Improvement and treatment of infarct damage

Abstract

Infarction damage is ameliorated or treated through administration of mitochondrial degradation inhibitors, such as those that act by inhibiting the translocation of one or more molecules across the mitochondrial membrane, such as the BAX / BNIP3 complex that is involved in mitochondrial degradation. Preventing mitochondrial degradation allows for more efficient oxygenation of the infarcted area, allowing for a greater degree of cell survival and more successful recovery. Such treatments can reduce cell death signaling resulting from destabilized mitochondria, thereby reducing cell death signaling that may otherwise accompany the intervention.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This document claims the benefit of priority to U.S. Provisional Application No. 63 / 276,028, filed November 5, 2021, the contents of which are incorporated by reference in their entirety herein. [Background technology]

[0002] background Side effects are an ongoing concern in the broad field of patient interventions. A recurring concern is that an intervention may inadvertently or secondarily harm the patient by eliciting a secondary patient response that is inconsistent with the intended effect.

[0003] These secondary responses are particularly problematic when they result in cell signaling that can lead to the initiation of cell death. Several interventions independently pose this risk. These range from chemotherapy intended to target proliferating cancer cells, accidental trauma associated with surgery, peripheral nerve injury, compression of body parts that cause muscle destruction, burns, and debilitating diseases such as or associated with cancer, pulmonary, neurological and hematological diseases, or reoxygenation of myocardial or brain tissue in individuals suffering from circulatory disorders such as heart attack or stroke, among others.

[0004] A common theme of these interventions is the risk of initiating any number of programmed cell death pathways in the cells of the individual intended to be treated. These cell death pathways are often induced by mitochondria or involve abnormalities in mitochondrial components, such as calcium ion or reactive oxygen species accumulation, changes in inner membrane polarization, pore formation in the outer membrane, release of cytochrome c, mitochondrial swelling, or even lysis.

[0005] Treating acute circulatory disorders, for example, presents several challenges. During circulatory disorders, cells, cell populations, tissues or organs (collectively, affected areas) are starved of oxygen. This has a major negative impact on cellular respiration in mitochondria. However, rapid reintroduction of oxygen to the affected area induces several responses in mitochondria, such as the collapse of mitochondrial membranes, which counterintuitively leads to the initiation of cell death pathway signaling, which indicates cardiac dysfunction.

[0006] Similarly, chemotherapy that targets the selective killing of the cells or tumors that drive malignant tumors is also generally problematic.Either the target cells themselves or other cells of the patient may respond to treatment by initiating cell death pathways that involve changes in mitochondrial status, such as mitochondrial collapse.This can often signal a similar response in related cells, which can exacerbate the harmful side effects of chemotherapy, or even limit its effectiveness by preventing the use of chemotherapeutic agents at sufficient levels to target malignant or harmful cells.

[0007] Mitochondrial membrane collapse has been implicated as a final cell death pathway (Karch and Molkentin, 2015; Murphy et al., 2016; Hendgen-Cotta et al., 2019). Loss of mitochondrial inner membrane (MIM) impermeability to small solutes causes depolarization (Δψm) with cessation of ATP synthesis, mitochondrial swelling, subsequent membrane rupture and necrotic cell death (Bernardi et al., 2015; Kwong and Molkentin, 2015; Galluzzi et al., 2018). Pore formation in the mitochondrial outer membrane (MOMP) promotes cytosolic release of apoptotic proteins such as cytochrome c, apoptosis-inducing factor (AIF) and endonuclease G, triggering apoptosis (Ong and Gustafsson, 2012). Thus, damaged mitochondria contribute to various pathologies, including neurological and cardiovascular disorders (Hendgen-Cotta et al., 2008; Kloner et al., 2017; Nunnari and Suomalainen, 2012; Fuchs and Steller, 2011). Previous investigations into the reduction of mitochondria-driven cell death in humans have focused primarily on mitochondrial proteins or phospholipids, such as cyclophilin D and cardiolipin. However, this requires a direct interaction of therapeutic agents with mitochondria (Atar et al., 2015; Cung et al., 2015; Gibson et al., 2016; Schaller et al., 2010). Despite generally promising experimental evidence, such agents have failed to reduce cell death (Atar et al., 2015; Cung et al., 2015; Gibson et al., 2016; Hausenloy et al., 2017; Schaller et al., 2010). The death mitochondrial pathway has been proposed to be regulated by BCL-2 family members, among others ( Galluzzi et al., 2018 ), and the functional importance of BAX has been identified as a paramount effector ( Garner et al., 2019 ; Kalkavan and Green, 2018 ; Karch and Molkentin, 2015 ; Wei et al., 2001 ; Whelan et al., 2012 ).The precise molecular activators and mechanisms governing BAX translocation to mitochondria, its activation, mitochondrial interaction and pore formation in vivo are largely unknown ( Luna-Vargas and Chipuk, 2016 ).

[0008] Evidence from in vitro and ex vivo experiments suggests a pathogenic role for the BH3-only protein BNIP3, which is predominantly localized to mitochondria, in mitochondrial perturbation through homodimerization ( Diwan et al., 2007 ; Gustafsson, 2011 ; Hamacher-Brady et al., 2007 ; Karch and Molkentin, 2015 ; Kubasiak et al., 2002 ; Kubli et al., 2007 ; Regula et al., 2002 ; Vande Velde et al., 2000 ).

[0009] The recent finding that BNIP3 can interact with recombinant BAX and mitochondrial BAX in vitro, whose binding appears to be important for insertion into the MOM (Hendgen-Cotta et al., 2017), suggests the possibility of BNIP3 as a regulator of BAX. This may implicate BNIP3 / BAX as an important potential cytosolic therapeutic target for mitochondrial and cytoprotection. Elucidation of BNIP3 activity on cell death remains challenging due to the incomplete structural basis of BNIP3 function. Therefore, we investigated the precise mechanism of the deleterious function of BNIP3 on BAX and mitochondria-driven cell death and to develop therapeutic options, with the aim to understand mitochondrial signaling, as well as approaches to modulate the effects of treatments on mitochondria-induced cell death signaling to prevent mitochondrial damage with downstream signaling that may result in both cellular dysfunction and cell death, and manifest cardiac dysfunction, cardiotoxicity, and cardiac weakness. Summary of the Invention [Means for solving the problem]

[0010] Abstract Disclosed herein are methods, compositions, compositions for use and treatment regimens for improving the side effects of intervention.Some of these are methods, compositions, compositions for use and treatment regimens that include administering mitochondrial protection or stabilization agents, such as mitochondrial interaction and import inhibitors.Without being bound by theory, such treatments can reduce cell death signaling that arises from destabilized mitochondria, thereby reducing cell death signaling that may otherwise accompany intervention.By implementing the disclosures herein, the harm that arises from the methods, compositions, compositions for use and treatment regimens of intervention can be reduced.

[0011] Some exemplary interventional events are consistent with the disclosure herein, including reoxygenation in response to circulatory impairment to ameliorate infarct injury or risk of infarct injury, such as may occur in response to reoxygenation of oxygen-deprived tissue, for example, as may occur by application of a stent or other circulatory restoration procedure.

[0012] Further interventional events include the administration of chemotherapeutic agents, such as radiotherapy agents or chemotherapy agents that target cancerous cells, cell populations or tumors. Some such chemotherapeutic agents can cause off-target or unintended cell death, such as myocardial cell death or dysfunction, resulting in, for example, cardiac dysfunction or weakening (with or without chemotherapy or cancer treatment). Exemplary chemotherapeutic agents used to target cancer cells or tumors include alkylating agents such as, for example, altretamine, busulfan, carboplatin, carmustine, cisplatin, cyclophosphamide, dacarbazine, ifosfamide, lomustine, melphalan, temozolomide, trabectedin; antimetabolites such as 5-fluorouracil, 6-mercaptopurine, azacitidine, capecitabine, clofarabine, cytarabine, floxuridine, fludarabine, gemcitabine, methotrexate, pemetrexed, pentostatin, pralatrexate, trifluridine, and tipiracil; plant alkaloids such as vincristine, vinblastine, vinorelbine, paclitaxel, docetaxel, etoposide, teniposide, irinotecan, topotecan, and the like. alkyloids; HER2 inhibitors, such as trastuzumab, pertuzumab, margetuximab, immune checkpoint inhibitors, such as antibodies, such as nivolumab (anti-PD-1), avelumab (anti-PD-L1), ipilimumab (anti-CTLA-4), leratlimab (anti-LAG-3); anti-tumor agents, such as daunorubicin, doxorubicin, liposomal doxorubicin, epirubicin, idarubicin, and valrubicin. Exemplary chemotherapeutic agents include anthracyclines, such as daunorubicin, doxorubicin, liposomal doxorubicin, epirubicin, idarubicin, and valrubicins, such as doxorubicin.

[0013] Some mitochondrial stabilizing factors, such as mitochondrial interaction and import inhibitors, consistent with the disclosure herein, inhibit or block BNIP3 activity.The mitochondrial indications that are beneficially affected by the inhibitors disclosed herein variously include at least one indication selected from the list including DOX cell death induction, mitochondrial membrane potential destabilization, mitochondrial pore opening prevention, mitochondrial calcium ion overload prevention, mitochondrial ROS or accumulation prevention.The administration of mitochondrial stabilizing agents, such as interaction inhibitors and import inhibitors, in various embodiments, can stabilize mitochondria to attenuate cardiomyocyte dysfunction, death or cardiotoxicity, inflammatory response, cardiac dysfunction or cardiac weakness, preserve cardiac function, increase mitochondrial fitness, preserve autophagy flux, and otherwise inhibit mitochondria-mediated cell dysfunction and death signaling, and inflammatory response.

[0014] Compositions consistent with the disclosure herein may be contacted with an individual via a wide variety of routes, including ingestion, transdermal administration, or injection, among others.

[0015] Exemplary mitochondrial protective and / or stabilizing agents include mitochondrial interaction and import inhibitors, such as the BNIP3 fragments or BAX fragments disclosed herein and other agents in the art.

[0016] Consistent with the above and elsewhere in this disclosure, compositions for use in ameliorating the adverse effects of treatment are also disclosed herein. Some such compositions include oxygen or chemotherapeutic agents, such as mitochondrial protective and / or stabilizing agents, such as mitochondrial import inhibitors.

[0017] Some such compositions and methods disclosed herein further include identifying a patient to whom a mitochondrial protection and / or stabilization agent, such as a mitochondrial interaction and import inhibitor, is co-administered. The patient can be most directly identified as suffering from a circulatory disorder, such as a heart attack or stroke. Such a patient can be subjected to a treatment to restore circulation and oxygen exposure to one or more tissues, and a mitochondrial protection and / or stabilization agent, such as a mitochondrial interaction and import inhibitor, can be co-administered to address circulatory interruption. Such treatment can include administering a medicine to the patient, or subjecting the patient to a circulatory deblocking process, such as applying a stent, or both.

[0018] The patient may be further identified by being a candidate for cancer treatment, such as chemotherapy or radiation therapy, or may be an individual who is scheduled or expecting a surgical intervention, or may be a patient undergoing or having undergone such an intervention, such as an intervention that may result in the patient's bruising or thrombus formation. Additional patient identification criteria include status as a human patient with elevated or substantial chronic infarction risk, such as the risk associated with certain conditions. Examples include, among others, diabetes, hypertension, hyperlipidemia, obesity, genetic disorders, stress, smoking or smoking-related lung or other tissue damage, heart disease, stroke, burns, crush injuries, peripheral nerve injury, cardiac weakness, cachexia, sarcopenia and muscle wasting, such as in cancer, pulmonary disease, neurological disease, viral disease, blood disease, renal disease and liver disease, or (auto)inflammatory diseases. Additional patient identification criteria are contemplated and consistent with the disclosure herein.

[0019] Consistent with the above, compositions and methods are disclosed herein for treating and ameliorating intervention-related cell death, such as infarct injury, which occurs upon interruption of oxygenation of a cell population in a mammal. By implementing the disclosure herein, harm resulting from infarction can be mitigated by administration after, concurrently with, or prior to an infarct event or infarct intervention of a composition comprising a pharmaceutical agent for modulating mitochondrial activity or viability, such as a mitochondrial interaction inhibitor or mitochondrial transport inhibitor, or other mitochondrial integrity preserving agent.

[0020] Some compositions are consistent with the disclosure herein.Exemplary compositions include regulators of mitochondrial activity or viability.Some such regulators act by inhibiting mitochondrial membrane potential decomposition, inhibiting cytochrome c release, or otherwise inhibiting mitochondrial decomposition, such as mitochondrial decomposition that is involved in or signals cell apoptosis, necrosis, or other cell death signaling pathways.Some such regulators stabilize mitochondria, maintain mitochondrial homeostasis, or inhibit mitochondrial interaction, or mitochondrial import or mitochondrial signaling.

[0021] Some such compositions effect this inhibition by blocking signaling to mitochondria, for example by inhibiting some or all of the interaction with or translocation across at least one mitochondrial membrane, or by inhibiting both interaction with and translocation across the mitochondrial membrane. In some cases, the block is general, but in alternative cases, certain molecules, complexes, or signaling moieties are prevented from translocating across the membrane. For example, some compositions act by inhibiting protein complexes, such as the BAX / BNIP3 complex, from protein-protein interactions, insertion into the membrane, or translocation across the membrane.

[0022] Some such compositions effect this inhibition by blocking signaling to mitochondria, for example by inhibiting the formation or compositional changes of signaling or catalytic molecular complexes in the cytoplasm. In some cases, the blocking is general, but in alternative cases, specific molecules, complexes, or signaling moieties are prevented from forming or undergoing conformational changes. For example, some compositions act by inhibiting protein complexes, such as the BAX / BNIP3 complex, from forming or undergoing conformational changes, such as changes in response to translocation across a membrane or changes in prerequisites for translocation across a membrane.

[0023] Exemplary compositions inhibit proteins or protein complexes through several mechanisms, for example by presenting a portion of one of the protein components to interfere with the ability of a binding partner to interact with the corresponding region of the native protein of which the inhibitor comprises a portion. The portion of the protein component may be wild-type or unaltered. Alternatively, some compositions include proteins or polypeptides or peptides that exhibit some identity to one of the protein components of the complex, but have one or more regions, for example, modified at one or more amino acid residue positions, for example to improve stability, binding, complex inhibition or other activity.

[0024] Some such compositions inhibit or affect the BAX / BNIP3 complex to affect mitochondrial interaction and translocation and include segments of the complex components. Exemplary segments include BNIP3 segments such as the N-terminal segment of BNIP3, for example, the region drawn from MSQSGEENLQGSWVELHFSN of BNIP3, particularly WVELHFSN of BNIP3. Some such compositions present unmodified segments of BNIP3, while alternative compositions present modified BNIP3 segments such as polypeptides or peptides containing the sequence WVELHFFN, sometimes referred to herein as the PepB sequence. Additional BNIP3 segments spanning 5, 6, 7, 8, 9, 10, 15, 20, 25, 30 or more than 30 residues of BNIP3, or any number within the range defined by these numbers, are contemplated. Also contemplated are polypeptides that include all or part of BNIP3 as disclosed above, and further exhibit percent identity at least, at most or exactly 99%, 95%, 90%, 80%, 75%, 70%, 50%, or within the range defined by these percentages, or differ from full-length BNIP3, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30 residues or more BNIP3 fragments as identified by conventional local alignment. Additional variant polypeptides are listed elsewhere herein and are contemplated as part of this disclosure. Other fragments or full-length proteins are also contemplated, as are other mitochondrial stabilizing or protecting molecules.

[0025] Thus, compositions and methods are disclosed herein that relate to reducing the harm of infarction.Some such methods relate to improving infarction or infarction damage to subjects at risk of infarction or infarction damage risk events.Alternatively, or in combination, some such methods relate to improving infarction damage to subjects who suffer from or have experienced infarction events, such as acute infarction events.

[0026] Similarly, compositions and methods are disclosed herein for reducing the harm caused by chemotherapy or radiotherapy.Some such methods are related to improving the effect of secondary chemotherapy on the subject undergoing chemotherapy or radiotherapy, and the subject is at risk of infarction or infarction injury risk event.Alternatively, or in combination, some such methods are related to improving infarction injury on the subject suffering from or experiencing infarction event, such as acute infarction event.

[0027] The methods variously include administering a mitochondrial membrane interaction and translocation inhibitor, or other mitochondrial integrity preserving agent. Some such mitochondrial membrane interaction and translocation inhibitors inhibit BAX translocation to or through the mitochondrial membrane. Some such mitochondrial membrane interaction and translocation inhibitors inhibit BNIP3 interaction with the mitochondrial membrane, translocation to or through the mitochondrial membrane, or both BAX and BNIP3 interaction with the mitochondrial membrane, translocation to or through the mitochondrial membrane.

[0028] Infarct injury risk events include heart attack, stroke, kidney failure, liver failure, organ transplant, chemotherapy or other cancer treatments, surgery, burns, crush injuries, peripheral nerve injuries, cardiac weakness, cachexia, sarcopenia and wasting diseases, such as pulmonary diseases, neurological viral diseases, and hematological diseases, or any disorder associated with or resulting from insufficient oxygenation of an organ, tissue or cell population, particularly acute circulatory failure, or any disorder associated with disruption of mitochondrial activity, stability or homeostasis, such as those that may result in cellular dysfunction and cell death signaling.

[0029] Interaction and translocation inhibitors, or other mitochondrial integrity preserving agents, are often administered intravenously or via catheter, for example, at an injection or introduction site proximal to the site of potential infarct damage, either continuously or in a single or multiple bolus dose. Alternative routes of administration include injection, via catheter percutaneous administration, or ingestion.

[0030] Interaction inhibitor, translocation inhibitor or other mitochondrial integrity preserving agent is administered before infarction injury risk event, during or even after infarction injury risk event, or for a period after infarction injury risk event, for example, during surgery, organ transplantation, contusion or other injury.In various embodiments, administration is immediately before reperfusion, for example, 30, 20, 10, 5 4, 3, 2 or 1 minutes before reperfusion, most illustratively 5 minutes before reperfusion.Administration is alternately or additionally performed after reperfusion, for example, on one or more days up to 1, 2, 3, 4, 5, 6 or 7 days after reperfusion to all of these days.

[0031] In particular, in some cases, the interaction and migration inhibitor or other mitochondrial integrity preserving agent is administered before or at the same time as an acute event, such as surgery (such as surgery associated with infarction risk). Similarly, the interaction and migration inhibitor is administered to patients who have suffered an acute event, such as trauma, which is associated with a higher subsequent risk of infarction events. Alternatively, as described above, in some cases, the interaction and migration inhibitor is administered after an infarction event, such as an acute infarction event. In some cases, the interaction and migration inhibitor or other mitochondrial integrity preserving agent is administered before or at the same time as a cancer therapeutic intervention, such as a chemotherapy treatment regimen. The interaction and migration inhibitor administered after an infarction event, such as an acute infarction event, is optionally administered for a period of time before, at the same time as, or in addition to, reoxygenation of the infarcted tissue or cell population.

[0032] The interaction and migration inhibitors are variously administered in carriers that lack available oxygen molecules or have insufficient concentrations of oxygen molecules to effect reoxygenation in these cases. Alternatively, in some cases, the interaction and migration inhibitors are administered simultaneously with or in carriers that contain oxygen molecules at concentrations sufficient to effect reoxygenation of the infarcted tissue or cell population, or at levels consistent with preparation of the infarcted tissue or cell population for subsequent oxygenation.

[0033] The interaction and translocation inhibitors or other mitochondrial integrity preserving agents are variously administered continuously, via multiple regular or irregular doses, or in a single dose.

