, in particular, refers to anthracycline-based chemotherapy, such as doxorubicin therapy.In one specific modality, the subject suffers from cardiotoxicity induced by anthracycline-based chemotherapy, such as chemotherapy with doxorubicin. A reperfusion-related disorder can generally refer to a disorder involving reperfusion injury. Reperfusion injury is tissue damage caused when, for example, blood supply is restored to a tissue after a period of ischemia, where the restoration of circulation results in inflammation and oxidative damage through the induction of oxidative stress rather than the restoration of normal function. Accordingly, the invention provides a method for treating reperfusion injury in a subject, comprising administering the peptide or pharmaceutical composition according to the invention to the subject in a therapeutically effective amount. The invention also provides a method for treating acute myocardial infarction, myocardial reperfusion injury, or heart failure in a subject, comprising administering to the subject one or more of the peptides or pharmaceutical compositions disclosed herein in an amount effective to treat acute myocardial infarction, myocardial reperfusion injury, or heart failure in a subject in need thereof. Preferably, the one or more peptides or pharmaceutical composition are administered in an amount effective to inhibit the dimer / oligomer activity of BNIP3, BAX, or BNIP3 / BAX, respectively, in a subject. In certain modalities, treatment includes relief and / or prevention of reperfusion and mitochondrial-related injury. In additional modalities, treatment includes relief and / or prevention of cancer therapy-induced cardiotoxicity. The subject can be, for example, a mammal, and is preferably a human being. As used herein, "treating" or "treating" a disease or disorder means to alleviate, improve, or eliminate a sign or symptom of the disease or disorder being treated. When the peptides or composition are administered to a subject before or at the onset of the disease or disorder, the peptides or composition may prevent or reduce the severity of the disease or disorder. For example, administration of the peptides or composition to a subject may prevent or reduce the severity of cancer therapy-induced cardiotoxicity, such as cardiotoxicity induced by chemotherapy, radiotherapy, targeted therapy, or immunotherapy. In these modalities, the peptide according to the invention may be administered before, during, and / or after cancer therapy. Administration of the peptides may include preventive and / or therapeutic administration. The peptides and compositions of the present invention can be administered to subjects using routes I bCQnn / 77n7 / q / YIAI of administration known in the art. Administration can be systemic or localized to a specific site. Routes of administration include, but are not limited to, intravenous, intramuscular, intracardiac, intrathecal or subcutaneous injection, oral or rectal administration, and injection into a specific site. In specific embodiments, the peptide or pharmaceutical composition according to the invention is administered to the subject during or after the event of impaired blood supply, ischemia, or vessel occlusion, particularly before reperfusion of the tissue affected by the vessel occlusion. In certain embodiments, the peptide or pharmaceutical composition is administered within a 6-hour period before reperfusion, particularly within a 4-hour, 2-hour, or 1-hour period before reperfusion. In specific embodiments, the peptide or pharmaceutical composition is administered within a 45-minute period, particularly within a 30-minute period before reperfusion. All combinations of the various elements described herein are within the scope of the invention unless otherwise stated herein or clearly contradicted by the context. The method may include expressing an effective amount of the peptide according to the invention in a cell, wherein apoptosis, necrosis, or a combination thereof, is altered in the cell compared to a control cell. Expression may include, for example, introducing a polynucleotide encoding the peptide into the cell. The cell may be ex vivo or in vivo, and may be a cardiac cell. Apoptosis, necrosis, or a combination thereof may be reduced in the cell. The present invention provides a method comprising administering to a subject in need an effective amount of a composition comprising a polynucleotide encoding the peptide according to the invention, wherein apoptosis, necrosis, or a combination thereof, is increased in the subject. The administration may include the administration of the polynucleotide to cardiac tissue, brain tissue, hepatic tissue, and renal tissue. The subject may have signs of, or be at risk of, a selected disease such as acute infarction, hypoxia, ischemia, stroke, or vascular disease. The method may result in a reduction of a sign of disease. As used herein, the term polynucleotide refers to a polymeric form of nucleotides of any length, whether ribonucleotides or deoxynucleotides, and includes both double-stranded and single-stranded RNA and DNA. A polynucleotide can be obtained directly from a natural source or prepared using recombinant, enzymatic, or chemical techniques. A polynucleotide can be linear or circular in topology. A polynucleotide can be, for example, a portion of a vector, such as an expression or cloning vector, or a fragment. A polynucleotide can include nucleotide sequences with different functions, including, for example, coding regions and non-coding regions such as regulatory regions. As used herein, gene refers to a nucleotide sequence that codes for an mRNA. A gene has a transcription start site at its 5' end and a transcription terminator at its 3' end. As used herein, a target gene refers to a specific gene whose expression is inhibited by a polynucleotide as described herein. As used herein, a target mRNA is an mRNA encoded by a target gene. Unless otherwise noted, a target gene may result in multiple mRNAs distinguished by the use of different combinations of exons. These related mRNAs are known as splicing variants or transcript variants of a gene. I bCQnn / 77n7 / q / YIAI As used herein, the terms coding region and coding sequence are used interchangeably and refer to a sequence of nucleotides that codes for a polypeptide and, when placed under the control of appropriate regulatory sequences, expresses the encoded polypeptide. The boundaries of a coding region are generally determined by a translation start codon at its 5' end and a translation stop codon at its 3' end. A regulatory sequence is a sequence of nucleotides that regulates the expression of a coding sequence to which it is operationally linked. Non-limiting examples of regulatory sequences