[0034] Alternatively, some methods herein relate to the treatment of chronic infarction risk conditions. In these cases, the interaction and migration inhibitor or other pharmaceutical agent is administered alone or as part of a treatment regimen administered to a mammal, such as a human patient, with elevated or substantial chronic infarction risk, such as the risk associated with a particular condition. Examples include, among others, wasting diseases such as diabetes, hypertension, hyperlipidemia, renal failure, liver failure, obesity, genetic disorders, stress, smoking or smoking-related lung or other tissue damage, heart disease, stroke, burns, crush injury, peripheral nerve injury, cachexia, sarcopenia and muscle wasting, pulmonary disease, neurological disease, viral disease, hematological disease, cardiac weakness, or (auto)inflammatory disease. In many cases, these embodiments of the present disclosure include repeated or regular administration of the inhibitor in a manner somewhat similar to regular insulin administration for blood glucose level management, or by injection, such as through a port.

[0035] Some preferred interaction and translocation inhibitors or other mitochondrial integrity preserving agents include chimeric proteins, polypeptides or peptides, such as a segment having at least 75% identity to at least 8 consecutive residues of BNIP3, e.g., a segment having at least 8 consecutive residues of BNIP3, the seventh of which includes a phenylalanine residue. In some cases, these eight consecutive residues include residues having at least 75% identity to residues corresponding to residues 13-20 of BNIP3. In some cases, the eight consecutive residues include residues having at least 87.5% identity to residues corresponding to residues 13-20 of BNIP3.

[0036] Larger protein complex fragments are also contemplated as components of the compositions used in the compositions and methods disclosed herein.For example, turning again to the BAX / BNIP3 complex targeting approach, the composition variously comprises BAX or BNIP as a segment that has at least 87.5% identity or at least 75% identity to 50 residues or less of the complex component, such as BNIP3.In representative embodiments, the BNIP3 fragment does not include a BH3 motif, and / or does not include a PESTQ motif, and / or does not include a transmembrane domain or a C-terminal BNIP3 domain.In many cases, the protein segment comprises a segment that binds to at least one of a BAX binding motif, a BNIP3 binding motif, or BAX and BNIP3.

[0037] Protein or peptide segments for use in the methods herein often include constituent segments from multiple sources; i.e., protein or peptide segments contemplated for use herein are often part of chimeric proteins in combination with a cellular import or localization signal, such as, for example, the HIV TAT cellular import signal motif.

[0038] The methods contemplated herein may include inhibiting mitochondrial interaction and translocation, for example, using the above protein or peptide segments or other compositions consistent with the disclosure herein to show efficacy in inhibiting mitochondrial translocation and subsequently reducing damage from a simultaneous or previous infarction event.In various cases, the translocation inhibitor or other mitochondrial integrity preserving agent herein reduces damage from a subsequent infarction event by at least 10%, at least 20%, at least 30%, at least 40%, at least 50% or at least 75%.Similarly, in various cases herein, the interaction and translocation inhibitor reduces the recovery time from a subsequent infarction event by at least 10%, at least 20%, at least 30%, at least 40%, at least 50% or at least 75%.The various methods herein reduce BAX mitochondrial concentration by at least 30%, or for example at least 10%, at least 20%, at least 30%, at least 40%, at least 50% or at least 75% compared to untreated baseline compared to untreated baseline. Various methods herein reduce BNIP mitochondrial concentration by at least 30%, or for example at least 10%, at least 20%, at least 30%, at least 40%, at least 50% or at least 75% compared to untreated standard.Various methods herein reduce mitochondrial swelling by at least 30%, or for example at least 10%, at least 20%, at least 30%, at least 40%, at least 50% or at least 75% compared to untreated standard.Various methods herein reduce BAX activity concentration by at least 30%, or for example at least 10%, at least 20%, at least 30%, at least 40%, at least 50% or at least 75% compared to untreated standard.Various methods herein reduce cytochrome c release by at least 30%, or for example at least 10%, at least 20%, at least 30%, at least 40%, at least 50% or at least 75% compared to untreated baseline.Various methods herein improve Inf / AAR by at least 50%, or for example at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% compared to untreated baseline.Various methods herein reduce caspase activity by at least 30%, or for example at least 10%, at least 20%, at least 30%, at least 40%, at least 50% or at least 75% compared to untreated baseline. Various methods herein reduce membrane depolarization by at least 30%, or, for example, by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, or at least 75%, compared to an untreated baseline.

[0039] Some methods disclosed herein further include monitoring infarct damage recovery, monitoring mitochondrial status, or both infarct damage recovery and mitochondrial status, often so that the impact of treatment on the mammal, such as recovery of a human patient, can be determined.

[0040] Also disclosed herein are compositions and methods for identifying molecules suitable for ameliorating, reducing or treating infarction or infarction-related cell or tissue damage. Some such methods relate to the binding of BAX / BNIP3 oligomeric complexes as an indicator of the role of the binding molecule in inhibiting BAX / BNIP3-mediated necrosis and / or apoptosis.

[0041] Some such methods for identifying molecules suitable for ameliorating, reducing or treating infarction or infarction-related cell or tissue damage include assaying for binding of the molecule to BAX / BNIP3 oligomeric complexes, and in particular, binding of the molecule to BAX / BNIP3 oligomeric complexes indicates efficacy in ameliorating infarction. Assaying for various bindings includes assaying for co-localization of the molecule and BAX / BNIP3 oligomeric complexes, for example as determined by fluorescence microscopy. Alternatively or in combination, the assay includes assaying for co-migration of the molecule and BAX / BNIP3 oligomeric complexes, and in many cases, the co-migration is performed under conditions to maintain the integrity of the oligomeric BAX / BNIP3 oligomeric complexes, for example under non-denaturing conditions. Co-migration is determined, for example, using either gel electrophoresis, SDS-PAGE or Western blot analysis. Assaying for binding can additionally or alternatively include assaying for co-precipitation of the molecule and BAX / BNIP3 oligomeric complexes, for example using co-immunoprecipitation. Any of the above assays can be performed on cells or cell extracts, such as cells or cell extracts that have been subjected to oxygen deprivation, and optionally immediately after oxygen deprivation, such as within 10 minutes of oxygen deprivation, or alternatively within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30 minutes, or 30 minutes or more after oxygen deprivation. Any of these assays can be performed at baseline, or after ischemia and 5, 10, 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 16 hours, 24 hours or more after reperfusion.

[0042] Some such methods for identifying molecules suitable for ameliorating, reducing or treating infarction or infarction-related cell or tissue damage include assaying the localization of the BAX / BNIP3 complex upon contact with the molecule. The contacting may occur, for example, in cells subjected to oxygen deprivation or in extracts of cells subjected to oxygen deprivation, particularly extracts containing functional mitochondrial membranes or functional mitochondria. In some such assays, retention of BAX / BNIP3 outside the mitochondria of cells indicates efficacy in ameliorating infarction. Similarly, inhibition of BAX / BNIP3 translocation to the mitochondria of cells may indicate efficacy in ameliorating infarction. Inhibition of BAX translocation to the mitochondria of cells may indicate efficacy in ameliorating infarction. In some cases, inhibition of BNIP3 translocation to the mitochondria of cells may indicate efficacy in ameliorating infarction. Assaying for BAX, BNIP3 or BAX / BNIP3 localization may include fluorescent assays, such as may be performed using fluorescently labeled antibodies or fluorescently labeled proteins, or epitope-tagged BAX, BNIP3 or BAX and BNIP3 proteins. Alternatively, or in combination, molecular localization may be determined using immunofluorescence or detection, or radiolabeling, and overlaying such signals on an image of the cell or mitochondrion in question. Alternatively, or in combination, localization of the complex includes one or more of an assay of mitochondrial integrity, an assay of mitochondrial swelling, an assay of cytochrome c release, or an assay of caspase-3 activity. Any of the above assays are performed on cells or cell extracts, such as cells or cell extracts subjected to oxygen deprivation, and optionally immediately after oxygen deprivation, such as within 10 minutes of oxygen deprivation, or alternatively within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30 minutes, or 30 minutes or more after oxygen deprivation. Any of these assays may be performed at baseline, or after ischemia, 5 minutes, 10 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 16 hours, 24 hours, or more after reperfusion.

[0043] Incorporation by Reference All publications, patents, and patent applications mentioned herein are incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.In particular, International Publication No. 02 / 02743, published on January 10, 2002, is incorporated by reference in its entirety herein.Similarly, International Publication No. 2020 / 229632, published on November 19, 2020, is incorporated by reference in its entirety herein. BRIEF DESCRIPTION OF THE DRAWINGS [Brief description of the drawings]

[0044] [Figure 1A] BNIP3 oligomer expression.

[0045] [Figure 1B] BNIP3 oligomer expression.

[0046] [Figure 1C] Schematic diagram of assay progression.

[0047] [Figure 1D] TAT-BNIP3 restores wild-type infarct ratio.

[0048] [Figure 1E] Delta-TM administration returns troponin I to sham-treated levels.

[0049] [Figure 1F] Delta TM reduces or delays ApopTag positive events compared to vehicle-treated infarcted tissue.

[0050] [Figure 1G] Delta-TM reduces the rate of mitochondrial depolarization.

[0051] [Figure 1H]Delta-TM reduces mitochondrial swelling to sham-treated levels as indicated by increased OD540 values.

[0052] [Figure 1I] Images showing that Delta-TM reduces mitochondrial swelling to sham-treated levels.

[0053] [Figure 1J] Delta-TM reduces relative mitochondrial BAX levels to nearly Sham levels.

[0054] [Figure 1K] Delta-TM reduces relative mitochondrial BNIP3 levels to nearly Sham levels.

[0055] [Figure 1L] Delta-TM reduces relative active BAX concentrations to near basal levels at I / R30.

[0056] [Figure 1M] TAT-BNIP3 induces mitochondrial BAX accumulation in a Bnip3- / - genetic background.

[0057] [Figure 1N] BAX and BNIP3 blots.

[0058] [Figure 1O] Delta-TM maintains relative cytochrome c concentrations near basal levels at I / R30.

[0059] [Figure 1P] Delta-TM restores ATP to basal levels.

[0060] [Figure 1Q] Delta-TM maintains relative BNIP3 phosphorylation at near basal levels.

[0061] [Figure 2A] BAX and BNIP3 bind in a spot assay.

[0062] [Figure 2B] Treatment schematic.

[0063] [Figure 2C] BAX peptide binding heat map.

[0064] [Figure 2D] BNIP3 folding model.

[0065] [Figure 2E] BNIP3 N-terminus.

[0066] [Figure 2F] BNIP3 secondary structure prediction.

[0067] [Figure 2G] BNIP3-BAX binding prediction.

[0068] [Figure 3A] TAT-20A MSQGEENLQGSWVELHFSN

[0069] [Figure 3B] TAT-20C LDAQHESGRSSSKSSHCDSP

[0070] [Figure 3C] FIG. 20A shows the decrease in relative BNIP3 mitochondrial concentration.

[0071] [Figure 3D] 20A restores basal relative caspase 3 activity.

[0072] [Figure 3E] 20A improves percent Inf / AAR compared to vehicle or 20C.

[0073] [Figure 3F] Human protein blot.

[0074] [Figure 3G] Mouse / human alignment

[0075] [Figure 3H] 20A Truncation Fluorescence Assay for N-Terminal Truncation. WVELHFSN shows elevated levels.

[0076] [Figure 3I] Substitution fluorescence assay.

[0077] [Figure 3J] Binding pocket prediction.

[0078] [Figure 3K] Interaction heatmap.

[0079] [Figure 3L] Interaction model.

[0080] [Figure 3M] Folding prediction.

[0081] [Figure 3N] TAT-WVELHFFN binds to BNIP3 and BAX

[0082] [Figure 3O] Time course model.

[0083] [Figure 3P] PLA positive events per cell.

[0084] [Figure 3Q] Comparison of TAT-peptide mutants.

[0085] [Figure 4A] Treatment regimen.

[0086] [Figure 4B] Peptide localization.

[0087] [Figure 4C] Peptide localization images.

[0088] [Figure 4D] TAT-WVELHFFN counteracts the TAT-WVELAASN increase in relative mitochondrial BNIP3 concentration.

[0089] [Figure 4E] TAT-WVELHFFN counters the TAT-WVELAASN increase in relative mitochondrial BAX concentration.

[0090] [Figure 4F] TAT-WVELHFFN counters the TAT-WVELAASN increase in relative mitochondrial swelling.

[0091] [Figure 4G] TAT-WVELHFFN counteracts the TAT-WVELAASN increase in relative active BAX concentration.

[0092] [Figure 4H] TAT-WVELHFFN counteracts the TAT-WVELAASN increase in relative cytochrome c concentrations.

[0093] [Figure 4I] TAT-WVELHFFN counteracts the TAT-WVELAASN increase in relative caspase-3 activity.

[0094] [Figure 5A] BNIP3 accumulation.

[0095] [Figure 5B] BAX accumulation.

[0096] [Figure 5C] BAX accumulation.

[0097] [Figure 5D] BAX accumulation.

[0098] [Figure 5E] BAX and BNIP3 coimmunoprecipitation results.

[0099] [Figure 5F] Consequences of BAX and BNIP3 accumulation.

[0100] [Figure 5G] BNIP3 accumulation affected by TAT-WVELHFFN.

[0101] [Figure 5H] Cumulative results.

[0102] [Figure 5I] TAT-WVELHFFN migration in baseline and I / R tissues.

[0103] [Figure 6A] Schematic of the assay time course.

[0104] [Figure 6B] AAR / LV% for different treatments.

[0105] [Figure 6C] TAT-WVELHFFN improves Inf / AAR%.

[0106] [Figure 6D] TAT-WVELHFFN improves Inf / AAR% at concentrations below 20 nmol.

[0107] [Figure 6E] TAT-WVELHFFN reduces ApopTag events.

[0108] [Figure 6F] Treatment images.

[0109] [Figure 6G] Delta TM and Delta TM / TAT-WVELHFFN decrease Inf / AAR%.

[0110] [Figure 6H] TAT-WVELHFFN reduces Inf / AAR and Inf / LV in porcine infarct events.

[0111] [Figure 6I] TAT-WVELHFFN reduces Inf / AAR in porcine ischemic stroke events.

[0112] [Figure 6J-L] (FIG. 6J) Treatment course.

[0113] (FIG. 6K) PepB treatment reduces Inf / AAR.

[0114] (FIG. 6L) Before treatment, cardiac function was preserved more than in the control.

[0115] [Figure 6M] Treatment progress.

[0116] [Figure 6N] Additional post-treatment improves cardiac function over controls.

[0117] [Figure 7A] Assay flow diagram related to Figure 1B.

[0118] [Figure 7B] TAT-BNIP3 reduces the signal of the Bnip3− / − background relative to FIG. 1D.

[0119] [Figure 7C] Delta TM reduces Inf / AAR compared to vehicle, relevant to FIG. 1E.

[0120] [Figure 8A] Mitochondrial depolarization assay related to Figure 1G.

[0121] [Figure 8B] Mitochondrial swelling and damage assays related to Figure 1H and Figure 1I.

[0122] [Figure 8C] Flow cytometry results related to Figure 1G.

[0123] [Figure 8D] Mitochondrial images related to Figure 1H and Figure 1I.

[0124] [Figure 8E] Heart and cardiomyocyte protein blots related to Figure 1J and Figure 1K.

[0125] [Figure 8F] BNIP3 mRNA accumulation levels related to Figure 1J and Figure 1K.

[0126] [Figure 8G] Relative BNIP3 protein concentrations related to Figure 1J and Figure 1K.

[0127] [Figure 8H] Subcellular fractionation studies of BNIP3 monomers and dimers. The dimers show increased mitochondrial localization relative to Figure 1J and Figure 1K.

[0128] [Figure 8I] Mouse heart localization images related to Figure 1J and Figure 1K.

[0129] [Figure 9A] Relative cytochrome c, AF, active BAX accumulation levels for vehicle, DIDS, BAX and Delta™, and associated assay schematics for FIG. 1L.

[0130] [Figure 9B] Assay schematic related to Figure 1L.

[0131] [Figure 9C] Assay schematic related to Figure 1L.

[0132] [Figure 9D] Relative BNIP3 phosphorylation and active BAX concentrations are affected by AKT inhibition as related to FIG. 1Q.

[0133] [Figure 10A] Mutational alanine scan heat map related to Figure 3H.

[0134] [Figure 10B] WVELAASN folding prediction related to Figure 3L.

[0135] [Figure 11A] Proteinase K accumulation associated with Fig. 4B .

[0136] [Figure 11B] The effect of WVELHFFN on ejection fraction and left ventricular fractional shortening related to Figure 4B.

[0137] [Figure 11C] TAT-WVELAASN and TAT-WVELHFFN have comparable effects on cell viability related to Figure 4B.

[0138] [Figure 12A] Confirmatory synthesis assay.

[0139] [Figure 12B] Confirmatory synthesis assay.

[0140] [Figure 12C] Confirmatory synthesis assay.

[0141] [Figure 12D] Confirmatory synthesis assay.

[0142] [Figure 13A] DOX and TAT-WVELHFFN uptake.

[0143] [Figure 13B] TAT-WVELHFFN counteracts DOX-induced cell death.

[0144] [Figure 14A] TAT-WVELHFFN counteracts DOX-induced cell death at high DOX concentrations.

[0145] [Figure 14B] TAT-WVELHFFN reduces the number of RLUs when 10 uM DOX is administered.

[0146] [Figure 15A] TAT-WVELHFFN counteracts DOX-induced loss of mitochondrial membrane potential.

[0147] [Figure 15B] TAT-WVELHFFN counteracts DOX-induced loss of mitochondrial membrane potential.

[0148] [Figure 16A] TAT-WVELHFFN antagonizes DOX-induced mPTP opening.

[0149] [Figure 16B] TAT-WVELHFFN antagonizes DOX-induced mPTP opening.

[0150] [Figure 17A] TAT-WVELHFFN counteracts DOX-induced calcium ion accumulation.

[0151] [Figure 17B] TAT-WVELHFFN counteracts DOX-induced calcium ion accumulation.

[0152] [Figure 18A] TAT-WVELHFFN counteracts DOX-induced reactive oxygen species (ROS) accumulation.

[0153] [Figure 18B] TAT-WVELHFFN counteracts DOX-induced reactive oxygen species (ROS) accumulation.

[0154] [Figure 19] Schematic diagram of treatment regimen.

[0155] [Figure 20-1] Survival curves, body weight change measurements, tibia total length and heart weight measurements indicate that TAT-WVELHFFN does not affect the long-term survival of healthy mice. [Figure 20-2] Survival curves, body weight change measurements, tibia total length and heart weight measurements indicate that TAT-WVELHFFN does not affect the long-term survival of healthy mice.

[0156] [Figure 21] TAT-WVELHFFN increases cardiac troponin I / body weight and creatine kinase / bondy weight, but does not increase LDH / body weight or BNP / body weight for the indicated treatment regimen.

[0157] [Figure 22A] For non-zero time points, TAT-WVELHFFN (right data points) restores cardiac function to vehicle levels (left data points) countering the negative effects of DOX (middle data points).

[0158] [Figure 22B] For non-zero time points, TAT-WVELHFFN (right data points) restores cardiac function to vehicle levels (left data points) countering the negative effects of DOX (middle data points).

[0159] [Figure 22C] For non-zero time points, TAT-WVELHFFN (right data points) counteracts the effects of DOX (middle data points) and restores cardiac function to vehicle levels (left data points).

[0160] [Figure 22D] TAT-WVELHFFN and DOX have no effect on heart rate.

[0161] [Figure 22E] TAT-WVELHFFN opposes the effect of DOX in preventing cardiomyocyte death to vehicle levels.

[0162] [Figure 23A] TAT-WVELHFFN opposes the effect of DOX to maintain respiratory reserve capacity.

[0163] [Figure 23B] TAT-WVELHFFN opposes the effect of DOX in maintaining maximal respiratory capacity.

[0164] [Figure 23C] TAT-WVELHFFN opposes the effect of DOX to maintain basal respiration at vehicle levels.

[0165] [Figure 23D] TAT-WVELHFFN opposes the effect of DOX to maintain complex II stimulated respiration at vehicle levels as a measure of spare capacity.