include promoters, enhancers, transcription start sites, translation start sites, translation stop sites, and transcription terminators.The term "operationally linked" refers to a juxtaposition of components such that they are in a relationship that allows them to function in their intended manner. A regulatory sequence is operationally linked to a coding region when it is joined in such a way that the expression of the coding region is achieved under conditions compatible with the regulatory sequence. A polynucleotide that includes a coding region may include heterologous nucleotides flanking one or both sides of the coding region. As used herein, heterologous nucleotides refer to nucleotides not normally present flanking a coding region that is present in a wild-type cell. For example, a coding region present in a wild-type microbe that codes for a polypeptide is flanked by homologous sequences, and any other nucleotide sequences flanking the coding region are considered heterologous. Examples of heterologous nucleotides include, but are not limited to, regulatory sequences. Typically, heterologous nucleotides are introduced into a polynucleotide of the present invention through the use of standard genetic and / or recombinant methodologies well known to a person skilled in the art.A polynucleotide of the present invention can be included in a suitable vector. The presence of heterologous nucleotides flanking one or both sides of a polynucleotide described herein results from human manipulation. The terms complement and complementary, as used herein, refer to the ability of two single-stranded polynucleotides to base-pair with each other, where an adenine in one strand of a polynucleotide will base-pair with a thymine or uracil in one strand of a second polynucleotide, and a cytosine in one strand of a polynucleotide will base-pair with a guanine in one strand of a second polynucleotide. Two polynucleotides are complementary to each other when a nucleotide sequence in one polynucleotide can base-pair with a nucleotide sequence in a second polynucleotide. For example, 5'-ATGC and 5'-GCAT are complementary. The term substantial complement and cognates thereof, as used herein, refer to a polynucleotide that is capable of selectively hybridizing to a specified polynucleotide under strict hybridization conditions.Strict hybridization can occur under a number of pH, salt, and temperature conditions. The pH can range from 6 to 9, preferably from 6.8 to 8.5. The salt concentration can range from 0.15 M sodium to 0.9 M sodium, and other cations can be used provided their ionic strength is equivalent to that specified for sodium. The hybridization reaction temperature can range from 30 °C to 80 °C, preferably from 45 °C to 70 °C. Additionally, other compounds can be added to a hybridization reaction to promote specific hybridization at lower temperatures, such as [a] or [a] approaching [a]. I bCQnn / 77n7 / q / YIAI room temperature. Among the compounds considered to reduce temperature requirements is formamide. Consequently, a polynucleotide is usually substantially complementary to a second polynucleotide if hybridization occurs between the polynucleotide and the second polynucleotide. As used herein, specific hybridization refers to hybridization between two polynucleotides under strict hybridization conditions. A polynucleotide that includes a coding region may include heterologous nucleotides flanking one or both sides of the coding region. As used herein, heterologous nucleotides refer to nucleotides not normally present flanking a coding region that is present in a wild-type cell. For example, a coding line present in a wild-type microbe that codes for a polypeptide is flanked by homologous sequences, and any other nucleotide sequences flanking the coding region are considered heterologous. Examples of heterologous nucleotides include, but are not limited to, regulatory sequences. Typically, heterologous nucleotides are introduced into a polynucleotide of the present invention through the use of standard genetic and / or recombinant methodologies well known to a person skilled in the art.A polynucleotide of the present invention can be included in a suitable vector. The presence of heterologous nucleotides flanking one or both sides of a polynucleotide described herein results from human manipulation. The present invention further provides a method for preventing cell damage or cell death, comprising bringing the cell into contact with the peptide according to the invention. The method can be performed in vitro or in vivo, and in particular, it is performed ex vivo. The present invention further provides a method for screening a compound suitable for the prevention of reperfusion injury and / or mitochondrial-related disorders and / or cancer therapy-induced cardiotoxicity, comprising (i) providing one or more candidate compounds; (ii) determine the ability of the candidate compounds to interfere with the binding of BNIP3 and BAX; (iii) select those candidate compounds that interfere with the binding of BNIP3 and BAX. Candidate compounds can be any suitable compound, especially including peptides and small molecule compounds. EXAMPLES Example 1: BNIP3 represents a therapeutic target for l / R injury A combination of necrotic and apoptotic cardiomyocyte death is a hallmark of the early phase of reperfusion injury. Mitochondria play an important role in both processes. The BH-only BCL2 family member BNIP3 is a potential activator of mitochondria-driven necrotic and apoptotic cell death cascades in cell culture and isolated rat hearts.31,32,36,38 BNIP3 has previously been implicated in left ventricular remodeling following acute myocardial infarction and in heart failure with preserved ejection fraction.3a,39,4 To investigate the involvement of BNIP3 in left ventricular injury and whether BNIP3 provides an attractive target for therapeutic intervention, BNIP3-deficient and wild-type mice were subjected to 24 h of reperfusion. I bCQnn / 77n7 / q / YIAI after occlusion of the left anterior descending coronary artery in a clinically relevant in vivo model4144 (Figure 1A). To delineate the affected area from the unaffected zone, Evans blue dye was injected into the aorta and coronary arteries. To demarcate non-viable myocardium, the infarcted area within the at-risk zone, heart sections were stained with triphenyltetrazolium chloride (Figure 1B). Consistent with a previous study in isolated rat hearts using a well-established dominant-negative inhibitor of BNIP331, genetic ablation of BNIP3 resulted in a significantly reduced infarct size by 46% compared to wild-type mice (Figure 10). Therefore, restoring BNIP3 to BNIP3-deficient mice resulted in infarct sizes comparable to wild-type mice in a dose-dependent manner and ruled out potential side effects of BNIP3 genetic deletion (Figure 10). These results indicate that BNIP3 plays an important role in infarct development in vivo. Example 2: BNIP3 is a mediator of BAX-induced cell death in l / R lesions The pro-death BAX family member BCL-2 appears to be positioned at the intersection of mitochondrial-dependent necrosis and apoptosis as an effector protein.5 For this, the translocation of BAX from the cytosol to the mitochondria is the critical step.37,45,46 A basal interaction of BAX with BNIP3 that occurs in the mitochondrial membrane (MOM) of cardiomyocytes in vivo has recently been demonstrated.35 To determine whether the translocation of BAX to the mitochondria as the most relevant step in cell death occurs in the MOM and is dependent on BNIP3, early changes in mitochondrial BAX and BNIP3 concentrations were investigated. After 10 min of reperfusion following vessel occlusion, the level of BAX in the MOM increases markedly, along with the increased mitochondrial concentration of BNIP3 (Figures 2A and 2B). Co-immunoprecipitation revealed that BNIP3 and BAX form heterodimers in the MOM (Figure 2C).Taking advantage of BNIP3-deficient mice, we then examined whether BNIP3 mediates BAX translocation in the 5' direction during reperfusion injury. No increase in mitochondrial BAX was observed in the early reperfusion phase following BNIP3 genetic ablation, demonstrating a direct impact of BNIP3 (Figure 2D). To verify that this effect is indeed achieved by BNIP3, BNIP3 was administered to BNIP3-deficient mouse hearts 5 min before initiating the myocardial infarction procedure. The addition of BNIP3 restored BAX translocation in BNIP3-deficient mouse hearts in the early reperfusion phase and ruled out potential side effects of BNIP3 genetic deletion (Figure 2D). Example 3: Identification of the critical BNIP3 sequence required for BNIP3 inhibition in l / R injury Since the interaction of BNIP3 with BAX is recognized as a substantial activity in in vivo reperfusion injury assessed by co-immunoprecipitation, the α5, α6, α7, and α8 helices of BAX were identified as potential binding sites by evaluating a peptide microarray with a library of 13 synthesized BAX peptides (Figures 3A and 3B). For this study, the in situ 3D structure of BNIP3 was predicted by homology modeling using Modeller 9.1547 (Figure 3C). The resulting model was energy-minimized using NAMD2.9 and the CHARMM36 force field. The BNIP3 model represents nine α-helices of varying lengths (64%), followed by random coils (19%), and non-structural elements. I bCQnn / 77n7 / q / YIAI identified 17%, which was confirmed by circular dichroism spectroscopy (Figure 3D). Computational docking simulations suggested the BAX helices α5, α6, a7 and a8 and the BNIP3 MSQSGEENLQGSWVELHFSN sequence (amino acids 1-20; SEQ ID NO: 20) as interaction sites (Figure 3E), while the first 10 amino acids alone failed to bind to BAX (Figure 3F). It was hypothesized that amino acids 1-20 of BNIP3 might be sufficient to antagonize BNIP3 activity, so a cell-permeable peptide, TAT-BNIP3-20A peptide, was designed, composed of the HIV-1 TAT protein transduction domain (PTD, GRKKRRQRRRPQ (SEQ ID NO: 31), Figure 3G)4849 linked by a covalent bond to 20 amino acids derived from amino acids 1-20 of BNIP3 (Figure 3H). Amino acids 42-61 of BNIP3 were used to generate the control peptide, TAT-BNIP3-20C (Figure 3I).The peptide fragments were capped at the N-terminus with an acetyl group and at the C-terminus with an amide group. Treatment of wild-type mice with TAT-BNIP3-20A demonstrated that the peptide is taken up by the myocardium (Figure 3J) and interacts there with endogenous BNIP3. To examine whether TAT-BNIP3-20A, administered 5 minutes before reperfusion—a time point relevant to clinical practice—has the ability to antagonize BNIP3 activity and reduce reperfusion injury in vivo, the predetermined myocardial infarction model was used in mice (Figure 4A). Treatment with TAT-BNIP3-20A, but neither the vehicle nor TAT-BNIP3-20C, resulted in a 37% reduction in infarct size (Figure 4B). This was attributed to the prevention of BNIP3 translocation to the mitochondria by the peptide fragment in TAT-BNIP3-20A (Figure 40), which leads to markedly inhibited caspase-3 activity, a key event in reperfusion injury following mitochondrial membrane disruption (Figure 4D). Example 4: The peptide fragment TAT-BNIP3-20A inhibits the death of apoptotic and necrotic human cardiomyocytes To address the translational need, reoxygenation experiments were performed on human ventricular cardiomyocytes derived from human induced pluripotent stem cells (HICS) (Figure 5A). The human HSCs were exposed to 2 h of reoxygenation following hypoxia. It should be noted that even in HICS-derived TAT-BNIP3-20A, the protective properties of BNIP3-20A are present. BNIP3-20A is capable of preventing the translocation of human BNIP3 to the mitochondria (Figure 5B), resulting in considerable mitochondrial protection evidenced by low mitochondrial inner membrane depolarization (Figure 5C) and fewer apoptotic and necrotic cells (Figure 5D). Example 5: Identification and design of a peptide fragment inhibitor of BNIP3 / BAX activity N-terminal truncations of the BNIP3-20A peptide sequence followed by single residue swapping revealed that a peptide containing amino acids 13-20 in combination with a Ser to Ph substitution at position 19 showed substantially higher BNIP3 binding in peptide microarrays (see Table 1). Table 1: Truncated BNIP3-20A fragments showing the highest bonding capabilities to BNIP3 I bCQnn / 77n7 / q / YIAI Sequence (SEQ ID NO) Fluorescence intensity MSQSGEENLQGSWVELHFSN (21) 1040.33 SGEENLQGSWVELHFSN (25) 2656.33 SWVELHFSN (29) 4889.33 I bCQnn / 77n7 / q / YIAI Based on these results, the peptide BNIP3-8B was designed, composed of the PTD covalently linked to eight amino acids derived from amino acids 13–20 of BNIP3 with the substitution of Ser-19 to Phe-19 (WVELHFFN (SEQ ID NO: 8); Figure 6A). To demonstrate the requirement of the phenylalanine residue in the peptide sequence, Phe-18 was substituted to Ala-18, and His-17 to Ala-17, generating the peptide BNIP3-8C (Figure 6B). The circular dichroism spectrum of BNIP3-8B showed that the peptide exhibits a random coil conformation (Figure 6C). In addition, BNIP3 / BNIP3 interaction studies were performed with the BNIP3-20A peptide in which individual residues of the wild-type BNIP3 1-20 sequence were replaced with 18 neutral amino acids. BNIP3 peptides with specific amino acid substitutions showed increased binding capacity to BNIP3 (example data shown in Table 2). Table 2: Substituted BNIP3-20A peptides showing the highest binding capabilities to BNIP3 Sequence (SEQ ID NO) Amino acid substitution Fluorescence intensity