[0166] [Figure 24A] Protein blot.

[0167] [Figure 24B] TAT-WVELHFFN partially restores LC3II / I levels in response to chloroquine addition to DOX samples.

[0168] [Figure 24C] TAT-WVELHFFN counteracts DOX-induced BNIP3 accumulation.

[0169] [Figure 24D] TAT-WVELHFFN counteracts DOX-induced p62 accumulation. [Diagram 25] Not specified [Figure 26A] Not specified [Figure 26B-1] Not specified [Figure 26B-2] Not specified [Figure 26B-3] Not specified [Figure 26B-4] Not specified [Figure 26B-5] Not specified DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0170] Detailed Description In order to improve the outcome of individuals who suffer from or are at risk of infarct injury due to interruption of oxygenation in affected area, the inventors have developed methods and compositions for treating or improving the symptoms of interruption of oxygenation to affected area.This research has then been expanded to include a wide range of approaches for improving secondary injury by intervention, particularly by mitochondrial stabilization, for example, by inhibiting mitochondrial interaction and import involved in mitochondrial destruction signaling.

[0171] The inventors' research has identified systems, methods and compositions for improving or treating ischemia / reperfusion (I / R) injury.In particular, the inventors herein disclose compositions and methods for treating I / R injury symptoms, for preventive treatment of individuals at risk for I / R injury, and for additional post-treatment of individuals at risk for the consequences of I / R injury, so as to reduce the harm that may result from subsequent I / R events in such individuals at risk.The inventors further disclose compositions and methods for improving some interventions related to the harmful effects resulting from mitochondrial destabilization.

[0172] We used unbiased biochemical approaches, computational modeling, and in vivo models of myocardial and cerebral I / R injury caused by a mixture of necrotic and apoptotic cell death as a clinically relevant and sophisticated system. Our results identify interactions and translocation across mitochondrial membranes as a mechanism to perturb to reduce mitochondrial cell death signaling in response to reperfusion and other causes of secondary adverse effects. We further identify cytosolic BNIP3 as a key regulator of BAX in its heteromeric state for both mitochondrial necrotic and apoptotic signaling in mammalian cells, including human cardiomyocytes, as a specific example validating this mechanism.

[0173] As a specific example related to the general concept of mitochondrial stabilization, we found that cytosolic oligomers containing BNIP3 / BAX heterodimers are a key mitochondrial membrane attack complex with BNIP3 phosphorylation as a master switch for necrotic and apoptotic cell death-inducing activity. By combining in silico BNIP3 3D modeling with structural and binding behavior investigations of BNIP3 and BAX derived peptides, we developed a BNIP3 octapeptide antagonist that prevents the identified mitochondrial membrane attack of oligomeric BNIP3 / BAX heterodimers. This effectively reduces cell death in human cardiomyocytes, mouse and pig tissues in I / R injury by 40-60%. These results were found to be generalizable to other causes of secondary adverse effects, e.g. those associated with chemotherapy interventions, where mitochondrial stabilization by import inhibition led to similar improvements in cell death outcomes in human cardiomyocytes, mouse and pig tissues.

[0174] Our results have implications for understanding the molecular mechanisms of mitochondrial membrane disruption leading to necrosis and apoptosis, and for identifying the therapeutic potential of BNIP3 antagonists for I / R injury conditions such as myocardial infarction and stroke in humans. Our results further have implications for methods of treatment and compositions for use in treatment, in that incorporation of mitochondrial protective and / or stabilizing agents, such as the mitochondrial interaction and import inhibitors disclosed herein, can have a substantial positive effect on reducing secondary cell death.

[0175] Mitochondria are essential end effectors in physiological and pathological cell death induction (Kwong and Molkentin, 2015). When damaged, they transform the injury into lethal signaling pathways (Nunnari and Suomalainen, 2012). Previous studies hypothesized that BNIP3 is a potential upstream regulator of BAX that disrupts the integrity of the MOM and MIM and induces necrotic and apoptotic cell death (Hamacher-Brady et al., 2007; Kubli et al., 2007, 2008). Notably, BNIP3 has been suggested to exert its death-promoting activity as a stable homodimer that localizes to the MOM by coordinating BAX activation (Kubli et al., 2007). However, fundamental questions regarding the underlying mechanism remained unresolved, partly due to the lack of a complete structure of BNIP3. Since spin labeling followed by pulsed electron paramagnetic resonance spectroscopy confirmed that BNIP3 and BAX can heterodimerize (Hendgen-Cotta et al., 2017), we investigated the activity of BNIP3 / BAX in a potential heterodimeric state that can cause mitochondrial collapse. Based on further insights into the underlying mechanisms, we developed a general strategy to prevent mitochondria-induced necrosis and apoptosis. We found that BNIP3 and BAX form inactive heterodimers in the cytosol, which assemble into high molecular weight complexes under native and I / R conditions. Thereby, the first 20 amino acids, including the conserved region, represent a functional domain in binding to BAX. These oligomeric BNIP / BAX heterodimers are essential for interaction with mitochondria and subsequently induce depolarization of the MIM. Unexpectedly, BAX activation and pore formation did not occur simultaneously. This is important because BAX activation is required for the apoptotic signaling cascade (Tang et al., 2019). These observations therefore make post-translational modifications of BNIP3 a potential stimulus that stimulates BAX activation through conformational changes.Phosphorylation modifies proteins from hydrophobic non-polar to hydrophilic polar, allowing proteins to change conformation when interacting with other molecules (Ardito et al., 2017). Our data represent the first report of phosphorylation of BNIP3 in regulating BAX activation and pore formation. During ischemia and early stages of reperfusion, ATP synthesis is highly limited (Chen and Zweier, 2014), which explains the timeline of BNIP3 / BAX interaction-induced necrosis and apoptosis in I / R. Taken together, our findings suggest that phosphorylation of BNIP3 may convert BAX conformation to form MOMP. Protection of mitochondria to avoid mitochondria-driven cell death-limiting tissue injury is currently an unmet therapeutic goal (Nunnari and Suomalainen, 2012). For example, the prominent impact of BNIP3 / BAX heterodimer activity under the control of BNIP3 on cell death in response to reperfusion therapy (Heusch and Rassaf, 2016; Kloner et al., 2017), which is essential to rescue patients during ischemic events, means that the BNIP3 / BAX heterodimer is an excellent novel target for the development of small peptide antagonists. Guided by newly modeled 3D structural information and specific N-terminal sequence behavior investigation of BNIP3, we discovered the key amino acids and engineered the cell-permeable octapeptide TAT-WVELHFFN, which inhibits the oligomeric mitochondrial membrane attack complex containing BNIP3 / BAX heterodimer in the cytosol as a defense against mitochondrial damage and cell death. Treatment with the BNIP3 octapeptide antagonist TAT-WVELHFFN shows excellent tissue protection in myocardial infarction and stroke. Highlighting the translational potential of this approach, the octapeptide TAT-WVELHFFN prevents cell death in mouse, porcine and human cardiomyocytes.

[0176] Thus, disclosed herein are compositions, compositions for use, and methods for ameliorating I / R injury, for example, by administration of compositions comprising mitochondrial membrane interaction and transport inhibitors or other mitochondrial integrity preserving agents.More generally, disclosed herein are compositions, compositions for use, and methods for ameliorating secondary injury caused by interventional regimens such as chemotherapeutic agents, radiation therapy, surgical intervention, or chronic conditions, among others.

[0177] Some mitochondrial membrane interaction and translocation inhibitors or other mitochondrial integrity preserving agents are consistent with the disclosure herein. Some mitochondrial membrane interaction and translocation inhibitors or other mitochondrial integrity preserving agents disclosed or contemplated herein share the effect of protecting mitochondria from degradation upon reintroduction of oxygen after an ischemic event. Others share the property of improving the measurements of mitochondrial state changes shown in Figures 13A-24D, such as ROS accumulation, calcium ion accumulation, mitochondrial mPTP opening, mitochondrial membrane integrity preservation, caspase-3 activation or cytochrome c release. Protection from degradation includes at least one of the following: maintaining mitochondrial membrane potential, preventing cytochrome c release into the cytoplasm, reducing mitochondrial swelling, preventing proteins such as BAX protein or BNIP3 from interacting with and translocating to the outer membrane, or other mitochondrial degradation. The effect of this protection is that there is no subsequent initiation of cell death pathways in the affected cells, so that reperfusion or regimen application of oxygen or therapeutic compounds can be achieved without the simultaneous induction of mitochondrial degradation and cell death.

[0178] Some examples of mitochondrial stabilizing factors that inhibit cytochrome c release are described in Bombrun et al., 3,6-dibromocarbazole piperazine derivatives of 2-propanol as first inhibitors of cytochrome c release via BAX channel modulation. J. Med. Chem. 46, 4365-4368 (2003), which is incorporated by reference in its entirety. Additional inhibitors of mitochondrial apoptosis consistent with the present disclosure are described in Peixoto, PM, Ryu, SY, Bombrun, A., Antonsson, B., and Kinnally, KWMAC inhibitors suppress mitochondrial apoptosis. Biochem. J. 423, 381-387 (2009), which is incorporated by reference in its entirety. Small molecule allosteric inhibitors of BAX protect against doxorubicin-induced cardiomyopathy (Nature Cancer | VOL 316 1 | March 2020 | 315-328, incorporated herein by reference in its entirety). Exemplary mitochondrial stabilizing factors include small molecules, proteins, protein fragments, and molecules that share some degree of identity with known proteins or protein fragments, such as at least 70%, 80%, 90%, 95% or 95% or more, to known protein fragments involved in mitochondrial stability. Some examples of mitochondrial stabilizers include some or all of PepB disclosed herein, and in some cases incorporate some or all of PepB into a larger molecular pharmaceutical.

[0179] Of particular interest are specific protein, polypeptide or peptide mitochondrial membrane interaction and transport inhibitors or other mitochondrial integrity preserving agents, however, several protein, hormone and small molecule effectors are consistent with the methods disclosed herein.

[0180] Representative mitochondrial integrity preserving agents are found in a number of mitochondrial membrane transport inhibitors derived from BNIP3 near the N-terminal fragment disclosed herein and previously disclosed in PCT Publication No. WO2020 / 229362, published November 19, 2020, which is incorporated herein by reference in its entirety.

[0181] Exemplary segments described herein include BNIP3 segments such as the N-terminal segment of BNIP3, e.g., the region drawn from MSQSGEENLQGSWVELHFSN of BNIP3, particularly WVELHFSN of BNIP3. Some such compositions represent unmodified segments of BNIP3, while alternative compositions represent modified BNIP3 segments, such as a polypeptide or peptide comprising the sequence WVELHFFN. These proteins, polypeptides or peptides optionally include a cellular localization signal, such as the HIV TAT localization signal, at their N- or C-terminus.

[0182] Several administration routes are consistent with the methods and compositions herein. Exemplary administration routes include direct administration of mitochondrial interaction and translocation inhibitors or other mitochondrial integrity preserving agents in a biocompatible solution to a mammalian subject, such as a human patient. Methods often include intravenous administration via a peripheral or central vein directly to the affected area, such as the affected ventricle, intraarterial administration via a peripheral artery, intraarterial administration via a catheter, or intraarterial administration via a catheter into the corresponding one or more coronary arteries and the peritoneal cavity. The administration route often correlates with the affected area in which I / R injury is ameliorated, and may be different for target areas, such as the heart, brain, kidney, or other affected areas.

[0183] Consistent with these routes of administration, several carriers are contemplated as part of the disclosure herein. NaCl solution, Ringer's solution, and most solutions consistent with human administration are compatible with the compositions and methods herein. Solutions are optionally selected or engineered to have a desired molecular oxygen concentration. In some cases, solutions are selected to be administered prior to reperfusion or reoxygenation and are formulated to have an oxygen concentration at least as low as the local affected area concentration. Alternatively, some solutions are formulated to achieve "pre-oxygenation" by having an oxygen concentration that is elevated but lower than the oxygen concentration of the subsequently applied high oxygen reperfusion composition. Alternatively, some compositions are delivered in solutions with high or reperfusion levels of dissolved oxygen to optionally achieve simultaneous mitochondrial translocation inhibition and oxygenation.

[0184] In addition to the carrier, the interaction and mitochondrial transport inhibitor or other mitochondrial integrity preserving agent is optionally administered simultaneously or in a composition containing one or more additional factors such as anesthetics, sedatives, anticoagulants, antibiotics, or amelioration of infarction events, prevention of further infarction events, patient recovery or remission of treatment, or any other reason that brings about a beneficial outcome. Exemplary anticoagulant cofactors include, among others, heparin, GPIIb / IIIa antagonists, any kind of platelet inhibitors. Concurrently administered pain relieving factors include morphine and / or morphine derivatives, or any other drugs that inhibit / relieve pain, such as fentanyl, hydromorphone, morphine, oxycodone, oxymorphone, dilaudid, tramadol, as well as butorphanol, NSAIDs (non-steroidal anti-inflammatory drugs, such as ibuprofen, acetaminophen, ketorolac, or other analgesics that are consistent with liquid formulations). The composition may alternatively or additionally include a sedative such as propofol or benzodiazepines. Likewise, the composition may alternatively or additionally include an antibiotic.

[0185] Methods of administration optionally include co-administration via alternative routes of complementary medicines, such as oral administration or inhalation. Examples include orally administered anticoagulants (such as warfarin), orally administered painkillers (such as opiates or NSAIS, among others), or inhaled or orally administered anesthetics, or antibiotics.

[0186] As disclosed herein, mitochondrial interaction and translocation inhibition has beneficial effects in individuals suffering from infarction events or undergoing therapeutic intervention, and can be administered before, at the same time, or after the start of reoxygenation or application of therapeutic drugs.The composition is variously administered up to 48 hours, up to 24 hours, up to 12 hours, up to 6 hours, up to 5 hours, up to 4 hours, up to 3 hours, up to 3 hours, up to 1 hour, up to 30 minutes, up to 15 minutes, up to 10 minutes, or up to 5 minutes before reoxygenation.The administration is a single dose, or includes a dosing regimen of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 doses.The dose includes sequential administration of equivalent pharmaceutical amounts, or includes changes in dose volume or pharmaceutical concentration, so that the effective composition can "rise" from an initial low level before oxygenation. In various embodiments, administration is immediately prior to reperfusion, e.g., 30, 20, 10, 5 4, 3, 2, or 1 minutes prior to reperfusion, most illustratively 5 minutes prior to reperfusion. Administration is alternated or additionally performed after reperfusion, e.g., on one or more up to and including 1, 2, 3, 4, 5, 6, or 7 days following reperfusion.

[0187] Administration is often performed in a hospital environment, such as an operating room. Alternatively, administration may be performed before the patient enters the hospital or before surgery. For example, paramedics may administer the compositions herein as soon as they see the patient or as soon as acute myocardial infarction is diagnosed or suspected. Early administration has the advantage of shortening the time to infarction surgery and extending the time that mitochondrial interaction and transfer inhibitors or other mitochondrial integrity preserving agents have to act before reoxygenation.

[0188] Administration is also performed before or at the same time as reperfusion of the ischemic myocardium. For example, percutaneous coronary intervention is performed using an interventional catheter system, and the blocked coronary artery is reopened using a wire, balloon and / or stent system. This procedure is often performed using anticoagulants such as heparin to inhibit blood clotting, using platelet inhibitors such as aspirin, clopidogrel, cangrelor, ticagrelor, prasugrel, GPIIB / IIIa antagonists, using drugs to inhibit / relieve pain (e.g., morphine or its derivatives, etc.), using drugs to sedate and relax the patient (e.g., propofol, benzodiazepines, etc.), and using different concentrations of oxygen before, during and / or after the procedure. Mitochondrial interaction and translocation inhibitors are administered simultaneously, before, after, or even for a period of time after such procedures.

[0189] In some cases, myocardial infarction is treated with fibrinolysis. Fibrinolysis is an important reperfusion strategy in situations where primary PCI cannot be provided in a timely manner, preventing 30 early deaths per 1000 patients treated within 6 hours after symptom onset. The greatest absolute benefit is seen among the highest risk patients, including the elderly, when treatment is provided less than 2 hours after symptom onset. Mitochondrial interaction and translocation inhibitors are administered simultaneously, before, after, or even for a period of time after such procedures.

[0190] In some cases, the operating room treats myocardial infarction with CABG (coronary artery bypass grafting) surgery. Emergency CABG is considered for patients with a patent infarct-related artery but anatomy unsuitable for PCI, with large myocardial territories at risk, or cardiogenic shock. In patients with MI-related mechanical complications requiring coronary revascularization, CABG is recommended at the time of repair. In STEMI patients with failed PCI or coronary artery occlusion unsuitable for PCI, emergency CABG is rarely performed, as the benefit of surgical revascularization in this setting is uncertain. Again, mitochondrial interaction and translocation inhibitors are administered simultaneously with, before, after, or even for a period of time following such procedures.

[0191] Alternatively, some methods disclosed herein include administration to patients at risk of infarction events.For example, infarction events, such as stroke, are substantial risks to individuals undergoing surgery or suffering from trauma (e.g., bruises) that may result from falls or accidents.Many surgeries, such as cardiac surgery (valve surgery, heart failure surgery, coronary artery surgery or other cardiac surgery or aortic surgery, etc.), are associated with higher infarction risk, and prolonged surgery in general can affect the heart and lead to organ damage.

[0192] Individuals who suffer an acute infarct event, such as a heart attack, are sometimes at increased risk of a second such event, such as a stroke, due to the destruction of blood clotting factors, often in response to treatment.

[0193] Further acute events with increased risk of infarction events include, for example, myocarditis, SARS-Cov2 or other viral infections, such as infections that lead to myocardial damage, especially in ICU patients. Similarly, cardiac trauma (e.g., accidents involving chest compression), hypoxia (e.g., asphyxiation, ICU treatment, such as may be suffered by high altitude mountaineers), anemia, such as bleeding or blood disease or cancer, or hypertensive crisis, or other conditions mentioned herein, may be suitable for carrying out the method or administration of the composition herein. It is understood that further acute conditions may result in infarction or cardiac damage requiring reperfusion, each of which may be ameliorated by treatment with the composition or implementation of the method herein.

[0194] Tissue or organ transplantation often increases the risk of infarction or myocardial injury. Transplanting the heart and transporting the organ to the heart transplant recipient often results in injury and cell death, which can be improved by implementing the disclosure herein. The composition can be added to the preservation solution during transportation or when given to the patient before or after the heart is transplanted. Apart from the heart, the composition disclosed herein can protect cells and therefore organ death in kidney, liver, lung or other transplants in some cases.

[0195] Similarly, some individuals present a higher risk of infarction due to chronic conditions such as hypertension, high cholesterol, diabetes, malignant or benign cancer, obesity, or moderate or mild overweight, among other conditions that increase the risk of infarction. Furthermore, long-term treatment regimes, such as cancer therapy (radiation, chemotherapy, anticancer immunotherapy or other cancer treatments) or other treatment regimes that include a series of repeated treatments, may increase the risk of infarction and are therefore suitable for the co-implementation of the methods or administration of the compositions disclosed or contemplated herein. A partial list of chemotherapeutic agents that are consistent with mediating mitochondrial degradation risk or would benefit from mitochondrial stabilization as a co-step of treatment includes radiotherapy or chemotherapeutic agents, such as chemotherapeutic agents that target cancerous cells, cell populations, or tumors. Some such chemotherapeutic agents cause off-target or unintended cell death, such as cardiomyocyte death. Exemplary chemotherapeutic agents used to target cancer cells or tumors include alkylating agents such as, for example, altretamine, busulfan, carboplatin, carmustine, cisplatin, cyclophosphamide, dacarbazine, ifosfamide, lomustine, melphalan, temozolomide, trabectedin; antimetabolites such as 5-fluorouracil, 6-mercaptopurine, azacitidine, capecitabine, clofarabine, cytarabine, floxuridine, fludarabine, gemcitabine, methotrexate, pemetrexed, pentostatin, pralatrexate, trifluridine, and tipiracil; The present invention includes compounds selected from the list consisting of ER2 inhibitors, such as trastuzumab, pertuzumab, margetuximab, immune checkpoint inhibitors, such as antibodies, such as nivolumab (anti-PD-1), avelumab (anti-PD-L1), ipilimumab (anti-CTLA-4), relatlimab (anti-LAG-3), etc.; plant alkaloids, such as vincristine, vinblastine, vinorelbine, paclitaxel, docetaxel, etoposide, teniposide, irinotecan, topotecan, etc.; and antitumor agents, such as daunorubicin, doxorubicin, liposomal doxorubicin, epirubicin, idarubicin, and valrubicin.Exemplary chemotherapeutic agents include anthracyclines, such as daunorubicin, doxorubicin, liposomal doxorubicin, epirubicin, idarubicin, and valrubicins, such as doxorubicin.