MSQSGEENLQGSWVELHFSN (21) - 1040.33 MSQSGEENLQGSWVELCFSN (70) H17->C 6787.00 MSQSGEENLQGCWVELHFSN (71) S12->C 7038.00 MSQSGEENLQGSWVCLHFSN (72) E15->C 7149.00 MSQSGEENLQGSWVELYFSN (73) H17->Y 7988.00 MSQSGYENLQGSWVELHFSN (74) E6->Y 13122.50 MSQSGEENLQYSWVELHFSN (75) G11->Y 10230.33 MSQSGEENLQGSWVELHFFN (22) S19-> F 15000.00 Example 6: In vitro and in vivo effects of TAT-BNIP3-8B peptide fragment uptake, toxicity, and stability First, the distribution and presence of BNIP3-8B in the intracardiac injection at the time of the desired effect in vivo were evaluated. Then, the pharmacokinetic profile of TAT-BNIP3-8B in human serum, plasma, and whole blood was assessed, and toxicity was evaluated in isolated adult cardiomyocytes. TAT-BNIP3-8B is taken up by the heart, spleen, and liver and is present in plasma (Figure 7A). Consequently, TAT-BNIP3-8B is present in the heart 10 minutes after reperfusion, the time point when the BNIP3-BAX-mitochondrial triangle cell death cascade occurs; cardiomyocytes showed no signs of overt toxicity (Figure 7B), and the half-life of TAT-BNIP3-8B in human serum, plasma, and whole blood supports its inhibitory capacity in vivo (Figures 8A to 8C). The incubation of TAT-BNIP3-8B with proteinase K served as a control. Example 7: Mechanism of action of TAT-BNIP3-8B peptide It was hypothesized that TAT-BNIP3-8B binds to BNIP3 and BAX monomers and homodimers, as well as to BNIP3 / BAX heterodimers and heterooligomers, to disrupt their activity. To evaluate the interaction behavior of TAT-BNIP3-8B, BNIP3LGAT-BNIP3-8B, and BAX / TAT-BNIP3-8B, overlap assays and coupling simulations were performed. Both results suggested that TAT-BNIP3-8B binds to BNIP3 and BAX (data not shown). Specifically, in the myocardial infarction model (Figure 9A), 5 minutes after reperfusion, endogenous BNIP3 and BAX mono-, homo-, and hetero-dimers were co-immunoprecipitated with fluorescently labeled TATBNIP3-8B (Figure 9B). Furthermore, reperfusion induced BNIP3 and BAX to oligomerize, as evidenced by the large-scale formation of oligomers consisting of BNIP3 and 3 BAX (Figure 9C). SDS-PAGE and co-immunoprecipitation experiments revealed the heterointeractions of BNIP3 / BAX in higher-order oligomeric complexes, and this oligomerization was strongly inhibited by treatment with TAT-BNIP3-8B (Figure 9C). Example 8: TAT-BNIP3-8B reduces myocardial infarction size The efficacy and effect of TAT-BNIP3-8B treatment, administered 5 min before reperfusion, were then studied in the appropriate myocardial infarction model (Figure 9A). Specifically, TAT-BNIP3-8B reduced infarct size in a dose-dependent manner by up to 40% compared to the vehicle and to TAT-B-Gal treatment (Figures 9D and 9E). Treatment with TAT-BNIP3-8C did not appreciably affect infarct size compared to TAT-BNIP-8B, indicating the importance of the phenylalanine residue (Figure 9D). Example 9: TAT-BNIP3-8B reduces mitochondrial perturbation and the cell death cascade Mitochondrial damage can result from disruption of the inner mitochondrial membrane (IMM) and the mitochondrial membrane (MOM). The critical mitochondrial event in necrosis is the early opening of the mitochondrial permeability transition pore (mPTP) in the IMM, which causes a time-dependent dissipation of the electrical potential difference across the IMM, followed by mitochondrial swelling and cell disruption. The key mitochondrial event in apoptosis is the activation of BAX, which induces its exposure to the transmembrane domain and MOM permeabilization, allowing the release of apoptogens, such as cytochrome c, and subsequent caspase activation.TAT-BNIP3-8B injected into the left ventricle 5 minutes before reperfusion in the l / R model administered in vivo prevents the translocation of BNIP3 and BAX to the mitochondria, resulting in the inhibition of the cell death machinery in the 3' direction, which includes mitochondrial swelling, BAX activation, cytochrome c release, and caspase 3 activity. Example 10: TAT-BNIP3-8B inhibits the death of apoptotic and necrotic cardiomyocytes We tested whether TAT-BNIP3-8B can inhibit cell death in human induced pluripotent stem cells. Human induced pluripotent stem cells were exposed to 2 h of reoxygenation after hypoxia (Figure 11A). BNIP3-8B markedly inhibits the death of necrotic and apoptotic cells (Figure 11B) and the loss of mitochondrial inner membrane potential (Figure 11C). Example 11: TAT-BNIP3-8B reduces cerebral infarct size Since BNIP3 is suggested to play a critical role in cerebral ischemia38, it is also I bCQnn / 77n7 / q / YIAI evaluated the effects of the BNIP3-8B peptide on clinical outcome in a mouse model of focal cerebral reperfusion. Mice underwent 24 h of reperfusion following a 30-minute transient middle cerebral artery occlusion (tMCAO). Treatment with BNIP3-8B immediately after tMCAO markedly reduced infarct size by 52% (Figure 12). Example 12: TAT-BNIP3-8B reduces myocardial infarct size in pigs The efficacy and effect of TAT-BNIP3-8B treatment administered 5 min before reperfusion was then studied in a porcine model of myocardial infarction. Pigs underwent 60-minute occlusion of the left anterior descending coronary artery followed by 4 h of reperfusion. Notably, TAT-BNIP3-8B significantly reduced infarct size by 56% compared to the vehicle (Figure 13). Example 13: TAT-BNIP3-8B protects against doxorubicin-induced mitochondrial injury. The effects of the BNIP3-8B peptide on doxorubicin-induced mitochondrial injury in HL-1 cells were evaluated. To monitor mitochondrial swelling, HL-1 cells were treated with 5 μM doxorubicin, replicating the peak plasma concentration achieved by standard infusion in patients. Mitochondrial swelling was measured by optical density at 540 nm, where increased mitochondrial volume due to swelling results in reduced optical density. TAT-BNIP3-8B administered concurrently with doxorubicin prevents mitochondrial swelling, as indicated by the increased optical density (Figure 14). These data suggest that TAT-BNIP3-8B may protect mitochondria against damage mediated by chemotherapies such as anthracyclines. Example 14: Methods The chemicals were obtained from Sigma Aldrich. Antibodies against BNIP3, tubulin, cytochrome c, and BAX were obtained from Abeam, and activated BAX from Enzo. Animals. Male mice of similar age (12±3 weeks) and an average body weight of 30 g were used. Wild-type C57BL / 6 mice were obtained from Jackson Laboratory (Bar Harbor, ME, USA) and kept for one week in the local animal house for acclimatization. C57BL / 6J-7g / 7 (Βηίρΐ7) mice were obtained from Prof. Gerald W. Dorn, Center for Molecular Cardiovascular Research and Department of Pediatrics, University of Cincinnati, Cincinnati, Ohio, USA. The mice were generated by replacing exons 2 and 3 with a neomycin resistance cassette.33 The mice were bred and housed at the local animal house of the University Hospital of Essen. All experiments were approved by the local ethics committee in accordance with the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes (Directive 2010 / 63 / EU). In vivo mouse model of myocardial infarction. Wild-type and Bnip3-deficient (Bnip3 / ) mice were anesthetized by intraperitoneal injection of ketamine (100 mg / kg) and xylazine (10 mg / kg) and intubated. Mechanical ventilation parameters were set at a tidal volume of 2.1 to 2.5 mL and a respiratory rate of 140 breaths per minute using a mouse miniventilator. Deep anesthesia was maintained by adding 2% by volume of isoflurane to the ventilation gas. The thorax was opened via a lateral thoracotomy (a 1-cm left lateral incision between the 3rd and 4th ribs). A 6-0 prolene suture was placed around the left coronary artery (LCA), and a piece of soft silicone tubing was inserted over the thoracic cavity. The artery was occluded. Coronary occlusion was achieved by tightening and tying the suture. After 30 minutes of occlusion, the silicon tubing was removed, and the suture was left in place. For longer reperfusion times, the chest was closed using 4-0 prolene. BNIP3 (2, 6, and 9 nmol in 50 μL of 0.9% sodium chloride) was injected into the left ventricular cavity 5 minutes before vessel occlusion. Peptides containing 20 amino acids (2 nmol / 50 μL) and peptides containing 8 amino acids (8 nmol / 50 μL) were injected in NaCl 5 minutes before reperfusion. Sodium chloride injection (50 μL) served as a control treatment. In vivo model of myocardial infarction in pigs. After induction of anesthesia, a small incision was made over the femoral artery and vein; the vessels were isolated. A small opening was made in the artery and a liner was introduced. In addition, a liner was placed in the femoral vein or another appropriate vein to allow for the administration of emergency drugs if needed. A blood sample was drawn prior to heparin administration and used to establish a baseline activated clotting time (ACT), which was recorded. Subsequently, as directed by the surgeon, heparin (250 to 350 IU / kg) was administered as needed to achieve and maintain an ACT that is twice the baseline ACT level. After the initial heparin bolus, the ACT was recorded again and monitored at least every 60 minutes thereafter until the end of surgery. An appropriate guide catheter was advanced into the ostium of the left anterior descending (LAD) artery using fluoroscopic guidance. Non-ionic contrast was used for all procedures. A balloon catheter was advanced through the guide catheter into the left anterior descending (LAD) coronary artery. The balloon was advanced into the coronary arteries through the guide catheter to a suitable location above the first diagonal branch of the LAD. The balloon was then inflated to a pressure sufficient to ensure complete occlusion of the artery. Occlusion was verified using fluoroscopy. After occlusion was verified, the balloon remained inflated in the artery for 60 minutes. Incidence support drugs and defibrillation were recorded in the study log.The peptide and vehicle were administered intravenously 5 minutes before reperfusion. At the end of the vessel occlusion period, the balloon was deflated, and the ischemic area was allowed to reperfuse. Complete balloon deflation was verified fluoroscopically. At the end of the procedure, all catheters were removed, the artery and vein were ligated, and the incision was closed using standard techniques. The animals were euthanized 4 hours after reperfusion. Infarct size measurement. For infarct size analysis, mice were sacrificed after 24 hours of reperfusion; hearts were removed and perfused with PBS for 5 min. After perfusion, the LOA was re-ligated at the same location as described above. Evans blue dye (1 ml of a 1% solution) was injected into the aorta and coronary arteries for delineation of the ischemic AR from the non-ischemic zone. The tissue was wrapped in transparent food wrap and stored for one hour in a freezer at -20°C. The heart was then sectioned serially perpendicular to the long axis into 1-mm pieces, and each piece was weighed. The sections were incubated in 1% TTC for 5 min at 37°C for demarcation of viable and non-viable myocardium within the at-risk zone.Infarct size, aortic arch area (AAR), and non-ischemic left ventricle were assessed using computer-assisted planimetry by an observer blinded to sample identity. Myocardial infarct size was expressed as a percentage of the AAR. I bCQnn / 77n7 / q / YIAI Immunoblotting. Human stem cells and tissue stem cells were lysed in RIPS buffer (50 mM Tris-HCl, 150 mM NaCl, 0.5 mM EDTA, 1% NP-40, and protease and phosphatase inhibitor, pH 7.4). Isolated mitochondria were lysed in Mito-lyse buffer (200 mM sucrose, 10 mM HEPES, 1 mM EGTA, 1% Triton X100, and protease and phosphatase inhibitor, pH 7.4). Scavenged samples were removed by centrifugation (20,000 x g, 15 min, 4 °C). Protein concentrations in the supernatant were measured using the DC protein assay (Bio-Rad). Samples were diluted in 4 x LDS sample buffer and 10 x reducing agent (Invitrogen) and prepared for SDS-PAGE by heating at 95 °C for 5 min. Equivalent amounts of protein were separated using 4-12% Bis-Tris Gels (Invitrogen), transferred to nitrocellulose, and immunoblotted with primary antibodies.The secondary antibodies used were horseradish peroxide-conjugated anti-rabbit or anti-mouse goat IgG (Invitrogen). Immunoblotting was detected by ECL (Thermo Scientific) and an image was generated on an Imager 600 (Amersham). Interaction studies. For protein-peptide interaction studies, peptide libraries were synthesized and immobilized on microarray slides. Recombinant BNIP3 was used at concentrations of 1 pg / ml. Peptides were synthesized and immobilized for: (i) the BNIP3 / BAX interaction study; (ii) the BNIP3 / BNIP3 interaction study, in which C, N, or C / N-terminal truncations were made to the wild-type sequence of BNIP3 1-20; and (iii) the BNIP3 / BNIP3 interaction study, in which individual residues of the wild-type sequence of BNIP3 1-20 were exchanged for 18 neutral amino acids. Microarray. For protein-peptide binding studies, recombinant BNIP3 (Cusabio) and BAX (MyBioSource) were used at a concentration of 10 pg / ml or 1 pg / ml, respectively. A DyLight microscale antibody labeling kit (Thermo) with the Dylight 650 label was used. The assay was performed using the automated TECAN HS4800 microarray processing station. Microarrays were incubated with customer-supplied samples diluted in blocking buffer for 2 h at 30 °C. Before each step, the microarrays were washed with washing buffer. The microarrays were scanned using a high-resolution fluorescence scanner. The