[0196] Thus, some methods include prophylactic or concomitant administration of mitochondrial transport inhibitors or other mitochondrial integrity preserving agents to subjects at risk for an infarct event or otherwise at risk for secondary effects from mitochondrial destabilization. Such administration can either directly ameliorate the harm or help promote reperfusion in response to the event by preparing the affected tissue or the entire subject for reperfusion in the affected area.

[0197] Administration is optionally acute, such as prior to surgery or in response to trauma or other isolated infarct risk event.

[0198] The composition is variously administered up to 48 hours, 24 hours, 12 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, 1 hour, 30 minutes, 15 minutes, 10 minutes, or 5 minutes before the approaching infarction risk event. Alternatively, the composition is variously administered at least or within 96 hours, 72 hours, 48 ​​hours, 24 hours, 12 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, 1 hour, 30 minutes, 15 minutes, 10 minutes, or 5 minutes after the infarction risk event. The administration is a single dose or includes a dosing regimen of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 doses. Dosage may include continuous administration of an equivalent amount of pharmaceutical agent, or in some cases, may include changes in dose volume or concentration of pharmaceutical agent, such that the effective composition may be "up-regulated" from an initial low level prior to a risk event, such as surgery.

[0199] Alternatively, administration is regular or sporadic in response to a chronic condition with a high risk of infarction events. Administration is variously monthly, weekly, daily, or at other regular or irregular intervals. Administration is often at a regular dose, but irregular dosages or time intervals are also contemplated herein. Irregular administration, such as in response to a biometric output, such as insulin levels, blood glucose levels, cholesterol levels, or other biometric readouts, is also contemplated herein. Administration may be alone or in combination with, for example, cholesterol-lowering or cholesterol-regulating pharmaceuticals.

[0200] The compositions and methods herein improve outcomes in mammals, such as human patients, who have suffered from an infarct event or are subject to a treatment regime that involves the risk of secondary mitochondrial destabilization. Improvement is measured in any number of ways, starting with increased survival rates and extending to more specific measurements of increased performance among surviving individuals. Improvement is measured, in some cases, by MRI visualization or measurement of infarct size, or measurement of salvage myocardium using risk area and infarct size. Measurement is also easily achieved by SPECT, PET, CT or other scanning techniques. Measurement can also be achieved by measurement of infarct size using contrast agents, left ventricular function (regional and global), or echocardiography relying on strain analysis.

[0201] Specific assessment of cardiac infarction recovery may depend on EKG:ST segment disappearance. Assays may also be accomplished by using ventriculography in an angiography room with contrast agents to determine left ventricular function.

[0202] Laboratory analyses or blood draw-based assays include measurements of troponin, high-sensitivity troponin, creatine kinase, and creatine kinase Mb.

[0203] The performance of the method or composition is often evaluated as cell viability or infarct area. Higher cell viability correlates with smaller infarct size. Infarct size is associated with prognosis and correlates with mortality. Higher cell viability is expected to correlate with smaller infarct size in MRI, higher myocardial salvage in MRI, lower infarct size in CT, PET, SPECT, better and faster ST-segment resolution, lower release of troponin, high sensitivity troponin, creatine kinase, creatine kinase Mb, lower infarct size in echocardiography, better left ventricular function in echo, normal strain / strain rate in echo, better left ventricular function in ventriculography. Cell viability is improved, in some cases, by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more than 90% compared to the control.

[0204] The performance of the method or composition may also be measured at the cellular level by effects on mitochondrial membrane translocation, such as BNIP3 or BAX, mitochondrial swelling, BAX mitochondrial activity concentration, cytochrome c release, Inf / AAR ratio, or caspase activity. Reductions in any of these parameters are, in some cases, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or greater than 90%.

[0205] Controls for these proportions are variously selected from measurements of outcomes in previous untreated individuals or mammals, measurements of experimental controls, fixed values ​​known in the art, values ​​measured from control or untreated populations, or other reference parameters.

[0206] Also disclosed herein are methods and compositions for identifying additional candidate molecules for preventing, ameliorating or treating infarction or infarction-related damage, or for preventing, ameliorating or treating secondary effects associated with mitochondrial destabilization or mitochondrial degradation, such as mitochondrial import-associated degradation, and associated signal transduction. Without being bound by theory, some such methods relate to the discovery that BAX / BNIP3 complexes are oligomeric in the cytoplasm, and that binding to such oligomeric complexes or inhibition of translocation of BAX, BNIP3, or both BAX and BNIP3 to mitochondria indicates candidate molecules for preventing, ameliorating or treating infarction or infarction-related damage.

[0207] The method is carried out by contacting a composition containing a candidate molecule with at least one cell, cell extract or tissue and assaying the effect of the molecule on molecular interaction or localization of the molecule, or on the localization of BAX, BNIP3 or BAX / BNIP3 heterodimers or oligomers.

[0208] The cells, cell extracts or tissues are subjected to oxygen deprivation in response to the induction of an infarction event or the simulation of the effect of an infarction event. Several oxygen starvation regimes are consistent with the disclosure herein, and such assays are performed within 10 minutes of oxygen deprivation, or within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30 minutes, or more than 30 minutes after oxygen deprivation, or for a longer period after oxygen deprivation. Any of these assays can be performed at baseline, or 5 minutes, 10 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 16 hours, 24 hours or more after reperfusion after ischemia. In some cases, the oxygen deprivation is absolute, but in the alternative, oxygen levels are reduced compared to healthy conditions, or in some cases remain unchanged or even increased compared to healthy conditions. Exemplary degrees of oxygen reduction include a reduction of 100%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5% or less than 5% compared to healthy or pre-assay conditions.

[0209] Alternatively, cells, cell extracts or tissues are subjected to radiation therapy, chemotherapy or other treatments in response to induction of secondary mitochondrial destabilization.Several treatment regimes are consistent with the disclosure herein, and such assays are performed within 10 minutes of treatment, or within or more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30 minutes after treatment, or for a longer period after treatment.Any of these assays can be performed at baseline, or after 5 minutes, 10 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 16 hours, 24 hours, or longer after treatment.A wide range of mitochondrial status outputs are available for assay, such as those used to generate the figures.

[0210] In some embodiments, intact cells are preferred, but alternatively, cell extracts are used, such as cell extracts having intact mitochondria or mitochondrial membranes, or cell extracts produced without substantially disrupting oligomeric complexes, such as the BAX / BNIP3 complex.

[0211] Molecules are assayed for their ability to bind to BAX, BNIP3, BAX / BNIP3 dimers, or in exemplary cases, BAX / BNIP3 oligomers. Without being bound by theory, binding to BAX, BNIP3, BAX / BNIP3 dimers, or in exemplary cases, BAX / BNIP3 oligomers indicates the efficacy or potential efficacy of the candidate molecule for preventing, ameliorating, or treating infarction or infarction-related damage.

[0212] Some binding assays are consistent with the disclosure herein. Assaying for various bindings includes assaying for co-localization of the molecule and the BAX / BNIP3 oligomeric complex, for example as determined by fluorescence microscopy. Alternatively or in combination, the assay includes assaying for co-migration of the molecule and the BAX / BNIP3 oligomeric complex, and in many cases, the co-migration is performed under conditions to maintain the integrity of the oligomeric BAX / BNIP3 oligomeric complex, for example under non-denaturing conditions. Co-migration is determined, for example, using gel electrophoresis, SDS-PAGE or Western blot analysis. Assaying for binding can additionally or alternatively include assaying for co-precipitation of the molecule and the BAX / BNIP3 oligomeric complex, for example using co-immunoprecipitation.

[0213] Alternatively, or in combination, the molecule is assayed for its effect on BAX / BNIP3 complex localization when contacted with the molecule. The contacting can occur, for example, in cells subjected to oxygen deprivation or in extracts of cells subjected to oxygen deprivation, particularly extracts containing functional mitochondrial membranes or functional mitochondria. In some such assays, retention of BAX / BNIP3 outside the mitochondria of cells indicates efficacy in ameliorating infarction. Similarly, inhibition of BAX / BNIP3 translocation to the mitochondria of cells may indicate efficacy in ameliorating infarction. Inhibition of BAX translocation to the mitochondria of cells may indicate efficacy in ameliorating infarction. In some cases, inhibition of BNIP3 translocation to the mitochondria of cells may indicate efficacy in ameliorating infarction. Assaying for BAX, BNIP3 or BAX / BNIP3 localization may include fluorescent assays, such as those that can be performed using fluorescently labeled antibodies or fluorescently labeled proteins, or epitope-tagged BAX, BNIP3 or BAX and BNIP3 proteins. Alternatively, or in combination, molecular localization may be determined using immunofluorescence or detection, or radiolabeling, and overlaying such signals on an image of the cell or mitochondrion in question. Assaying BAX, BNIP3, or BAX and BNIP3 in the mitochondrial fraction is an alternative approach to determine localization.

[0214] Alternatively, or in combination, complex localization assays may be supported or replaced by assays for mitochondrial integrity, mitochondrial swelling, cytochrome c release, caspase-3 activity, or other assays for mitochondrial activity or necrosis- or apoptosis-associated mitochondrial degradation.

[0215] Alternatively, or in combination, efficacy can be measured by the ability to improve the effect of the treatment on mitochondrial integrity, stability, or cell death signaling.

[0216] Numbered embodiments. The disclosure is further described with reference to the following partial list of numbered embodiments: 1. A method of ameliorating infarct injury to a subject at risk of an infarct injury risk event, comprising administering a mitochondrial membrane transport inhibitor. 2. The method of any of the previous embodiments, wherein the mitochondrial membrane transport inhibitor inhibits BAX transport to the mitochondrial membrane. 3. The method of any of the previous embodiments, wherein the mitochondrial membrane transport inhibitor inhibits BNIP3 transport to the mitochondrial membrane. 4. The method of any of the previous embodiments, wherein the infarct injury risk event comprises a heart attack. 5. The method of any of the previous embodiments, wherein the infarct injury risk event comprises a stroke. 6. The method of any of the previous embodiments, wherein the infarct injury risk event comprises renal failure. 7. The method of any of the previous embodiments, wherein the infarct injury risk event comprises acute circulatory failure. 8. The method of any of the previous embodiments, wherein the infarct injury risk event comprises organ transplantation. 9. The method of any of the previous embodiments, wherein the infarct injury risk event comprises surgery. 10. The method of any of the previous embodiments, wherein the transport inhibitor is administered intravenously. 11. The method of any of the previous embodiments, wherein the migration inhibitor is administered before an infarct injury risk event. 12. The method of any of the previous embodiments, wherein the migration inhibitor is administered before an infarct injury risk event. 13. The method of any of the previous embodiments, wherein the infarct injury risk event comprises surgery. 14. The method of any of the previous embodiments, wherein the migration inhibitor is administered after an infarct injury risk event. 15. The method of any of the previous embodiments, wherein the migration inhibitor is administered before reperfusion. 16. The method of any of the previous embodiments, wherein the infarct injury risk event comprises contusion. 17. The method of any of the previous embodiments, wherein the infarct injury risk event comprises heart failure. 18. The method of any of the previous embodiments, wherein the infarct injury risk event comprises a physiological response to stress. 19. The method of any of the previous embodiments, wherein the infarct injury risk event comprises a physiological response to diabetes. 20. The method of any of the previous embodiments, wherein the infarct injury risk event comprises a physiological response to hypertension. 21. The method of any preceding embodiment, wherein said infarct injury risk event comprises a physiological response to hyperlipidemia.22. The method of any of the previous embodiments, wherein the infarct injury risk event comprises a physiological response to obesity. 23. The method of any of the previous embodiments, wherein the infarct injury risk event comprises a physiological response to a genetic disorder. 24. The method of any of the previous embodiments, wherein the infarct injury risk event comprises a physiological response to a lung disorder. 25. The method of any of the previous embodiments, wherein the infarct injury risk event comprises an inflammatory response. 26. The method of any of the previous embodiments, wherein the infarct injury risk event comprises an autoinflammatory response. 27. The method of any of the previous embodiments, wherein the BAX translocation inhibitor is administered in multiple doses. 28. The method of any of the previous embodiments, wherein the multiple doses are administered at regular intervals. 29. The method of any of the previous embodiments, wherein the BAX translocation inhibitor comprises a chimeric peptide. 30. The method of any of the previous embodiments, wherein the chimeric peptide comprises a segment having at least 75% identity to at least 8 consecutive residues of BNIP3. 31. The method of any preceding embodiment, wherein said at least 8 contiguous residues of BNIP3 comprise a phenylalanine residue at position 7 of said at least 8 contiguous residues of BNIP3. 32. The method of any preceding embodiment, wherein said 8 contiguous residues comprise residues having at least 75% identity to residues corresponding to residues 13-20 of BNIP3. 33. The method of any preceding embodiment, wherein said 8 contiguous residues comprise residues having at least 87.5% identity to residues corresponding to residues 13-20 of BNIP3. 34. The method of any preceding embodiment, wherein said chimeric protein comprises a segment having 75% identity to no more than 50 residues of BNIP3. 35. The method of any preceding embodiment, wherein said chimeric protein comprises a segment having 87.5% identity to no more than 50 residues of BNIP3. 36. The method of any preceding embodiment, wherein said chimeric protein does not comprise a BH3 motif. 37. The method of any preceding embodiment, wherein said chimeric protein does not comprise a PESTQ motif. 38. The method of any preceding embodiment, wherein said chimeric protein comprises a BAX binding motif. 39. The method of any preceding embodiment, wherein said migration inhibitor reduces damage from a subsequent infarction event by at least 10%.40. The method of any of the previous embodiments, wherein the migration inhibitor reduces damage from a subsequent infarct event by at least 20%. 41. The method of any of the previous embodiments, wherein the migration inhibitor reduces damage from a subsequent infarct event by at least 30%. 42. The method of any of the previous embodiments, wherein the migration inhibitor reduces damage from a subsequent infarct event by at least 40%. 43. The method of any of the previous embodiments, wherein the migration inhibitor reduces damage from a subsequent infarct event by at least 50%. 44. The method of any of the previous embodiments, wherein the recovery of infarct damage is monitored. 45. A method of reducing damage of an infarct event to a subject suffering from an infarct event, comprising administering a mitochondrial membrane migration inhibitor. 46. The method of any of the previous embodiments, wherein the migration inhibitor is administered prior to reoxygenation following treatment of the infarct event. 47. The method of any of the previous embodiments, wherein the peptide is administered at least 5 minutes prior to reperfusion. 48. The method of any of the previous embodiments, wherein the peptide is administered up to 5 minutes prior to reperfusion. 49. The method of any preceding embodiment, wherein said peptide is administered at least 1 day prior to an infarction risk event. 50. The method of any preceding embodiment, wherein said peptide is administered at least 2 days prior to an infarction risk event. 51. The method of any of the previous embodiments, wherein the peptide is administered at least 3 days prior to an infarction risk event. 52. The method of any of the previous embodiments, wherein the peptide is administered at least 7 days prior to an infarction risk event. 53. The method of any of the previous embodiments, wherein the migration inhibitor is administered simultaneously with reoxygenation following treatment of the infarction event. 54. The method of any of the previous embodiments, wherein the treatment reduces BAX mitochondrial concentration by at least 30% compared to untreated baseline. 55. The method of any of the previous embodiments, wherein the treatment reduces BNIP3 mitochondrial concentration by at least 30% compared to untreated baseline. 56. The method of any of the previous embodiments, wherein the treatment reduces mitochondrial swelling by at least 30% compared to untreated baseline. 57. The method of any of the previous embodiments, wherein the treatment reduces BAX activity concentration by at least 75% compared to untreated baseline. 58. The method of any of the previous embodiments, wherein the treatment reduces cytochrome c release by at least 75% compared to untreated baseline. 59. The method of any of the previous embodiments, wherein said treatment improves the Inf / AAR ratio by at least 50% compared to untreated baseline. 60. The method of any of the previous embodiments, wherein said treatment reduces caspase activity compared to untreated baseline. 61. The method of any of the previous embodiments, wherein said treatment reduces membrane depolarization compared to untreated baseline. 62. The method of any of the previous embodiments, wherein said infarct injury risk event comprises a heart attack. 63. The method of any of the previous embodiments, wherein said infarct injury risk event comprises a stroke. 64. The method of any of the previous embodiments, wherein said migration inhibitor is administered intravenously. 65. The method of any of the previous embodiments, wherein said migration inhibitor is administered via a catheter. 66. The method of any of the previous embodiments, wherein said migration inhibitor comprises a chimeric peptide. 67. The method of any of the previous embodiments, wherein said chimeric protein comprises a segment having at least 75% identity to at least 8 consecutive residues of BNIP3. 68. The method of any preceding embodiment, wherein said at least eight consecutive residues of BNIP3 comprise a phenylalanine residue at position 7 of said at least eight consecutive residues of BNIP3.69. The method of any preceding embodiment, wherein the 8 contiguous residues comprise residues corresponding to residues 13-20 of BNIP3. 70. The method of any preceding embodiment, wherein the chimeric protein comprises a segment having 75% identity to no more than 50 residues of BNIP3. 71. The method of any preceding embodiment, wherein the chimeric protein does not comprise a BH3 motif. 72. The method of any preceding embodiment, wherein the chimeric protein does not comprise a PESTQ motif. 73. The method of any preceding embodiment, wherein the chimeric protein comprises a BAX binding motif. 74. The method of any preceding embodiment, comprising monitoring recovery of infarct injury. 75. A method of evaluating a molecule for infarction amelioration, wherein the method comprises assaying for binding of the molecule to a BAX / BNIP3 oligomeric complex. 76. The method of any preceding embodiment, wherein binding of the molecule to the BAX / BNIP3 oligomeric complex indicates efficacy in ameliorating infarction. 77. The method of any preceding embodiment, wherein said assaying comprises assaying for co-localization of the molecule and the BAX / BNIP3 oligomeric complex. 78. The method of any preceding embodiment, wherein said co-localization is assayed by fluorescence microscopy. 79. The method of any preceding embodiment, wherein said assaying comprises assaying for co-migration of the molecule and the BAX / BNIP3 oligomeric complex. 80. The method of any preceding embodiment, wherein said co-migration is performed under conditions that maintain the integrity of oligomeric BAX / BNIP3 oligomeric complexes. 81. The method of any preceding embodiment, wherein said co-migration is assayed using gel electrophoresis. 82. The method of any preceding embodiment, wherein said gel electrophoresis comprises SDS-PAGE. 83. The method of any preceding embodiment, wherein said gel electrophoresis comprises Western blot analysis. 84. The method of any preceding embodiment, wherein said co-migration is performed under non-denaturing conditions. 85. The method of any preceding embodiment, wherein said assaying comprises assaying for co-precipitation of said molecule and a BAX / BNIP3 oligomeric complex. 86. The method of any preceding embodiment, wherein said co-precipitation comprises immunoprecipitation.87. The method of any of the previous embodiments, wherein said assay comprises contacting said molecule with cells subjected to oxygen deprivation. 88. The method of any of the previous embodiments, wherein said assay comprises contacting said molecule with cells within 10 minutes of subjecting cells to oxygen deprivation. 89. A method of evaluating a molecule for ameliorating infarction, said method comprising contacting said molecule with cells subjected to oxygen deprivation and assaying for localization of BAX / BNIP3 complex. 90. The method of any of the previous embodiments, wherein retention of BAX / BNIP3 outside of mitochondria of said cells indicates efficacy in ameliorating infarction. 91. The method of any of the previous embodiments, wherein inhibition of BAX / BNIP3 translocation to mitochondria of said cells indicates efficacy in ameliorating infarction. 92. The method of any of the previous embodiments, wherein inhibition of BAX translocation to mitochondria of said cells indicates efficacy in ameliorating infarction. 93. The method of any of the previous embodiments, wherein inhibition of BNIP3 translocation to mitochondria of said cells indicates efficacy in ameliorating infarction. 94. The method of any preceding embodiment, wherein assaying for BAX / BNIP3 complex localization comprises immunofluorescence. 95. The method of any preceding embodiment, wherein assaying for BAX / BNIP3 complex localization comprises assaying for mitochondrial integrity. 96. The method of any preceding embodiment, wherein assaying for BAX / BNIP3 complex localization comprises assaying for mitochondrial swelling. 97. The method of any preceding embodiment, wherein assaying for BAX / BNIP3 complex localization comprises assaying for cytochrome c release. 98. The method of any preceding embodiment, wherein assaying for BAX / BNIP3 complex localization comprises assaying caspase-3 activity. 99. The method of any preceding embodiment, wherein said assaying comprises contacting said molecule with said cells within 10 minutes of subjecting said cells to oxygen deprivation. 100. A method of ameliorating a side effect of a treatment, comprising administering a mitochondrial protectant and / or stabilizer. 101. The method of any preceding embodiment, wherein the treatment comprises administering a chemotherapeutic agent. 102. The method of any preceding embodiment, wherein the mitochondrial protecting and / or stabilizing agent comprises a mitochondrial interaction inhibitor and an import inhibitor. 103. The method of any preceding embodiment, wherein the chemotherapeutic agent targets cancerous cells. 104. The method of any preceding embodiment, wherein the chemotherapeutic agent targets tumors. 105. The method of any preceding embodiment, wherein the chemotherapeutic agent causes cardiomyocyte death. 106. The chemotherapeutic agent is an alkylating agent such as altretamine, busulfan, carboplatin, carmustine, cisplatin, cyclophosphamide, dacarbazine, ifosfamide, lomustine, melphalan, temozolomide, trabectedin, etc.; antimetabolites such as 5-fluorouracil, 6-mercaptopurine, azacitidine, capecitabine, clofarabine, cytarabine, floxuridine, fludarabine, gemcitabine, methotrexate, pemetrexed, pentostatin, pralatrexate, trifluridine, and tipiracil; HER2 inhibitors, such as trastuzumab, pemetrexed, pentostatin, pralatrexate, trifluridine, and tipiracil, etc. 107. The method of any preceding embodiment, wherein the chemotherapeutic agent comprises a compound selected from the list consisting of daunorubicin, doxorubicin, liposomal doxorubicin, epirubicin, idarubicin, and valrubicin. 108. The method of any preceding embodiment, wherein the chemotherapeutic agent comprises a compound selected from the list consisting of daunorubicin, doxorubicin, liposomal doxorubicin, epirubicin, idarubicin, and valrubicin. 108. The method of any preceding embodiment, wherein said chemotherapeutic agent comprises an anthracycline. 109. The method of any preceding embodiment, wherein said anthracycline comprises doxorubicin. 110. The method of any preceding embodiment, wherein said inhibitor affects BNIP3 activity.111. The method of any of the previous embodiments, wherein said inhibitor affects at least one indication selected from the list comprising DOX cell death induction, mitochondrial membrane potential stabilization, mitochondrial pore opening prevention, mitochondrial calcium ion overload prevention, mitochondrial ROS or accumulation prevention. 112. The method of any of the previous embodiments, wherein said inhibitor attenuates cardiotoxicity. 113. The method of any of the previous embodiments, wherein said inhibitor attenuates cardiomyocyte death. 114. The method of any of the previous embodiments, wherein said inhibitor preserves cardiac function. 115. The method of any of the previous embodiments, wherein said inhibitor increases mitochondrial fitness. 116. The method of any of the previous embodiments, wherein said inhibitor preserves autophagy flux. 117. The method of any of the previous embodiments, wherein said inhibitor comprises a polypeptide. 118. The method of any of the previous embodiments, wherein said inhibitor comprises a BNIP3 fragment. 119. The method of any of the previous embodiments, wherein said inhibitor comprises a BAX fragment. 120. The method of any of the previous embodiments, wherein administering comprises injecting. 121. A composition for use in ameliorating adverse effects of treatment, comprising a chemotherapeutic agent and a mitochondrial interaction inhibitor and an import inhibitor. 122. A composition for use according to any of the previous embodiments, wherein said chemotherapeutic agent targets cancerous cells. 123. A composition for use according to any of the previous embodiments, wherein said chemotherapeutic agent targets tumors. 124. A composition for use according to any of the previous embodiments, wherein said chemotherapeutic agent causes cardiomyocyte death.125. The chemotherapeutic agent is an alkylating agent such as altretamine, busulfan, carboplatin, carmustine, cisplatin, cyclophosphamide, dacarbazine, ifosfamide, lomustine, melphalan, temozolomide, trabectedin, etc.; antimetabolites such as 5-fluorouracil, 6-mercaptopurine, azacitidine, capecitabine, clofarabine, cytarabine, floxuridine, fludarabine, gemcitabine, methotrexate, pemetrexed, pentostatin, pralatrexate, trifluridine, and tipiracil; HER2 inhibitors, for example, trastuzumab, pertuzumab, etc. 125. The composition for use according to any of the previous embodiments, wherein the chemotherapeutic agent comprises a compound selected from the list consisting of daunorubicin, doxorubicin, liposomal doxorubicin, epirubicin, idarubicin, and valrubicin. 126. The composition for use according to any of the previous embodiments, wherein the chemotherapeutic agent comprises a compound selected from the list consisting of daunorubicin, doxorubicin, liposomal doxorubicin, epirubicin, idarubicin, and valrubicin. 127. The composition for use according to any of the previous embodiments, wherein the chemotherapeutic agent comprises an anthracycline. 128. The composition for use according to any of the previous embodiments, wherein the anthracycline comprises doxorubicin. 129. The composition for use according to any of the previous embodiments, wherein the inhibitor affects BNIP3 activity. 130. The composition for use according to any of the previous embodiments, wherein the inhibitor affects at least one indication selected from the list comprising DOX cell death induction, mitochondrial membrane potential stabilization, mitochondrial pore opening prevention, mitochondrial calcium ion overload prevention, mitochondrial ROS or accumulation prevention. 131. The composition for use according to any of the previous embodiments, wherein the inhibitor attenuates cardiotoxicity.132. A composition for use according to any of the previous embodiments, wherein the inhibitor attenuates cardiomyocyte death. 133. A composition for use according to any of the previous embodiments, wherein the inhibitor preserves cardiac function. 134. A composition for use according to any of the previous embodiments, wherein the inhibitor increases mitochondrial fitness. 135. A composition for use according to any of the previous embodiments, wherein the inhibitor preserves autophagic flux. 136. A composition for use according to any of the previous embodiments, wherein the inhibitor comprises a polypeptide. 137. A composition for use according to any of the previous embodiments, wherein the inhibitor comprises a BNIP3 fragment. 138. A composition for use according to any of the previous embodiments, wherein the inhibitor comprises a BAX fragment. 139. A composition for use according to any of the previous embodiments, wherein administering comprises injecting. 140. A method of ameliorating harm to cardiac function, comprising identifying a patient at risk of harm associated with mitochondrial disruption and administering a mitochondrial import inhibitor to the patient. 141. A method according to any of the previous embodiments, wherein the harm associated with mitochondrial disruption is risk of harm to cardiac function. 142. The method of any preceding embodiment, wherein the patient at risk of harming cardiac function comprises a patient suffering from a myocardial infarction. 143. The method of any preceding embodiment, wherein the patient at risk of harming cardiac function comprises a patient suffering from a cardiac arrest. 144. The method of any preceding embodiment, wherein the patient at risk of harming cardiac function comprises a patient expected to undergo surgery. 145. The method of any preceding embodiment, wherein the patient at risk of harming cardiac function comprises a patient undergoing surgery. 146. The method of any preceding embodiment, wherein the patient at risk of harming cardiac function comprises a patient who has undergone surgery. 147. The method of any preceding embodiment, wherein the patient at risk of harming cardiac function comprises a patient undergoing a chemotherapeutic agent. 148. The method of any preceding embodiment, wherein the chemotherapeutic agent targets the cancer. 149. The method of any preceding embodiment, wherein the chemotherapeutic agent targets the tumor. 150. The method of any previous embodiment, wherein the chemotherapeutic agent and the mitochondrial import inhibitor are administered simultaneously. 151. The method of any preceding embodiment, wherein the chemotherapeutic agent and the mitochondrial import inhibitor are administered in a common composition. 152. The method of any preceding embodiment, wherein the chemotherapeutic agent and the mitochondrial import inhibitor are not administered simultaneously. 153. The method of any preceding embodiment, wherein the chemotherapeutic agent targets cancerous cells. 154. The method of any preceding embodiment, wherein the chemotherapeutic agent targets tumors. 155. The method of any preceding embodiment, wherein the chemotherapeutic agent causes cardiomyocyte death. 156. The chemotherapeutic agent is an alkylating agent such as altretamine, busulfan, carboplatin, carmustine, cisplatin, cyclophosphamide, dacarbazine, ifosfamide, lomustine, melphalan, temozolomide, trabectedin, etc.; antimetabolites such as 5-fluorouracil, 6-mercaptopurine, azacitidine, capecitabine, clofarabine, cytarabine, floxuridine, fludarabine, gemcitabine, methotrexate, pemetrexed, pentostatin, pralatrexate, trifluridine, and tipiracil; HER2 inhibitors such as trastuzumab, pemetrexed, pentostatin, pralatrexate, trifluridine, and tipiracil, etc. 157. The method of any preceding embodiment, wherein the chemotherapeutic agent comprises a compound selected from the list consisting of daunorubicin, doxorubicin, liposomal doxorubicin, epirubicin, idarubicin, and valrubicin. 158. The method of any preceding embodiment, wherein the chemotherapeutic agent comprises a compound selected from the list consisting of daunorubicin, doxorubicin, liposomal doxorubicin, epirubicin, idarubicin, and valrubicin. 158. The method of any preceding embodiment, wherein said chemotherapeutic agent comprises an anthracycline. 159. The method of any preceding embodiment, wherein said anthracycline comprises doxorubicin. 160. The method of any preceding embodiment, wherein said inhibitor affects BNIP3 activity.161. The method of any of the previous embodiments, wherein said inhibitor affects at least one indication selected from the list comprising DOX cell death induction, mitochondrial membrane potential stabilization, mitochondrial pore opening prevention, mitochondrial calcium ion overload prevention, mitochondrial ROS or accumulation prevention. 162. The method of any of the previous embodiments, wherein said inhibitor attenuates cardiotoxicity. 163. The method of any of the previous embodiments, wherein said inhibitor attenuates cardiomyocyte death. 164. The method of any of the previous embodiments, wherein said inhibitor preserves cardiac function. 165. The method of any of the previous embodiments, wherein said inhibitor increases mitochondrial fitness. 166. The method of any of the previous embodiments, wherein said inhibitor preserves autophagy flux. 167. The method of any of the previous embodiments, wherein said inhibitor comprises a polypeptide. 168. The method of any of the previous embodiments, wherein said inhibitor comprises a BNIP3 fragment. 169. The method of any of the previous embodiments, wherein said inhibitor comprises a BAX fragment. 170. The method of any of the previous embodiments, wherein administering comprises injecting.

[0217]

[0218] With reference to the drawings, the following can be seen:

[0219] Figure 1A-Q. Cytoplasmic BNIP3 sequentially determines necrotic and apoptotic cell death by regulating BAX in its heterodimeric state. (A) Immunoblot for cytosolic BNIP3 after blue native PAGE at baseline (n=5). (B) Immunoblot for cytosolic BNIP3 after size-exclusion chromatography (n=3) (Figure 7A). (C) Timeline and schematic of the in vivo I / R model of heart sections showing the non-ischemic area (remote; blue), ischemic area (area at risk, AAR; red), and infarct area (white, embedded in the AAR). (D) Wild-type mice and Bnip3 treated with TATBNIP3 as indicated. - / -Infarct size per area at risk (Inf / AAR) in mice (n=3–6), showing that increased TAT-Bnip3 administration reverts the Inf / AAR percent ratio to wild-type levels. (See Figure 7B) (E–L and N–Q) Wild-type mice were treated with vehicle or TAT-BNIP3-ΔTM. (E) Representative images of necrotic areas (blue), troponin I (red) and nuclei (blue) at 24 h of reperfusion. Scale bar, 1 mm and plasma troponin I after 24 h of reperfusion (n=4–12). Sham-operated mice served as controls. Delta-TM-treated mice show a substantial reversion to sham-treated troponin I levels (see Figure 7C). (F) Representative images of apoptotic cells (red) and nuclei (blue) in the AAR 4 h after I / R. Scale bars, 2,000 μm (left), 200 μm (right), and apoptotic events in whole hearts and apical-to-basal planar sections after 4 h of reperfusion (n=3–5). (G) Depolarized mitochondria at baseline and at the indicated reperfusion times (n=3–5) assessed by flow cytometry (see Figure 8A). I / R 10 In I / R, Delta-TM mice showed a return to basal levels of depolarized mitochondria. For 2D scatter plots, see FIG. 8C. (H) Mitochondrial swelling in the AAR after 10 min of reperfusion (OD, optical density; n=3-4). Sham-operated mice served as controls. 10 In the I / R study, Delta-TM mice showed a return to basal levels of swollen mitochondria. (I) Representative electron micrographs showing swollen mitochondria in the AAR at 10 min of reperfusion. Scale bars, 2 μm (left), 1 μm (right) of each pair (n=3). For fragmented mitochondria, see FIG. 8B and FIG. 8D. 10In Fig. 8, Delta-TM mice showed a return to basal levels of mitochondrial swelling. (J and K) Quantification and representative immunoblots (see Fig. 8E-I) of mitochondrial BAX concentrations in the area at risk (AAR) (n=5-7) at baseline and 10 min of reperfusion (AAR) (J), and mitochondrial BNIP3 levels (*p=0.0148 and *p=0.0185) (K). Delta-TM mice showed a return to basal levels for both assays. (L) Quantification and representative immunoblots of active BAX at baseline and the indicated reperfusion time points (n=5). I / R 30 In the control group, Delta-TM mice showed a return of active BAX concentrations to basal levels (see Figure 9A-C). (M) Untreated and TAT-BNIP3-treated Bnip3 mice - / - Quantification and representative immunoblot of mitochondrial BAX levels in the AAR of mice (n=3; *p=0.0152). I / R 10 In contrast, Delta-TM mice showed an opposite shift to untreated Bnip3- / - mice alone. (N) Western blot monitoring cytosolic BAX co-immunoprecipitated with BNIP3 (left) and cytosolic BNIP3 co-immunoprecipitated with BAX (right) at baseline and after 10 and 30 min of reperfusion (n=3). (O) Cytosolic cytochrome c in the AAR at baseline and the indicated reperfusion time points (n=5-6). I / R 30 In I / R, Delta-TM mice maintained basal levels of cytochrome c. (P) ATP in the AAR at baseline and 10 min of reperfusion (n=4; *p=0.0153 and *p=0.011). 10 In the control group, Delta-TM mice maintained basal levels of ATP. (Q) Serine-phosphorylated BNIP3 levels at baseline and the indicated reperfusion time points (n=5-8) (see FIG. 9D). Data represent mean±SEM. p-values ​​were determined by two-way ANOVA with Bonferroni correction. I / R 30 In mice, Delta-TM mice maintained basal levels of BNIP3 phosphorylation.

[0220] Figure 2A-G. The N-terminus is the interaction domain of BNIP3. A) Images of membranes spotted with BAX and BNIP3 and incubated with fluorescently labeled (Dy650) BNIP3 (left) and BAX (right). (B) Schematic of the protocol designed by JPT (Berlin, Germany). (C) Heatmap of BNIP3 incubation with the BAX peptide library, showing helices α5, α6 and α7+α8 as interaction sites. Color coding ranges from white (low or no intensity) to yellow (medium intensity) and red (high intensity), showing the structure of mouse BAX (PDB 4S0OZit) with BNIP3 interaction sites ribbon-displayed in blue (α5), green (α6), yellow (α7) and orange (α8). (D and E) 3D structural model of BNIP3 predicted by Modeller 9.15 with marked helices. (F) Circular dichroism (CD) spectroscopy of BNIP3. (G) Cartoon representation of the BAX / BNIP3 interaction with the indicated binding sites obtained from an in silico docking experiment using HADDOCK. BNIP3 is colored turquoise and BAX is colored green. Interaction sites are colored purple (BAX) and orange (BNIP3).

[0221] Figure 3A-Q. The N-terminus of BNIP3 is a key functional domain in human cardiomyocyte protection. (A) Structural formula of TAT-BNIP3-20A. (B) Structural formula of TAT-BNIP3-20C. (C-E) Mice were treated with TAT-BNIP3-20A (20A) or TAT-BNIP3-20C (20C). (C) Quantification and representative immunoblots of mitochondrial BNIP3 levels in the area at risk (AAR) at 10 min of reperfusion (n=6). 20A mice showed lower relative mitochondrial BNIP3 concentrations. (D) Quantification of caspase-3 activity in the AAR at 1 h of reperfusion (n=7). 20A mice showed lower relative caspase-3 activity, closer to basal levels. (E) Infarct size per AAR (Inf / AAR) in wild type mice treated with vehicle (NaCl), 20A or 20C for 24 hours of reperfusion (n=5-6). 20A mice showed lower INF / AAR percentage than either vehicle or 20C. (F) Immunoblot of BNIP3 in three human heart samples. (G) Alignment of human and mouse BNIP3 sequences. Red boxes indicate amino acids WVELHFSN, highest binding intensity represents conserved regions. (H and I) Peptide microarray of BNIP3 whose N-terminal sequence is BNIP3 (1-20aa). (H) N-terminal sequence truncation analysis elucidating BNIP3 / peptide interactions. VELHFSN showed substantially higher luminescence (see Figure 10A). (I) Single amino acid substitutions characterizing their binding ability. (J) Docking experiment on BAX (PDB code 4S0O) with TAT-WVELHFFN showing the interaction site in BAX. (K) Docking experiment on BAX (PDB code 4S0O) with TAT-WVELHFFN and TATWVELHFSN, and on a peptide microarray of BAX with the indicated peptide sequences. The addition of a third aromatic residue results in improved intrapeptide aromatic interactions, especially for WVELHFFN. (L) Space-filling model and structural formula of TAT-WVELHFFN (see Figure 10B). (M) Circular dichroism (CD) spectroscopy analysis of TAT-WVELHFFN.(N) Images of membranes spotted with BNIP3 (top) and BAX (bottom) and incubated with fluorescently labeled TAT-WVELHFFN (TAT-WVELHFFN-fluo). (O) Timeline of in vitro hypoxia / reoxygenation (H / R) study design in human ventricular cardiomyocytes derived from human induced pluripotent stem cells (human CMs). (P) BNIP3 / BAX interaction (n=5) in human CMs after 1 h hypoxia / 1 h reperfusion (PLA, proximity ligation assay) and representative staining confocal images. Cellular structures (green), nuclei (blue), PLA spots (red; marked by white arrows), 40x magnification. (Q) Depolarized mitochondria (green), healthy mitochondria (red), and nuclei (blue) after 1 h hypoxia / 1 h reoxygenation (upper panel). Apoptotic (green), necrotic (red) and healthy (blue) human CMs after 2 h hypoxia / 2 h reoxygenation treated with TAT-WVELAASN and TATWVELHFFN (lower panels). Scale bar, 200 μm. Data represent mean ± SEM. p values ​​were determined by two-way ANOVA with Bonferroni correction or two-tailed Student's t-test.