laser settings and resolution were identical for all measurements. The resulting images were analyzed and quantified using GenePix dot recognition software (Molecular Devices).For each point, the mean signal intensity (between 0 and 65535 arbitrary units) was extracted. For further data evaluation, the so-called MMC2 values ​​were determined. The MMC2 is equal to the mean of the three instances in the microarray, except when the coefficient of variation (CV), the standard deviation divided by the mean, is greater than 0.5. In this case, the mean of the two closest values ​​(MC2) is assigned to the MMC2. All steps were performed by JPT Peptide Technologies (Berlin, Germany). In silico three-dimensional (3D) structural modeling of BNIP3. The predicted in silico 3D structure of BNIP3 was obtained by homology modeling using Modeller9.15 and the resulting model was reduced to energy minimum using NAMD2.9 and the CHARMM36 force field. Circular dichroism (CD) spectroscopy. The CD spectra of the BNIP3 protein and the BNIP3-8B peptide were recorded on a Jasco J-715 spectrometer at 37°C, pH=7,4,1x PBS. I bCQnn / 77n7 / q / YIAI Docking Simulation. Experiments were performed using Autodock Vina52 and HADDOCK53. The structure of BAX (pdb-ID: 4S0O) was taken from the Protein Data Bank, while the structures of BNIP3 and the peptide BNIP3-8B were modeled using Modeller9.1547. The template structures correspond to PDB codes 2k7w and 2ka1. The resulting model was reduced in energy by auto-scanning NAMD2.954 and the CHARMM36 force field. Immunoprecipitation. Immunoprecipitation was performed using G protein-coupled Dynabeads (Invitrogen). 500 pg of used proteins were incubated overnight at 4 °C with shaking in PBS buffer containing 1 mM DTT, 0.005% Brij35, and protease-phosphatase inhibitors. The following day, 20 pg of Dynabeads were added, and the solution was incubated again for 1 h. The precipitated immune complex was washed twice and then resuspended in elution buffer containing LDS sample buffer (1:4) and reducing agent (1:10) (Invitrogen) in PBS and heated for 5 min at 95 °C. After removal of the Dynabeads, the eluate was analyzed by immunoblotting. Caspase 3 activity was measured using the Abeam Caspase 3 Assay Kit (#ab39401). Murine hearts were collected after 30 min of ischemia and 4 h of reperfusion, the at-risk area was isolated, slicked in containment buffer, and the assay was performed according to the manufacturer's instructions. Cell culture. Human iPSC (human induced pluripotent stem cell) derived ventricular cardiomyocytes were obtained from (axol) and cultured according to the manufacturer's specification. To simulate vessel occlusion, cells were incubated in buffer (NaCl 113 mM, KCl 4.7 mM, HEPES 12 mM, MgSO4 1.2 mM, taurine 30 mM-1.3 mM, pH 7.4) under 1% O2, 37°C. Reoxygenation was performed in buffer supplemented with 5.5 mM glucose under 21% O2, 37°C. HL-1 cells were cultured in Claycomb medium according to the manufacturer's protocol. The cells were treated with 5 pM doxorubicin and 2 nmol TAT-BNIP3-8B for 30 min. JC-1 Assay. To analyze the mitochondrial inner membrane potential in human induced pluripotent stem cells, cells were stained with 5',6,6'-tetrachloro-1,T,3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1). Cells were incubated with 6 pM JC-1 in medium at 37 °C for 30 min. After washing the cells with PBS buffer at 37 °C, they were fixed with 4% PFA for 15 min at room temperature. DAPI staining was performed, and the cells were analyzed using EVOS FL (Life Technologies). Swelling assay. Mitochondrial swelling was measured by light scattering at 540 nm on a FLUOstar Omega microplate absorbance reader (BMG Labtech) at room temperature. The final assay volume was 200 lp, containing mitochondria at 0.5 mg / ml in buffer containing 250 mM sucrose, 10 mM HEPES, 1 mM EGTA, pH 7.4. Peptides. The peptides were generated by the resin synthesis procedure (JPT International, Berlin, Germany). They were capped at the N-terminus with an acetyl group and at the C-terminus with an ameide. For administration, the peptides were covalently linked to the sequence TAT GRKKRRQRRRPQ (SEQ ID NO: 31). For uptake and binding studies, the peptides were labeled with I bCQnn / 77n7 / q / YIAI is a fluorophore. A complete list of peptides is attached as appendix 1. LITERATURE 1. Zipes, D., Libby, P., Bonow, R. & Mann, D. Braunwald's Heart Disease: A Textbook of Cardiovascular Medicine. Elsevier / Saunders 10th Edition (2015). 2. Yellon, D. M. & Hausenloy, D. J. Myocardial reperfusion injury. N Engl J Med 357,1121-1135, (2007). 3. Cahill, T. J. & Kharbanda, R. K. Heart failure after myocardial infarction in the era of primary percutaneous coronary intervention: Mechanisms, incidence and ¡dentification of patients at risk. World J Cardiol 9, 407-415, (2017). 4. Christia, P. & Frangogiannis, N. G. Targeting inflammatory pathways in myocardial infarction. Eur J Clin Invest 43, 986-995, (2013). 5. Whelan, R. S. et al. Bax regulates primary necrosis through mitochondrial dynamics. Proc Nati Acad Sci U S A 109, 6566-6571, (2012). 6. Avila, M. S. et al. Carvedilol for Prevention of Chemotherapy-Related Cardiotoxicity: The CECCY Trial. J Am Coll Cardiol 71,2281-2290, (2018). 7. Gulati, G. et al. Prevention of cardiac dysfunction during adjuvant breast cáncer therapy (PRADA): a 2 x 2 factorial, randomized, placebo-controlled, double-blind clinical trial of candesartan and metoprolol. Eur Heart J 37,1671-1680, (2016). 8. Pituskin, E. et al. Multidisciplinary Approach to Novel Therapies in Cardio-Oncology Research (MANTICORE 101-Breast): A Randomized Trial for the Prevention of Trastuzumab-Associated Cardiotoxicity. J Clin Oncol 35, 870-877, (2017). 9. Baines, C. P. The cardiac mitochondrion: nexus of stress. Annu Rev Physiol 72, 61-80, (2010). 10. Ong, S. B., Samangouei, P., Kalkhoran, S. B. & Hausenloy, D. J. The mitochondrial permeability transition pore and its role in myocardial ischemia reperfusion injury. J Mol Cell Cardiol 78, 23-34, (2015). 11. Kokoszka, J. E. et al. The ADP / ATP translocator is not essential for the mitochondrial permeability transition pore. Nature 427, 461-465, (2004). 12. Baines, C. P., Kaiser, R. A., Sheiko, T., Craigen, W. J. & Molkentin, J. D. Voltage->dependent anion channels are dispensable for mitochondrial-dependent cell death. Nat Cell Biol 9, 550-555, (2007). 13. Kwong, J. Q. et al. Genetic deletion of the mitochondrial phosphate carrier desensitizes the mitochondrial permeability transition pore and causes cardiomyopathy. Cell Death Differ 21, 1209-1217, (2014). 14. Basso, E. et al. Properties of the permeability transition pore in mitochondria devoid of Cyclophilin D. J Biol Chem 280,18558-18561, (2005). 15. Bonora, M. et al. Role of the c subunit of the FO ATP synthase in mitochondrial permeability transition. Cell Cycle 12, 674-683, (2013). 16. Giorgio, V. et al. Dimers of mitochondrial ATP synthase form the permeability transition pore. Proc Nati Acad Sci II S A 110, 5887-5892, (2013). I bCQnn / 77n7 / q / YIAI 17. Argaud, L. et al. Specific inhibition of the mitochondrial permeability transition prevenís lethal reperfusion injury. J Mol Cell Cardiol 38, 367-374, (2005). 18. Skyschally, A., Schulz, R. & Heusch, G. Cyclosporine A at reperfusion reduces infarct size in pigs. Cardiovasc Drugs Ther 24, 85-87, (2010). 19. Cung, T. T. et al. Cyclosporine before PCI in Patients with Acute Myocardial Infarction. N Engl J Med 373,1021-1031,(2015). 20. Gibson, C. M. et al. EMBRACE STEMI study: a Phase 2a trial to evalúate the safety, tolerability, and efficacy of intravenous MTP-131 on reperfusion injury in patients undergoing primary percutaneous coronary intervention. Eur Heart J 37,1296-1303, (2016). 21. Szeto, Η. H. First-in-class cardiolipin-protective compound as a therapeutic agent to restore mitochondrial bioenergetics. Br J Pharmacol 171,2029-2050, (2014). 22. Schaller, S. et al. TR040303, a new cardioprotective compound, inhibits mitochondrial permeability transition. J Pharmacol Exp Ther 333, 696-706, (2010). 23. Atar, D. et al. Effect of intravenous TR040303 as an adjunct to primary percutaneous coronary intervention for acute ST-elevation myocardial infarction: MITOCARE study results. Eur Heart J 36, 112-119, (2015). 24. Rupprecht, H. J. et al. Cardioprotective effects of the Na(+) / H(+) exchange inhibitor cariporide in patients with acute anterior myocardial infarction undergoing direct PTCA. Circulation 101, 2902-2908, (2000). 25. Chi, L. G. et al. Effect of superoxide dismutase on myocardial infarct size in the canine heart after 6 hours of regional ischemia and reperfusion: a demonstration of myocardial salvage. Gire Res 64, 665675, (1989). 26. Arai, M. et al. An anti-CD18 antibody limits infarct size and preserves left ventricular function in dogs with ischemia and 48-hour reperfusion. J Am Coll Cardiol 27,1278-1285, (1996). 27. Williams, F. M., Kus, M., Tanda, K. & Williams, T. J. Effect of duration of ischaemia on reduction of myocardial infarct size by inhibition of neutrophil accumulation using an anti-CD18 monoclonal antibody. BrJ Pharmacol 111, 1123-1128, (1994). 28. Wei, M. C. et al. Proapoptotic BAX and BAK: a requisite gateway to mitochondrial dysfunction and death. Science 292, 727-730, (2001). 29. Ow, Y. P., Green, D. R., Hao, Z. & Mak, T. W. Cytochrome c: functions beyond respiration. Nat Rev Mol Cell Biol 9, 532-542, (2008). 30. Oberst, A., Bender, C. & Green, D. R. Living with death: the evolution of the mitochondrial pathway of apoptosis in animáis. Cell Death Differ 15,1139-1146, (2008). 31. Hamacher-Brady, A. et al. Response to myocardial ischemia / reperfusion injury involves Bnip3 and autophagy. Cell Death Differ 14, 146-157, (2007). 32. Kubli, D. A., Quinsay, Μ. N., Huang, C., Lee, Y. & Gustafsson, A. B. Bnip3 functions as a mitochondrial sensor of oxidative stress during myocardial ischemia and reperfusion. Am J Physiol Heart Circ Physiol 295, H2025-2031, (2008). 33. Diwan, A. et al. Inhibition of ischemic cardiomyocyte apoptosis through targeted ablation of I bCQnn / 77n7 / q / YIAI Bnip3 restrains postinfarction remodeling in mice. J Clin Invest 117, 2825-2833, (2007). 34. Hochhauser, E. et al. Bax ablation protects against myocardial ischemia-ireperfusion injury in transgenic mice. Am J Physiol Heart Gire Physiol 284, H2351- 2359, (2003). 35. Hendgen-Cotta, U. B. et al. Cytosolic BNIP3 Dimer Interacts with Mitochondrial BAX Forming Heterodimers in the Mitochondrial Outer Membrane under Basal Conditions. Int J Mol Sci 18, (2017). 36. Ray, R. et al. BNIP3 heterodimerizes with Bcl-2 / Bcl-X(L) and induces cell death independent of a Bcl-2 homology 3 (BH3) domain at both mitochondrial and nonmitochondrial sites. J Biol Chem 275, 14391448, (2000). 37. Kubli, D. A., Ycaza, J. E. & Gustafsson, A. B. Bnip3 mediales mitochondrial dysfunctlon and cell death through Bax and Bak. Blochem J 405, 407-415, (2007). 38. Zhang, J. & Ney, P. A. Role of BNIP3 and NIX in cell death, autophagy, and mitophagy. Cell Death & Differentiation 16, 939-946, (2009). 39. Chaanine, A. H. et al. Potential role of BNIP3 in cardiac remodeling, myocardial stiffness, and endoplasmic reticulum: mitochondrial calcium homeostasis in diastolic and systolic heart failure. Gire Heart Fail 6, 572-583, (2013). 40. Chaanine, A. H. et al. FOXO3a regúlales BNIP3 and modulates mitochondrial calcium, dynamics, and function in cardiac stress. Am J Physiol Heart Circ Physiol 311, H1540-H1559, (2016). 41. Hendgen-Cotta, II. B. et al. Nitrite reducíase activity of myoglobin regúlales respiration and cellular viability in myocardial ischemia-reperfusion injury. Proc Nati Acad Sci U S A 105, 10256-10261, (2008). 42. Luedike, P. et al. Cardioprotection through S-nitros(yl)ation of macrophage migration inhibitory factor. Circulation 125, 1880-1889, (2012). 43. Rassaf, T. et al. Nitrite reducíase function of deoxymyoglobin: oxygen sensor and regulator of cardiac energetics and function. Circ Res 100,1749-1754, (2007). 44. Totzeck, M. et al. Nitrite regúlales hypoxic vasodilation vía myoglobin-dependent nitric oxide generation. Circulation 126, 325-334, (2012). 45. Wolter, K. G. et al. Movement of Bax from the cytosol to mitochondria during apoptosis. J Cell Biol 139, 1281-1292, (1997). 46. Hou, Q. & Hsu, Y. T. Bax translocates from cytosol to mitochondria in cardiac cells during apoptosis: development of a GFP-Bax-stable H9c2 cell Une for apoptosis analysis. Am J Physiol Heart Circ Physiol 289, H477-487, (2005). 47. Salí, A. & Blundell, T. L. Comparative protein modelling by satisfaction of spatial restraints. J Mol Biol 234, 779-815, (1993). 48. van den Berg, A. & Dowdy, S. F. Protein transduction domain delivery of therapeutic macromolecules. CurrOpin Biotechnol 22, 888-893, (2011). 49. Shoji-Kawata, S. et al. Identification of a candidate therapeutic autophagy-inducing peptide. Nature 494, 201-206, (2013). 50. Hausenloy, D. J. & Yellon, D. M. Myocardial ischemia-reperfusion injury: a neglected therapeutic target. J Clin Invest 123, 92-100, (2013). I bCQnn / 77n7 / q / YIAI 51. Gottlieb, R. A. Cell death pathways in acute ischemia / reperfusion injury. J Cardiovasc Pharmacol Ther 16, 233-238, (2011). 52. Trott, O. & Olson, A. J. AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J Comput Chem 31,455-461, (2010). 53. Domínguez, C., Boelens, R. & Bonvin, A. M. HADDOCK: a protein-protein docking approach based on biochemical or biophysical information. J Am Chem Soc 125,1731 -1737, (2003). 54. Phillips, J. C. et al. Scalable molecular dynamics with NAMD. J Comput Chem 26, 17811802, (2005).< / anls>