[0222] Figure 4A-H. WVELHFFN peptide functions in necrotic and apoptotic signaling prevention in vivo. (A) Timeline of in vivo ischemia / reperfusion model. (B) Uptake of labeled TAT-WVELHFFN in different organs at 10 min of reperfusion. TAT-WVELHFFN was administered 5 min before reperfusion (see Figure 11A-C). (C) Representative 3D visualization of TAT-WVELHFFN-Cy5.5 (red) distribution throughout mouse heart after I / R (blue knots) by intrinsic autofluorescence (green). (D-I) Mice were treated with TAT-WELVHFFN or TAT-WELAASN. Sham-operated mice served as control. (D) Mitochondrial BNIP3 (*p=0.0161) and (E) mitochondrial BAX levels in the area at risk (AAR) at 10 min of reperfusion. (n=5). (F) Swollen mitochondria (OD, optical density; n=5) from the AAR at 10 min of reperfusion. (G) Active BAX and (H) cytoplasmic cytochrome c levels are seen in the AAR at 30 min of reperfusion (n=5; *p=0.032). (I) Caspase-3 activity in the AAR at 1 h of reperfusion (n=8-12; *p=0.0164). Data represent mean ± SEM. p values ​​were determined by two-way ANOVA with Bonferroni correction. In each of these assays, TAT-WELVHFFN administration returned the measured output to nearly sham levels.

[0223] Figure 5A-I. WVELHFFN functions in the inhibition of BNIP3 / BAX heterodimers as components of mitochondrial membrane attack oligomers (A and B). Representative immunoblots for BNIP3 (A) and BAX (B) after SDS-PAGE at baseline and 10 min reperfusion. Mice were treated with TAT-WELVHFFN and TAT-WVELAASN. Sham-operated mice served as controls (n=3). (C) Representative BAX immunoblots after co-immunoprecipitation of BNIP3 as in (A) (n=3). (D) Fluorescent immunoblots at 5 min reperfusion. Mice were treated with vehicle (NaCl) and fluorescently labeled TAT-WELVHFFN. (E) BAX and BNIP3 immunoblots after co-immunoprecipitation of cytosolic BNIP3 and BAX and photometric fluorescence measurements of the cytosolic fraction (n=3). (F) Immunoblots of cytosolic BNIP3 and BAX in the area at risk (AAR) after blue native PAGE at baseline and 10 min reperfusion (n=3). (G) Immunoblots of cytosolic BNIP3 after size exclusion chromatography at 10 min reperfusion with and without TAT-WVELHFFN treatment (n=3). (H) Immunoblots of cytosolic BAX after size exclusion chromatography at baseline and 10 min reperfusion with and without TAT-WVELHFFN treatment (n=3). (I) Fluorescence measurement of TAT-WVELHFFN-fluo after size exclusion chromatography at 5 min reperfusion (n=3).

[0224] Figure 6A-N. TAT-WVELHFFN improves clinical outcomes. (A) Timeline of in vivo ischemia / reperfusion (I / R) model. Animals were injected with vehicle (sodium chloride), β-Gal, TAT-WVELAASN or TAT-WVELHFFN. (B) Area at risk (AAR) per left ventricle (LV) and (C) infarct size per AAR (Inf / AAR) at 24 h of myocardial reperfusion in wild-type mice treated as indicated (n=6). TAT-WELVHFFN administration substantially reduced infarct size. (D) Infarct size at 24 h of myocardial reperfusion in wild-type mice treated with the indicated TAT-WVELHFFN doses (n=3-7; *p=0.0172). TAT-WELVHFFN substantially reduced infarct size at the appropriate dose. (E) Apoptotic events in cardiac planar slices at 4 h of reperfusion. In all planar slices examined, TAT-WELVHFFN administration reduced the number of apoptotic events. (F) Representative images of apoptotic cells (red) and nuclei (blue) in the AAR at 4 h of reperfusion. (G) Inf / AAR at 24 h of myocardial reperfusion in wild-type mice treated with TAT-BNIP3-ΔTM (ΔTM) 5 min before ischemia and further treated with TAT-WVELHFFN 5 min before reperfusion (n=3–6 mice). (H) In vivo myocardial I / R in pigs subjected to 60 min of ischemia and 4 h of reperfusion and treated with vehicle or TAT-WVELHFFN (n=3). AAR / LV (H), Inf / AAR (I) and Inf / LV (*p=0.02) (J). Inf / AAR and Inf / LV ratios were significantly decreased by administration of TAT-WELVHFFN. (I) Brain Inf / AAR at 24 hours of reperfusion in wild-type mice treated with vehicle or TATWVELHFFN (n=5-7). Inf / AAR ratios were significantly decreased by administration of TAT-WELVHFFN. (J) Timeline before treatment. (K) Inf / AAR in treated vs control subjects. Inf / AAR ratios were significantly decreased by administration of TAT-WVELHFFN(PepB). (L) Ejection fraction (EF)% of control vs treated individuals assayed on D-7, D-1, D0, D1, D3 and D5. Treated individuals showed significantly higher EF% as indicated by asterisks.(M) Timeline after treatment. (N) Left ventricular ejection fraction (LVEF)% of control vs. treated animals assayed on d-1, d1, d3, d5, and d7. Treated animals showed recovery of LV function on day 3, as indicated by asterisks.

[0225] Figure 7A-7C. In vivo apoptosis and necrosis analysis, related to Figure 1. (A) Workflow size exclusion chromatography. (B) Wild type, Bnip3 after injection of TAT-BNIP3 fusion protein. - / - and Bnip3 - / - Confocal microscopy images showing mouse heart tissue from mice. Mitochondria were stained in red, nuclei in blue, and BNIP3 in green. (C) Infarct size per area at risk (Inf / AAR) in wild-type mice treated as indicated (n=3–6); vehicle (NaCl), TAT-BNIP3ΔTM, ΔTM (left). Representative images of TTC-stained heart sections from mice treated with vehicle (NaCl) or ΔTM at 24 h of reperfusion (center). AAR / left ventricle (LV) at 24 h of reperfusion in wild-type mice (n=3–6 mice). Data represent mean ± SEM. p values ​​were determined by one-way ANOVA with Bonferroni's correction.

[0226] Figure 8A-I. Characterization of mouse myocardial mitochondria and BNIP3. Related to Figure 1. (A) Quantification of depolarized mitochondria at baseline and indicated reperfusion times in mouse myocardial I / R injury by flow cytometry (n=5). (B) Quantification of intact, swollen and damaged mouse heart mitochondria at baseline and 10 min reperfusion (n=3-5) and representative electron micrographs. Scale bar, 1 μm. (C) Representative flow cytometry 2D dot plots of mitochondrial membrane potential using JC-1 dye. (D) Representative electron micrographs showing fragmented mitochondria. Scale bar, 5 μm (left), Scale bar, 1 μm (right). (E) Immunoblots of BNIP3 in whole mouse hearts and isolated cardiomyocytes. (F and G) Time course of ischemia / reperfusion injury: BNIP3 mRNA transcript levels (n=4) (F) and protein concentration (n=9) (G) at baseline and indicated reperfusion times. Representative immunoblots of BNIP3. (H) Western blot monitoring of subcellular localization of BNIP3. (I) Representative images of mitochondria (red), cytosolic BNIP3 (green; indicated by yellow arrows), mitochondrial BNIP3 (yellow; indicated by white arrows) and nuclei (blue) in mouse heart slices. Magnification 64x.

[0227] Figure 9A-D. Mouse mitochondria in cardiac health and disease, related to Figure 1. (A) Mitochondrial pore formation experiments in which isolated mitochondria were treated with cytoplasmic and inhibitors demonstrate the requirement of BNIP3 in BAX pore formation as shown by quantification of cytochrome c, AIF and active BAX concentrations (n=3). (B and C) Schematic and representative traces of mitochondrial swelling experiments. Mitochondria were treated with either ΔT and a buffer inducing mitochondrial swelling (mPTP buffer) (B) or mitochondrial outer membrane rupture (non-mPTP-inducing buffer) (C) as assessed by measuring optical density (OD). (D) Quantification of BNIP3 serine phosphorylation and active BAX concentrations in HL-1 cells exposed to hypoxia (24 h) and reoxygenation (1 h) (H / R) with or without treatment with AKT inhibitors (n=5 independent experiments). Data represent mean ± SEM. p-values ​​were determined by two-way ANOVA with Bonferroni's correction.

[0228] Figure 10A-B. Alanine scan, related to Figure 3. (A) Heatmap of alanine scan using the N-terminal sequence of BNIP3 (1-20 aa) incubated with recombinant BNIP3. Color coding ranges from white (low or no intensity) to yellow (medium intensity) and red (high intensity). (B) Space-filling model and structural formula of WVELAASN.

[0229] Figure 11A-C. Identification and in vitro / in vivo effects of advanced TAT-WVELHFFN peptides, related to Figure 4. (A) Western blot of fluorescently labeled TAT-WVELHFFN level profile in human serum, plasma and whole blood after incubation at 37°C for the indicated times. Proteinase K treatment was used as a control at 60 min. (B) Viability of isolated adult cardiomyocytes treated with TAT-WVELHFFN and TAT-WVELAASN after 24 h (n=2). (C) Echocardiograms measured at baseline (pre) and 5 h after injection of 8 nmol TAT-WVELHFFN (n=3).

[0230] Figure 12A-12D. TAT-peptides. HPLC (220 nm, C18, linear gradient) of newly synthesized chimeras (A) MSQSGEENLQGSWVELHFSN; (B) LDAQHESGRSSSKSSHCDSP; (C) WVELHFFN and (D) WVELAASN.

[0231] Figure 13A-B. TAT-WVELHFFN ameliorates DOX-induced cell death in human cardiomyocytes. In Figure 13(A) images are seen showing DOX and TAT-WVELHFFN uptake. Images are generated using confocal microscopy. Magnification: 40x. Scale bar: 20 μm. 10 μM DOX and 20 μM TAT-WVELHFFN are administered and cells are visualized at 22 hours. Dox is shown in yellow and TAT-WVELHFFN is stained using Cy5. In Figure 13(B) it can be seen that TAT-WVELHFFN ameliorates DOX-induced cell death in human cardiomyocytes. Dead cells are measured relative to the control (left) compared to administration of 10 μM DOX (middle) and 10 μM DOX+TAT-WVELHFFN (right). Administration of the mitochondrial interaction and import inhibitor TAT-WVELHFFN ameliorates the increased cell death induced by administration of the chemotherapeutic agent DOX, restoring cell death to near control levels.

[0232] Figure 14A-B. TAT-WVELHFFN ameliorates DOX-induced cell death in human cardiomyocytes. In Figure 14(A), it can be seen that TAT-WVELHFFN ameliorates DOX-induced cell death in human cardiomyocytes. From left to right, baseline-adjusted values ​​of percent attributable damage were measured for DOX concentrations of 0, 1, and 10 μM. For each concentration, results are shown without (left) and with (right) TAT-WVELHFFN. Cells were incubated for 4 hours and TAT-WVELHFFN was administered at 0 or 1.5 pM. The results show that the limited damage caused by a 10 μM dose of the chemotherapeutic agent DOX is ameliorated by the mitochondrial interaction and import inhibitor TAT-WVELHFFN. In Figure 14(B), it can be seen that TAT-WVELHFFN ameliorates DOX-induced cell death in human cardiomyocytes. RLU, indicating cell death, was measured for DOX concentrations of 1 and 10 μM, from left to right. For each concentration, results are shown without (left) and with (right) TAT-WVELHFFN. Cells were incubated for 4 h and TAT-WVELHFFN was administered at 0 or 1.5 pM. Results show that the limited damage indicated by the increase in RLU caused by a 10 μM dose of the chemotherapeutic agent DOX is ameliorated by the mitochondrial interaction and import inhibitor TAT-WVELHFFN.

[0233] Figure 15A-B. TAT-WVELHFFN ameliorates mitochondrial membrane potential destabilization by DOX in human cardiomyocytes. In Figure 15(A) it can be seen that WVELHFFN ameliorates mitochondrial membrane potential destabilization by DOX. Representative images are of donor-human cardiomyocytes (HCM). Incubation is 4 h 10 μM DOX. Magnification: 40x. Scale bar: 20 μM. Fluorescence on the left indicates that the membrane potential is preserved in the control. In the middle, the lack of fluorescence indicates that the membrane potential is destroyed by administration of 10 μM DOX, while on the right it can be seen that co-administration of TAT-WVELHFFN ameliorates the negative effect of DOX on the mitochondrial membrane potential. In Figure 15(B) a quantification of the results in Figure 15(A) can be seen. Results are shown for corrected whole cell fluorescence compared to control, for 4 h and 22 h incubation times. For each chart, the control is shown on the left, followed by 10 μM DOX, center and DOX+TAT-WVELHFFN. At both time incubations, there is a reduction compared to the control for DOX administration, and an amelioration of that reduction by co-administration of TAT-WVELHFFN. This indicates that the negative effect of chemotherapeutic agents on mitochondrial membrane potential is ameliorated by co-administration of TAT-WVELHFFN.

[0234] Figure 16A-B. TAT-WVELHFFN prevents mPTP opening by DOX in human cardiomyocytes. In Figure 16(A) it can be seen that TAT-WVELHFFN prevents mPTP opening by DOX. Representative images are of donor-human cardiomyocyt4es (HCM). Incubation is 4 hours 10 μM DOX. Magnification: 40x. Scale bar: 20 μM. On the left, calcein-AM fluorescence shows that mPTP opening is prevented in the control. In the center, the lack of fluorescence shows that mPTP opening is activated by administration of 10 μM DOX, while on the right it can be seen that co-administration of TAT-WVELHFFN prevents the negative effect of DOX on mPTP opening. In Figure 16(B) a quantification of the results in Figure 16(A) can be seen. Results are shown for total cell fluorescence corrected relative to control for a 22 hour incubation time. For each chart, the effect of DOX at 0 and 10 μM is shown without (left) and with (right) TAT-WVELHFFN. It can be seen that for 10 μM DOX administration, there is a decrease relative to control, and the decrease is prevented to near control levels upon co-administration of TAT-WVELHFFN. This indicates that the negative effect of chemotherapeutic agents on mPTP opening is prevented by co-administration of TAT-WVELHFFN.

[0235] Figure 17. TAT-WVELHFFN prevents mitochondrial calcium ion overload caused by DOX in human cardiomyocytes. In figure E, it can be seen that TAT-WVELHFFN prevents mitochondrial calcium ion overload caused by DOX. Representative images are of donor-human cardiomyocytes (HCM). Incubation is 4 hours 10 μM DOX. Magnification: 40x. Scale bar: 20 μM. On the left, low levels of calcium-rhodamine fluorescence indicate that no calcium overload occurs in the control. In the center, the increase in fluorescence indicates that mitochondrial calcium ion overload is induced by administration of 10 uM DOX, while on the right, it can be seen that co-administration of TAT-WVELHFFN prevents the negative effect of DOX on calcium ion overload.

[0236] Figure 18A-B. TAT-WVELHFFN ameliorates mitochondrial reactive oxygen species (ROS) generation caused by DOX in human cardiomyocytes. In Figure 18(A) it can be seen that TAT-WVELHFFN ameliorates mitochondrial ROS generation caused by DOX. Representative images are of donor-human cardiomyocytes (HCM). Incubation is 4 h 10 μM DOX. Magnification: 40x. Scale bar: 20 μM. On the left, the low level of ROS fluorescence indicates that ROS does not accumulate in the control. In the center, the increase in fluorescence indicates that ROS generation is induced by administration of 10 μM DOX, while on the right it can be seen that co-administration of TAT-WVELHFFN ameliorates the negative effect of DOX on ROS generation. In Figure 18(B) a quantification of the results in Figure 18(A) can be seen. Results are shown for corrected ROS generation compared to the control for 4 h and 22 h incubation times. For each chart, the control is shown on the left, followed by 10 μM DOX, center and DOX+TAT-WVELHFFN. At both time incubations, there is an increase compared to the control for DOX administration, and an amelioration of that increase by co-administration of TAT-WVELHFFN. This indicates that the negative effect of chemotherapeutic agents on ROS generation is ameliorated by co-administration of TAT-WVELHFFN. Figures 15-18: Taken together, these results show that the conclusions made previously apply generally to the treatment of human cells.

[0237] Figure 19. Time course of administration of chemotherapeutic agents and mitochondrial import inhibitors.

[0238] Figure 20. PepB(TAT-WVELHFFN) has no long-term adverse effects on mammalian recipients. Survival probability, change in body weight over time, tibia length and heart weight were measured and in each case no significant differences were observed upon pepB administration compared to NaCL vehicle control.

[0239] Figure 21. PepB (TAT-WVELHFFN) prevents cardiomyocyte cell death. In Figure 21, it can be seen that in the administration regimen on the left, PepB prevents cardiomyocyte cell death. From center to right, it can be seen that cardiac troponin I (cTNI) / body weight is significantly lower for DOX co-administration with PepB compared to DOX administration with NaCl control. Similarly, creatine kinase (CK) / body weight is significantly lower for DOX co-administration with PepB compared to DOX administration with NaCl control. Further to the right, it can be seen that LDH / body weight (center right) and BNP / body weight (right) were unchanged by administration of PepB.

[0240] 22A-E. Inhibition of BNIP3 preserves cardiac function. In FIG. 22(A), from the left, the effect of control, DOX, and DOX+PepB (TAT-WVELHFFN) administration on ejection fraction (EF)% at three time points T0, T1, and T2, respectively, is seen. At both T1 (16 days) and T2 (42 days), DOX has a negative effect on EF%, which is ameliorated by simultaneous PepB administration. In FIG. 22(B), from the left, the effect of control, DOX, and DOX+PepB administration on fractional shortening (FS)% at three time points T0, T1, and T2, respectively, is seen. At both T1 and T2, DOX has a negative effect on FS%, which is ameliorated by simultaneous PepB administration. In FIG. 22(C), from the left, we see the effect of control, DOX, and DOX+PepB administration on isovolumic relaxation time (IVRT) (ms) at three time points T0, T1, and T2, respectively. At both T1 and T2, DOX slows IVRT, and this effect is ameliorated by simultaneous PepB administration. In FIG. 22(D), from the left, we see the effect of control, DOX, and DOX+PepB administration on heart rate at three time points T0, T1, and T2, respectively. Heart rate was not affected by DOX administration alone or in combination with PepB. In FIG. 22(E), we see the effect of control, DOX, and DOX+PepB on cardiac troponin I (cTnl) (ng / l), expressed on a logarithmic scale. It can be seen that DOX has a dramatic positive effect on cTnl, and this effect is ameliorated by simultaneous administration of PepB.

[0241] 23A-D. Inhibition of BNIP3 upon DOX administration increases mitochondrial fitness. In FIG. 23(A), we see that DOX inhibits oxygen consumption rate (OCR) at spare capacity (pmol / min / 1.5ug protein) and PepB (TAT-WVELHFFN) ameliorates this inhibition. From left, control DOX and DOX+PepB. In FIG. 23(B), we see that DOX inhibits OCR at maximal respiration (pmol / min / 1.5ug protein) and PepB ameliorates this inhibition. From left, control DOX and DOX+PepB. In FIG. 23(C), we see that DOX inhibits basal respiration (as a percentage of control) and PepB ameliorates this inhibition. From left, control DOX and DOX+PepB. In FIG. 23(D), we see that DOX inhibits complex II stimulated respiration (as a percentage of control) and PepB ameliorates this inhibition. From left: control DOX and DOX+PepB.