Claims

1. A peptide characterized in that it comprises (i) a cell uptake signal; and (ii) a fragment of BNIP3 comprising positions 13 to 20 of BNIP3 or an amino acid sequence derived therefrom; wherein the peptide has a length of 50 amino acids or less.

2. The peptide according to claim 1, further characterized in that the BNIP3 fragment has a length of 12 amino acids or less, especially 10 amino acids or less, in particular 8 amino acids.

3. The peptide according to claim 1 or 2, further characterized in that the BNIP3 fragment comprises positions 13 to 20 of BNIP3, optionally comprising 1, 2 or 3 amino acid substitutions compared to positions 13 to 20 of BNIP3.

4. The peptide according to claim 3, further characterized in that the amino acid substitutions are present in one or more of the positions corresponding to positions 15, 17 and 19 of BNIP3.

5. The peptide according to claim 3 or 4, further characterized in that the amino acid substitutions are selected from (i) glutamic acid at position 15 of BNIP3 to phenylalanine, isoleucine, leucine, valine, tyrosine, cysteine, histidine, arginine or threonine, and (ii) histidine at position 17 of BNIP3 to valine, and (iii) serine at position 19 of BNIP3 to tyrosine, cysteine, phenylalanine or histidine.

6. The peptide according to any of claims 1 to 5, further characterized in that the BNIP3 fragment comprises a phenylalanine residue in the position corresponding to position 19 of BNIP3.

7. The peptide according to any one of claims 1 to 6, further characterized in that the BNIP3 fragment (i) comprises positions 12 to 20 of BNIP3, optionally comprising 1, 2, 3 or 4 amino acid substitutions compared to positions 12 to 20 of BNIP3; (ii) comprises positions 4 to 20 of BNIP3, optionally comprising 1, 2, 3, 4, 5 or 6 amino acid substitutions compared to positions 4 to 20 of BNIP3; or (iii) comprises positions 1 to 20 of BNIP3, optionally comprising 1, 2, 3, 4, 5 or 6 amino acid substitutions compared to positions 1 to 20 of BNIP3; or (iv) consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 3 to 30.

8. The peptide according to claim 1, further characterized in that the BNIP3 fragment comprises a D-reverse-reverse sequence of positions 13 to 20 of BNIP3 or of any of the amino acid sequences defined in claims 2 to 7.

9. The peptide according to any one of claims 1 to 8, further characterized in that the cell uptake signal I bCQnn / 77n7 / q / YIAI (i) is a peptide having a length of 5 to 30 amino acids, in particular 8 to 20 amino acids or 10 to 16 amino acids, especially 12 amino acids; (ii) is the protein transduction domain of the HIV TAT protein; and / or (iii) comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 31 to 50.

10. The peptide according to any of claims 1 to 8, further characterized in that the cell uptake signal comprises a D-reverse-reverse sequence of a cell-penetrating peptide, in particular a cell-penetrating peptide as defined in claim 9.

11. The peptide according to any of claims 1 to 10, comprising the cell uptake signal and the BNIP3 fragment, further characterized in that it optionally comprises a linker between the cell uptake signal and the BNIP3 fragment.

12. The peptide according to any of claims 1 to 11, further characterized in that it has a length of 40 amino acids or less, in particular 35 amino acids or less, especially 30 amino acids or less.

13. A pharmaceutical composition characterized in that it comprises the peptide according to any of claims 1 to 12.

14. The peptide according to any of claims 1 to 12 for use in the treatment of reperfusion-related and / or mitochondrial-related disorders, as well as cancer therapy-induced cardiotoxicity.

15. The peptide for use according to claim 14, wherein the reperfusion-related and / or mitochondrial-related disorder is selected from the group consisting of myocardial infarction, stroke, acute kidney injury, trauma, circulatory arrest, and cessation of blood flow during organ transplantation.

16. The peptide for use according to claim 14 or 15, wherein the peptide is adapted to be administered to the patient during or after the event of impaired blood supply, especially prior to reperfusion of the ischemic-affected tissue.

17. The peptide for use according to claim 16, wherein the peptide is adapted to be administered to the patient within a 2-hour period, particularly within a 30-minute period prior to reperfusion.

18. The peptide according to any of claims 1 to 12 for use in the treatment or prevention of tissue damage due to mitochondrial necrosis or apoptosis.

19. A method for screening a compound suitable for the prevention of reperfusion injury and / or mitochondrial-induced disorders and / or cancer therapy-induced cardiotoxicity, characterized in that it comprises (i) providing one or more candidate compounds; (ii) determining the ability of the candidate compounds to interfere with the binding of BNIP3 and BAX; (iii) selecting those candidate compounds that interfere with the binding of BNIP3 and BAX.