[0242] Figures 24A-D. Inhibition of BNIP3 upon DOX administration restores autophagic flux. In Figure 24(A), from the left, Western blots of proteins extracted from control, DOX-treated and DOX+PepB (TAT-WVELHFFN)-treated tissues are seen. For each protein source, the left two lanes are untreated, while the right two lanes are sample sources additionally treated with chloroquine (CQ). Electrophoresed proteins were probed with LC3-I and LC3-II. In Figure 24(B), it can be seen that DOX inhibits the CQ-mediated LC3II / I increase. From the left, control DOX and DOX+PepB (left without CQ treatment, or right with CQ treatment, respectively). DOX abolishes the CQ-induced increase, while the addition of PepB partially restores this increase. In Figure 24(C), it can be seen that DOX induces p62 accumulation and the addition of PepB counteracts this increase. P62 levels are shown as a percentage of control for control, DOX-treated samples and DOX+PepB-treated samples. In Figure 24(D), it can be seen that DOX induces BNIP3 accumulation and the addition of PepB counteracts this increase. BNIP3 levels are shown as a percentage of control for control, DOX-treated samples and DOX+PepB-treated samples.

[0243] definition A partial list of definitions follows:

[0244] As used herein, "about" a number refers to a range of less than 10% to more than 10% of that number. "About" a range refers to an expanded range from 10% below the lower limit of the recited range to 10% above the upper limit of the recited range.

[0245] As used herein, the phrase "at least one of A, B, and C" refers to a set that may include A, A and additional unlisted elements, A and B, A and B and additional unlisted elements, B, B and an unlisted element, AB and C, AB and an unlisted element, B and C, B and C and an unlisted element, C only, or C and an unlisted element only.

[0246] As used herein, the terms peptide, polypeptide and protein are sometimes used interchangeably in reference to molecules that contain multiple peptide bonds. EXAMPLES

[0247] Working Example Example 1. Cytosolic BNIP3 sequentially determines necrotic and apoptotic cell death by regulating BAX in its heterodimeric state We hypothesized that cytosolic BNIP3 acts directly on BAX in mitochondria-driven cell death. To investigate this, we first used blue native PAGE (BN-PAGE) on the cytosolic fraction of myocardium, followed by Western blotting. We detected BNIP3 in the large-scale formation of oligomers with apparent molecular weights of 272 kDa to 450 kDa by anti-BNIP3 antibodies (Figure 1A). Size-exclusion chromatography followed by SDS-PAGE / Western blotting (Figure 7A) verified the apparent presence of BNIP3 in oligomers with molecular weights of 522 kDa to 921 kDa, but also showed oligomers with molecular weights of up to 54 kDa with BNIP3 (Figure 1B).

[0248] Next, we assessed the behavior of BNIP3 in necrosis and apoptosis using a well-established mouse myocardial in vivo I / R model as a clinically relevant system (Hendgen-Cotta et al., 2008; Luedike et al., 2012; Rassaf et al., 2014; Merz et al., 2019). Ischemia was induced by occlusion of the left coronary artery for 30 min, followed by 24 h of reperfusion (Figure 1C; see STAR Methods). Bnip3-deficient (Bnip3 - / - By using 10-fold reduced infarct size in wild-type mice, we found that Bnip3 deletion generally protected cardiac tissue from I / R injury, as evidenced by a 51% reduction in infarct size compared to wild-type mice (Figure 1D). - / - BNIP3 reversion in mice (Figure 7B) dose-dependently restored infarct size to levels comparable to those in wild-type mice (Figure 1D). Infarct size was also reduced in mice with BNIP3 activity acutely affected by administration of a dominant-negative form of BNIP3, TAT-BNIP3ΔTM (Diwan et al., 2007; Hamacher-Brady et al., 2007). - / -The apoptotic cell death was dose-dependently reduced to levels similar to those in mice (-55%) (Figure 7C). This attenuation of cell death was accompanied by a decrease in cardiac troponin I release, an indicator of cardiomyocyte necrosis (Figure 1E). We also found an overall reduction in apoptotic cells at 4 hours of reperfusion (Figure 1F). Furthermore, the spatial distribution of apoptotic cells was minimized throughout the LV in mice with affected BNIP3 activity compared to control mice (Figure 1F).

[0249] At the intracellular level, we identified a time-dependent dissipation of the potential difference across the MIM during reperfusion, which led to mitochondrial depolarization and swelling at 10 min of reperfusion (Figures 8A and 8B). When we influenced BNIP3 activity by TAT-BNIP3ΔTM administration, only marginal MIM depolarization, as well as small mitochondrial swelling and fragmentation, occurred (Figures 1G-I, 8C and 8D).

[0250] Our results therefore raise the intriguing notion that BNIP3 regulates necroptosis signaling within the first 10 min of reperfusion. The transient opening of a pore in the MIM (mPTP) as a critical event for mitochondrial depolarization requires the translocation of BAX at the MOM, but not its activation (Whelan et al., 2012; Karch et al., 2013). We first observed that not only BAX but also BNIP3 translocated from the cytosol to the MOM (Figure 1J and Figure 1K). Already at 10 min of reperfusion, the time of loss of Δψm, there was a significant mitochondrial level increase of BNIP3 and BAX dimers (Figure 1J and Figure 1K), whereas activated BAX was undetectable (Figure 1L). We observed the presence of activated BAX at 30 min of reperfusion. Notably, we found that using TAT-BNIP3ΔTM strongly reduced large-scale BAX translocation and thus was dependent on BNIP3 (Figure 1J). This effect also held true for BNIP3 (Figure 1K). The cytosolic presence and constitutive expression of BNIP3 at the transcript and protein levels in cardiomyocytes during I / R demonstrated its translocation from the cytosol to mitochondria (Figures 8E-8I).

[0251] To further strengthen the notion of BAX dependency on cytosolic BNIP3, we performed I / R surgery in Bnip3- / - mice and obtained similar findings at 10 min of reperfusion (Figure 1M). Restoration of BNIP3 by injection of TAT-BNIP3 fusion protein was essential to achieve BAX translocation at the MOM in BNIP3-deficient mice (Figure 1M), suggesting that in native cardiomyocytes, cytosolic BNIP3 likely determines BAX translocation.

[0252] Co-immunoprecipitation experiments revealed that BAX and BNIP3 were physically associated in the cytosol at baseline and 10 min of reperfusion, and this association was DTT-resistant supporting their non-covalent heterodimeric structure (Figure 1N). With regard to apoptosis signaling, we found no cytochrome c release within the first 10 min of reperfusion, but a significant increase in cytosolic cytochrome c content at 30 min of reperfusion (Figure 1O). This release of apoptosis inducers was prevented in mice with affected BNIP3 activity, as demonstrated by attenuation of cytosolic cytochrome c levels at 30 min of reperfusion, which were similar to those at baseline and 10 min of reperfusion (Figure 1O). However, MOMP induction requires activation of BAX (Kalkavan and Green, 2018; Dewson and Kluck, 2009; Edlich et al., 2011). According to this, peak BAX activation occurred at 30 min of reperfusion, and this activation was strongly inhibited by loss of BNIP3 activity (FIG. 1L).

[0253] We confirmed the dependency of MOMP formation on cytosolic BNIP3 using isolated mouse cardiac mitochondria incubated with cytosol by inhibiting BNIP3, BAX and voltage-dependent anion channels (Westphal et al., 2011) (Figure 9A). BAX activation, accompanied by the release of cytochrome c and AIF, was reduced by its inhibition, but also when BNIP3 activity was affected (Figure 9A). BNIP3-associated MIM and MOM damage was further validated in isolated mouse mitochondria challenged with recombinant BNIP3 and BAX to simulate cytosolic proteins and TAT-BNIP3ΔTM. Both MIM perturbation and MOMP formation were reduced, as evidenced by limited cytochrome c release (Figure 9B and Figure 9C). This data tempted us to speculate that post-translational modifications of BNIP3 cause BAX activation. To test this, we investigated the transient phosphorylation of BNIP3 and its potential impact on BAX activation. Consistent with the decline in cardiac function of ATP synthesis demonstrated within the first 10 min of reperfusion (Fig. 1P), phosphorylation levels of BNIP3 were similar to baseline phosphorylation levels, whereas when BNIP3 drives BAX activation (Fig. 1N), BNIP3 was highly phosphorylated at 30 min of reperfusion (Fig. 1Q). We further confirmed the restriction of BNIP3 phosphorylation and BAX activation by exposing HL-1 cells to hypoxia / reoxygenation and AKT inhibition (Fig. 9D).

[0254] Taken together, our findings identify that cytosolic BNIP3 activity governs necrotic and apoptotic cell death in a time-dependent manner by regulating BAX in the heterodimeric state, whereby phosphorylation of BNIP3 can activate BAX and MOMP formation, constituting a master switch of BNIP3 / BAX cell death activity from necrosis to apoptosis.

[0255] Example 2. The N-terminus is the interaction domain of BNIP3. To map the interaction domain of BNIP3 and its possible binding site in BAX, we first confirmed the BNIP3 / BAX interaction seen in vivo by in vitro protein-protein overlay lacking cellular kinase (Figure 2A). To identify the possible binding site of BNIP3 in BAX, we employed protein-peptide interaction studies using a library of 13 synthetic BAX peptides immobilized on a microarray (Figure 2B). Fluorescently labeled recombinant BNIP3 was incubated on the peptide microarray, and helices α5, α6, α7 and α8 of BAX were identified as potential interaction sites with BNIP3 (Figure 2C). Because the 3D structure of BNIP3 has not been fully elucidated except for its transmembrane domain located at the C-terminus using nuclear magnetic resonance spectroscopy (Bocharov et al., 2007; Sulistijo and Mackenzie, 2009), we predicted the 3D structure of BNIP3 in silico (Figure 2D) by homology modeling using Modeller 9.15 (Eswar et al., 2006), followed by energy minimization using NAMD 2.9 (Phillips et al., 2005) and the CHARMM36 force field (Vanommeslaeghe and MacKerell, 2015). The template structure corresponded to PDB codes 2K7W (Gavathiotis et al., 2008) and 2KA1 (Sulistijo and Mackenzie, 2009). The 3D model features nine α-helices of variable length representing 64% of the secondary structure, followed by random coils (19%) and uncharacterized structures (17%) (Figure 2E). We confirmed the predicted secondary structure of BNIP3 by circular dichroism (CD) spectroscopy (Figure 2F).Computational docking simulations performed with Autodock Vina (Trott and Olson, 2010) and HADDOCK (Dominguez et al., 2003) also suggested BAX helices α5, α6, α7, and α8, as well as the BNIP3 sequence MSQSGEENLQGSWVELHFSN (amino acids 1–20) as interaction sites, although the first 10 amino acids alone were unable to bind BAX (Figure 2G). Thus, BNIP3 amino acids 1–20 appear to be important for BNIP3 binding to BAX.

[0256] Example 3. The N-terminus of BNIP3 is a key functional domain in human cardiomyocyte protection. Based on our prediction of amino acids 1-20 of BNIP3 as a BAX interaction site, we hypothesized that this sequence may be a functional domain of BNIP3 and sufficient to antagonize BNIP3 / BAX activity. We first designed a cell-penetrating peptide, TAT-BNIP3-20A, composed of the HIV-1 Tat protein transduction domain 48-59 (PTD; GRKKRRQRRRPQ) (Shoji-Kawata et al., 2013) covalently linked to 20 amino acids derived from amino acids 1-20 of BNIP3 (Figure 3A). BNIP3 amino acids 42-61 were used to generate a control peptide, TAT-BNIP3-20C (Figure 3B). To initially test the efficacy of the peptide, wild-type mice were subjected to I / R with TAT-BNIP 3-20 A injection. Consistent with our hypothesis, TAT-BNIP3-20A prevented BNIP3 translocation and counteracted BNIP3 activity, as demonstrated by a marked reduction in caspase-3 activity (Figure 3C and 3D). Notably, TAT-BNIP3-20A reduced infarct size by 41% compared to treatment with vehicle, whereas no difference was observed between TATBNIP3-20C and vehicle treatment (Figure 3E). To address aspects of translation, since BNIP3 is expressed in human myocardium (Figure 3F) (Chaanine et al., 2013), we assessed mouse / human BNIP3 sequence alignments, focusing on the N-terminal region in question. Comparison revealed that amino acids 9–20 are conserved residues (Figure 3G). To identify the critical amino acids required for activity, we next performed an N-terminal truncation analysis of the first 20 amino acids of BNIP3 and identified amino acid residues 13-20, WVELHFSN, within an evolutionarily conserved domain (Figure 3H, Table S1A). This truncated peptide yielded the strongest signal with a 14-fold signal increase compared to the 20 residues of the BNIP3-20A peptide in the BNIP3 / peptide microarray (Figure 2A). We then focused on substitution analysis (Table S1B).Peptides with double or triple alanine scans did not demonstrate stronger binding of the protein when compared to the wild-type sequence (Figure 10A). Specific amino acid substitutions with natural amino acids (A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y) in the truncated sequence identified that replacement of W13 and F18 with each of the canonical amino acids significantly impaired BNIP3 binding (Figure 3I). This assigns a functional role for tryptophan and phenylalanine in binding activity. Consistently, an S to F substitution at position 19 showed substantially higher BNIP3 binding, corroborating the critical role of phenylalanine (Figure 3I). Docking simulations of the discovered sequences with S to F substitutions, WVELHFFN and BAX, showed that peptide binding to the BAX interface was governed by the conformation of the peptide located on the N-terminus of helix α5 (Figure 3J and 3K). Furthermore, the resulting conformation suggests that the better binding of WVELHFFN to BAX compared to the native sequence WVELHFSN is associated with improved aromatic intrapeptide interactions (Martinez and Iverson, 2012). This was confirmed by BAX / BAX microarray evidenced by a four-fold increase in signal intensity associated with the native sequence (Figure 3K). Our findings indicate that these amino acids are required to antagonize BNIP3 / BAX activity. Therefore, we designed a peptide in which the PTD is linked via covalent bonds to eight amino acids derived from amino acids 13-20 of BNIP3, but with an S to F substitution (TATWVELHFFN; Figure 3L). The CD spectrum of TAT-WVELHFFN showed that the peptide exhibited a random coil conformation (Figure 3M). To design a dysfunctional peptide as a control, we substituted potential interface residues including the critical phenylalanine residues F19–S19 and F18–A18 and H17–A17, thereby generating the TAT-WVELAASN peptide (Figure 10B).BNIP3-TAT-WVELHFFN and BAX-TAT-WVELHFFN fluorescence-based overlay assays then confirmed that TAT-WVELHFFN bound to both BNIP3 and BAX (Figure 3N). To probe the translational aspects, we performed hypoxia / reoxygenation experiments on human ventricular cardiomyocytes derived from human induced pluripotent stem cells (human CM; Figure 3O), which recapitulated the BNIP3 / BAX interaction assessed by proximity ligation assay (Figure 3P). Upon treatment with TAT-WVELHFFN, apoptotic and necrotic cell death as well as MIM depolarization were reduced compared to control peptide treatment (Figure 3Q).

[0257] Example 4. WVELHFFN peptide functions in preventing necrotic and apoptotic signaling in vivo Our results on the binding ability of WVELHFFN to both BNIP3 and BAX, and the protective effect of TAT-WVELHFFN against necrotic and apoptotic death of cardiomyocytes, predict an inhibitory effect on the BNIP3 / BAX heterodimer-induced lethal signaling pathway. To test this, we used an established in vivo myocardial I / R model focusing on basal reperfusion times at 10 and 30 minutes and several complementary methods of downstream signaling (Figure 4A). As demonstrated, cell death signaling occurs during the first minutes of reperfusion (Figure 1). Studies have shown that application of cardioprotectants after the onset of reperfusion did not attenuate I / R injury (Davidson et al., 2019). Therefore, we decided to administer TAT-WVELHFFN or the control peptide TAWVELAASN 5 min before the end of ischemia, which is a feasible time point for drug application in clinical settings (Figure 4A). To confirm the uptake, distribution and temporal presence of the myocardial peptide after in vivo intracardiac injection, we first injected fluorescently labeled TAT-WVELHFFN into ischemic wild-type mice 5 min before reperfusion. We found that TAT-WVELHFFN was present in the heart at the time of BNIP3 / BAX heterodimer initialization of the cell death cascade (Figure 4B). Similar to light sheet microscopy analysis of the whole heart (Merz et al., 2019), the distribution of the peptide also occurred within the risk region (Figure 4C). We further evaluated the pharmacokinetic profile of TAT-WVELHFFN in human serum, plasma and whole blood and characterized its toxicity and effects on cardiac function in mouse cardiomyocytes and mice. The observed stability of TAT-WVELHFFN in human whole blood, plasma and serum over 60 min provides in vivo inhibitory potential (Figure 11A). Notably, we could not detect any obvious signs of toxicity in either cardiomyocytes or mice (Figures 11B and 11C). Consistent with our hypothesis, treatment with TAT-WVELHFFN significantly reduced BNIP3 and BAX translocation to mitochondria at 10 min of reperfusion (Figures 4D and 4E).As a result, necrotic and apoptotic signaling, characterized by mitochondrial swelling at 10 min of reperfusion, and BAX activation, cytochrome c release, and caspase-3 activity at 30 min of reperfusion, was significantly reduced (Figures 4F-I).

[0258] Example 5. WVELHFFN functions in inhibiting the BNIP3 / BAX heterodimer as a component of the mitochondrial membrane attack oligomer

[0259] Based on the knowledge of cytosolic non-covalently bound BNIP3 / BAX heterodimers at baseline and 10 min at reperfusion obtained by co-immunoprecipitation (Figure 1), we sought to determine the mode of action of WVELHFFN on BNIP3 / BAX heterodimers. Upon peptide treatment, neither BNIP3 nor BAX dimers were affected at 10 min of reperfusion (Figures 5A and 5B). Co-immunoprecipitation experiments confirmed the unaffected heterodimeric state of BNIP3 and BAX (Figure 5C). To investigate whether TAT-WVELHFFN interacts with BNIP3 / BAX heterodimers, we used fluorescently labeled TAT-WVELHFFN injected into wild-type mice subjected to I / R. Western blot analysis revealed binding of TAT-WVELHFFN-fluo to the dimers (Figure 5D). We further verified this by co-immunoprecipitation and photometric fluorescence measurements of cytosolic BNIP3 and BAX (Figure 5E). As expected, 5 min after reperfusion, cytosolic BNIP3 and BAX co-immunoprecipitated with TAT-WVELHFFN-fluo (Figure 5E). BN-PAGE and size-exclusion chromatography followed by SDS-PAGE / Western blotting confirmed the presence of BNIP3 and BAX in apparent oligomers with molecular weights ranging from 95 kDa to 1627 kDa, but also showed oligomers with BNIP3 of molecular weights up to 54 kDa in the cytosol at baseline and at 10 min reperfusion I / R with and without TAT-WVELHFFN treatment (Figure 1B and Figures 5F-5H). To discover which oligomeric complexes with BNIP3 / BAX subunits exert deleterious activity on mitochondrial membranes, wild-type mice were treated with fluorescently labeled TAT-WVELHFFN. Size-exclusion chromatography followed by fluorescence measurements revealed that higher oligomeric complexes yielded the most intense signal with a signal increase of up to 250% compared to fractions consisting of dimers / oligomers with molecular masses between 54 and 167 kDa (Figure 5I). Taken together, these data indicate that BNIP3 / BAX heterodimers are components of higher oligomeric structures.Binding of TAT-WVELHFFN to oligomers outlines their status as a mitochondrial membrane attack complex, since TAT-WVELHFFN prevents BNIP3 / BAX-induced necrotic and apoptotic signaling.

[0260] Example 6. TAT-WVELHFFN improves clinical outcomes. Infarct size determines prognosis after myocardial infarction and stroke. Therefore, we studied whether TAT-WVELHFFN could alter infarct size when administered during ischemia 5 min before reperfusion in wild-type mice subjected to myocardial I / R (Figure 6A), a clinically relevant time point. Notably, TAT-WVELHFFN dose-dependently reduced myocardial infarct size by 40% compared to treatment with vehicle, whereas no differences were observed between TAT-β-Gal, TAT-WVELAASN and vehicle treatment (Figure 6B-D). We also observed a lower progression of apoptotic cell death throughout the LV myocardium (Figure 6E and Figure 6F). Treatment of TAT-BNIP3ΔTM mice (with affected BNIP3 activity) with TAT-WVELHFFN failed to induce a further reduction in infarct size relative to TATBNIP3ΔTM treatment, indicating a TAT-WVELHFFN effect on BNIP3 (Figure 6G).

[0261] To investigate translational potential, we selected a porcine catheter-based myocardial infarction model due to the similarity of organ size, coronary anatomy, immunology, and physiology to humans (Lindsey et al., 2018; Milani-Nejad and Janssen, 2014). Pigs were exposed to 60 min of ischemia by balloon inflation in the left coronary artery, followed by 4 h of reperfusion. 5 min prior to reperfusion, TAT-WVELHFFN or sodium chloride was administered. Notably, a single intravenous bolus injection of 0.075 mg / kg of the peptide was sufficient to reduce infarct size by 60% in pigs relative to controls (Figure 6H). Because BNIP3 may play an important role in cerebral ischemia (Zhang et al., 2007), we also evaluated the effect of TAT-WVELHFFN on focal cerebral ischemia. We subjected mice to 30 min of transient middle cerebral artery occlusion and 24 h of reperfusion. Here, TAT-WVELHFFN treatment immediately before reperfusion significantly reduced infarct size by 52% compared to controls (Figure 6I). To examine the effect of pretreatment, we administered TAT-WVELHFFN to wild-type mice subjected to 30 min of ischemia on d-7, d-5, d-3, d-1, and 5 min before 24 h of reperfusion (Figure 6J). Here, multiple treatments reduced infarct size by 40% (Figure 6K). Treatment with TAT-WVELHFFN -7, -5, -3, -1 days before the onset of I / R preserves cardiac function (Figure 6L). Echocardiographic measurements were performed to evaluate the effect on cardiac function of TAT-WVELHFN administered 5 min before reperfusion and on days 1, 3, 5, and 7 after the start of reperfusion in wild-type mice exposed to 50 min of ischemia (Figure 6M). Notably, treated mice showed recovery of LV function on day 3 (Figure 6N). Taken together, our findings demonstrate that this peptide improves clinical outcomes of myocardial infarction and stroke.

[0262] Example 7: TAT-WVELHFFN ameliorates DOX-induced human cardiomyocyte death and preserves mitochondrial integrity. Anthracyclines remain the cornerstone of modern chemotherapy regimens for a variety of solid cancers and hematological malignancies. Anthracycline-induced cardiotoxicity (AIC) is a concerning side effect that limits their clinical use. The risk of AI heart failure increases with the cumulative dose administered, with its incidence reaching nearly 10%. Here, we studied whether TAT-WVELHFFN could reduce chemoteurapeutic DOX-induced cell death in human cardiomyocytes. Thus, we administered 0, 1, and 10 μM DOX to human cardiomyocytes. The cells were incubated for 4 h and 22 h, and TAT-WVELHFFN was administered at 0 or 1.5 pM. The results show that the debilitating damage caused by 10 μM dose of the chemotherapeutic agent DOX was ameliorated by the mitochondrial interaction and import inhibitor TAT-WVELHFFN (Figure 14A and Figure 14B). Next, we investigated the effect of TAT-WVELHFFN on DOX-induced mitochondrial damage, which is associated with mitochondrial membrane potential destabilization, mPTP opening in MIM, mitochondrial Ca2+ overload, and generation of mitochondrial ROS. Human cardiomyocytes were incubated with 10 μM DOX for 4 and 22 hours, respectively. TAT-WVELHFFN was co-administered. Mitochondrial membrane potential was disrupted and mPTP opening was activated by administration of 10 μM DOX, while co-administration of TAT-WVELHFFN improved these negative effects of DOX. This indicates that the negative effects of chemotherapeutic agents on mitochondrial membrane potential and mPTP opening are ameliorated by co-administration of TAT-WVELHFFN (Figure 15 and Figure 16). Furthermore, mitochondrial calcium ion overload was induced by administration of 10 μM DOX for a 4-h incubation time, and ROS generation was induced by administration of 10 μM DOX for 4-h and 22-h incubation times. Co-administration of TAT-WVELHFFN prevented the negative effect of DOX on calcium ion overload and ameliorated the negative effect of DOX on ROS generation.This indicates that the negative effects of chemotherapeutic agents on mitochondrial Ca2+ overload and mitochondrial ROS generation are ameliorated by coadministration of TAT-WVELHFFN.

[0263] Example 8 TAT-WVELHFFN preserves cardiac function by preventing cardiomyocyte death, protecting mitochondrial fitness, and restoring autophagy flux. In a first attempt, we investigated whether DOX with co-administration of TAT-WVELHFFN (PepB) for 42 days had long-term adverse effects on mouse recipients (Figure 19). No significant differences were observed in survival, body weight change over time, tibia length, and heart weight for Dox treatment with PepB co-administration compared to sodium chloride carrier control in wild-type mice (Figure 20). Cardiac troponin I (cTNI) / body weight was significantly lower for DOX co-administration with PepB compared to DOX administration with NaCl control. Similarly, creatine kinase (CK) / body weight was significantly lower for DOX co-administration with PepB compared to DOX administration with NaCl control. LDH / body weight and BNP / body weight were not altered by administration of PepB (Figure 21). Next, we investigated the effect of DOX treatment on cardiac function assessed using echocardiographic measurements at three time points (T0, T1 16 days, T2 42 days) (Figure 19).

[0264] At both T1 (16 days) and T2 (42 days), DOX negatively impaired ejection fraction (EF%), fractional shortening (FS%), and isovolumic relaxation time (IVRT), an effect that was ameliorated by simultaneous PepB administration. Heart rate was not affected by DOX administration alone or in combination with PepB. Notably, DOX had a dramatic positive effect on cardiomyocyte death, as evidenced by high cardiac troponin I (cTnl) levels, an effect that was ameliorated by simultaneous PepB administration. In a next step, we investigated the effect of PepB on DOX-induced mitochondrial dysfunction. DOX treatment inhibited respiratory spare capacity, and PepB ameliorated this inhibition. Furthermore, DOX also affected maximal and basal respiration, and PepB ameliorated these impairments. The inhibition of complex II-stimulated respiration was also reduced by PepB cotreatment. To elucidate whether co-administration of PepB could restore DOX-induced impairment of autophagic flux, we treated mice with DOX and PepB, as well as chloroquine (CQ). As autophagosome markers, we measured the expression of LC3-I and LC3-II. DOX abolished the CQ-mediated increase in LC3II / I, whereas addition of PepB partially restored this increase. DOX also induced the accumulation of p62 and BNIP3, and addition of PepB countered these increases.

[0265] Additional methods and materials were drawn from the following sources: Reagent or Resource Source Identifier Antibodies Anti-BNIP3 polyclonal antibody, rabbit Abcam catalog number ab38621, RRID:AB_725737 Anti-BNIP3 monoclonal antibody, mouse Abcam catalog number ab10433 RRID:AB_2066656 Anti-BAX monoclonal antibody [E63], rabbit Abcam catalog number ab32503, RRID:AB_725631 catalog number ab7977 RRID:AB_306191 Anti-ANT 1 monoclonal antibody [5F51BB5AG7], mouse Abcam catalog number ab110322, RRID:AB_10862212 Anti-Troponin I monoclonal antibody (1H11L19), ABfinity(TM), rabbit ThermoFisher Scientific Catalog No. 701585, RRID:AB_2532494 Anti-phosphoserine monoclonal antibody, clone 4A4, mouse Merck Millipore Catalog No. 05-1000, RRID:AB_11210897 Anti-TOMM22 Abcam Catalog No. ab57523 RRID:AB_945897 Anti-BAX (active monomer) monoclonal antibody (6A7) Enzo Life Sciences Catalog No. ALX-804-224-C100, RRID:AB_2050800 Anti-tubulin antibody Abcam Catalog No. ab15246 RRID:AB_302787 Anti-AIF antibody Santa Cruz Catalog No. Sc9416 RRID:AB_2224665 Anti-cytochrome C monoclonal antibody, unconjugated, clone 7H8.2C12, M Mouse Abcam Catalog No. ab13575, RRID: AB_300470 AlexaFluor 488-conjugated goat anti-rabbit secondary antibody Invitrogen Catalog No. A11029 RRID: AB_138404 Goat anti-rabbit HRP secondary antibody Invitrogen Catalog No. 32260 RRID: AB_1965959 Goat anti-mouse HRP secondary antibody Abcam Catalog No. AB 6789 RRID: AB_955439 AlexaFluor 594-conjugated goat anti-rabbit secondary antibody Invitrogen Catalog No. A-11080 RRID: AB_2534124 Bacterial and viral strain preparations DE3pLys competent cells Novagen / Merck Millipore 70624 Biological samples Human endomyocardial biopsy Ethics approval from the ethical committee of the University of Duisburg-Essen, No. 17-7392-BO N / A Chemicals, Peptides, and Recombinant Proteins Bax Mouse Recombinant (Bax Mouse) Novateinbio PT_39984 Recombinant Mouse BCL2 / Adenovirus E1B 19 kDa Protein-Interacting Protein 3 (Bnip3) Cusabio CSB-CF002766 Mo-50 TAT-BNIP3-20A JPT This paper TAT-BNIP3-20C JPT This paper TAT-WVELAASN JPT This paper TAT-WVELHFFN JPT This paper TAT-WVELHFFN-Fam JPT This paper TAT-WVELHFFN-Cy5.5 JPTThis paper TAT-BNIP3Full lengthThis paperTAT-BNIP3ΔTMThis paper Tat-βGalThis paper Critical Commercial AssaysThis paperDuo Link in Situ Starter Kit-Proximity Ligation Assay Sigma DUO92101-1KT Caspase 3 Assay KitColorimetricAbcam Ab39401 Apoptag red in Situ Detection Millipore S7165 ATP Detection Kit Abcam Ab83359 Apoptose / Necrose Assay KitAbcam ab176749 Troponin IUltrasensitive Elisa Life Diagnostic CTNI-1-US DyLight Antibody Labeling KitThermoFisher Scientific 84535 Ni-NTA Fast Start Kit Qiagen 30600Peptide MicroarrayJPT N / A JC-1 Kit ThermoFisher Scientific M34152 RNeasy Mini Kit Qiagen 74104 High Capacity RNA-to-cDNA Kit Life Technologies 4388950 Experimental model: Cell line Human iPSC-derived vCM Axol Bioscience Ax 2505 HL-1 cell line WCClaycomb Louisiana State University Medical Center, New Orleans, LA, USA, WCClaycomb Experimental model: Organism / strain C57BL / 6J-TgH(Bnip3- / -) Gerald W. Dorn Gerald Dorn C57BL / 6J wild type Jackson Laboratory 000664 Oligonucleotides 18S rRNA ThermoFisher Scientific Mm03928990_g1 Bnip3 ThermoFisher Scientific Mm00833810 Recombinant DNA TAT-Bnip33 / pTriEx-1.1-HT Labor Ruhruniversitat Bochum R.Stoll TAT-Bnip3 dTM / pTriEx-1.1-HT Labor Ruhruniversitat Bochum, R. Stoll pTAT-HA-β-Gal vector S.F. Dowdy (University of California, San Diego, CA, USA) Science 03 Sep 1999: Vol. 285, Issue 5433, pp. 1569-1572 DOI: 10.1126 / science.285.5 433.1569 Software and Algorithms ImageJ Schneider et al., 2012 https: / / imagej.nih.gov / ij / Inkscape https: / / inkscape.org / Vevo 2100 Imaging System FUJIFILM, VisualSonics, Netherland Imaris Software (BitPlane) BitPlane www.bitplane.com GraphPad Prism 7 GraphPad Other Superdex 200 Increase 10 / 300 GL GE Healthcare 28-9909-44. .

[0266] Experimental models and controls Details Mice All animal procedures were performed in accordance with the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes (Directive 2010 / 63 / EU), in accordance with institutional guidelines and approval from the local ethical committee. Male mice aged 12 ± 3 weeks with an average body weight of 30 g were used. C57BL / 6J wild-type mice were obtained from Jackson Laboratory (Barr Harbor, ME, USA) and housed in a local animal house for 1 week for acclimatization. C57BL / 6J-TgH(Bnip3- / -) mice were bred and maintained in the local animal house at the University Hospital Essen. All mice were housed under a 12-h light / dark cycle. Cell lines The HL-1 cell line was provided by WCClaycomb (Louisiana State University Medical Center, New Orleans, LA, USA) and cultured as previously described (Claycomb et al., 1998). Human CM were obtained from Axol Bioscience (UK) and cultured according to the manufacturer's instructions. Method details Expression of recombinant proteins TAT-BNIP3 and TAT-BNIP3ΔTM plasmids were provided by R. Stoll (Ruhr-University Bochum, Germany). The pTAT-HA-β-Gal vector was provided by S.F. Dowdy (University of California, San Diego, CA, USA). His-tagged BNIP3 and BNIP3ΔTM were cloned into the pTriEx-1.1 vector. BNIP3 (full length), BNIP3ΔTM (amino acids 1-163) and β-galactosidase, all fused to the HIV-1 TAT protein transduction domain (GRKKRRQRRRPQ), were grown in Escherichia coli (BL21) and expressed with 100 mM IPTG at 37 °C for 48 h. Bacteria were resuspended in PBS (pH 8.0) and subsequently incubated with 1 mg / mL lysozyme for 1 h at 4°C and sonicated on ice. After centrifugation at 20,000 × g for 50 min at 4°C, the supernatant was applied to a column containing Ni-NTA. Proteins were eluted with 250 mM imidazole in phosphate buffer.Recombinant BAX (rBAX, PT_39984) and recombinant BNIP3 (rBNIP3, CSB-CF002766) were obtained from Hоlzel Diagnostika (Cologne, Germany). In vivo I / R model In vivo mouse myocardial I / R For in vivo I / R, an open-chest model was used. Briefly, wild-type and Bnip3− / − mice were anesthetized with ketamine (100 mg kg−1 intraperitoneally, i.p.) and xylazine (10 mg kg−1 i.p.), intubated, and ventilated. Deep anesthesia was maintained with 1.2–2 vol% isoflurane in conjunction with a gas mixture of 0.2 l / min O2 and 0.2 l / min compressed air. A lateral thoracotomy was performed and left coronary artery (LCA) occlusion was achieved by tightening and tying off a 6-0 prolene suture. After 30 min, the LCA was reoccluded for the desired reperfusion time. The thoracic cavity was closed using 4-0 prolene sutures. Buprenorphine was injected every 8 hours for analgesia. Infarct size was assessed after 30 minutes of ischemia followed by 24 hours of reperfusion using Evans blue staining for delineation of the area at risk of ischemia (AAR) from the non-ischemic zone (remote area) and 1% 2,3,5 triphenyltetrazolium chloride (TTC) staining for the border of viable and non-viable myocardium within the AAR (Hendgen-Cotta et al., 2007). Infarct area, AAR, and non-ischemic left ventricle were assessed with computer-assisted planimetry by an observer blinded to the identity of the specimen. Myocardial infarct size is expressed as a percentage of the AAR. Treatment of mouse myocardial I / R in vivo To evaluate the (patho)physiological relevance of BNIP3 in vivo, mice were injected with sodium chloride (NaCl) as vehicle, TAT-BNIP3 (67 nmol kg-1, 200 nmol kg-1, or 300 nmol kg-1 in 50 μL of 0.9% NaCl) and TAT-BNIP3ΔTM (33 nmol kg-1, 67 nmol kg-1, or 133 nmol kg-1 in 50 μL of 0.9% NaCl). TAT fusion proteins were injected into the left ventricular (LV) cavity 5 min before ischemia.To evaluate the in vivo inhibitory effects and efficacy of TAT-BNIP3-20A and TAT-WVELHFFN, 5 min before reperfusion was treated with NaCl, TAT-β-Gal, TAT-BNIP3-20A (67 nmol kg-1 in 50 μL of 0.9% NaCl), TAT-BNIP3-20C (67 nmol kg-1 in 50 μL of 0.9% NaCl), TATWVELHFFN, TAT-WVELHFFN-Lys (5(6) carboxyfluorescein), and TAT-WVELHFFNCy5.5 (67 nmol kg-1, 133 nmol kg-1, 267 nmol kg-1, or 667 nmol kg-1 in 50 μL of 0.9% NaCl) or TAT-WVELAASN (267 nmol kg-1 in 50 μL of 0.9% NaCl). Mice were injected with 100 mg / kg of 1000 mg / kg of 1000 mg / kg. In vivo mouse brain I / R Focal cerebral ischemia was induced by transient middle cerebral artery occlusion (tMCAO) for 30 min. Briefly, mice were anesthetized with 2% isoflurane in O2. A servo-controlled heating blanket was used to maintain core body temperature close to 37 °C throughout the entire surgery. After a midline cervical incision, a standardized silicone rubber-coated No. 6.0 nylon monofilament (6023910PK10; Doccol, Sharon, MA, USA) was inserted into the right common carotid artery and advanced through the internal carotid artery to occlude the origin of the MCA. After 30 min, mice were re-anesthetized and the occlusion filament was removed to allow reperfusion. Stroke volume was assessed 24 h after tMCAO based on TTC staining. Mice were then randomly assigned to operations by an independent investigator who was not involved in data analysis. Investigators involved in the surgery and evaluation of all readout parameters were blinded to the experimental groups. Treatment in in vivo mouse cerebral I / R. Immediately before reperfusion, mice were injected with sodium chloride or TAT-WVELHFFN (267 nmol kg-1 in 50 μL of 0.9% NaCl).In vivo pig myocardial I / R Experiments were performed by Charles River Laboratories, Inc. (Matawan, MI, USA, Laboratory Research No. 2865-001) in accordance with the United States Department of Agriculture (USDA) The Animal Welfare Act (9 CFR parts 1, 2, and 3) and the Guide for the Care and Use of Laboratory Animals, Institute of Laboratory Animal Resources, National Academy Press, Washington, DC, 2011. For in vivo I / R, a closed-chest model was used. Naïve domestic Yorkshire crossbred pigs (fattening pigs, males, weight range 37.5 kg–40.5 kg) were placed in dorsal recumbency and the surgical site was prepared with alternating wipes of chlorhexidine scrub and solution. The animals were connected to a defibrillator and ECG device and monitored fully throughout the procedure. Following induction of anesthesia, a small incision was made over the femoral artery and femoral vein to isolate the vessels. A small opening was made in the artery and a sheath was introduced. Additionally, a sheath was placed in the femoral vein to allow for emergency drug administration if necessary. The appropriate guide catheter was advanced into the ostium of the left anterior descending artery (LAD) using visual guidance followed by fluoroscopy. Non-ionic contrast media was used for all procedures. A balloon catheter was introduced by advancing it through the guide catheter to the LAD coronary artery. The balloon was advanced into the coronary artery through the guide catheter to the appropriate location above the first diagonal bifurcation of the LAD. The balloon was then inflated to a pressure sufficient to ensure complete occlusion of the artery, which was confirmed by fluoroscopy. Once confirmed, the balloon was left inflated within the artery for 60 minutes. At the end of the ischemic period, the balloon was deflated and the ischemic area was allowed to reperfuse for 4 hours. Complete balloon deflation was confirmed by fluoroscopy. At the end of the procedure, all catheters were removed, the artery and vein were ligated, and the incision was closed in a standard manner. For assessment of infarct size, the heart was removed and flushed with heparinized lactated Ringer's solution until clear of blood. During the myocardial infarction procedure, the LAD was ligated at the site of balloon occlusion. Once tied, the LAD, LCX and RCA were cannulated.Evans blue dye was injected. Hearts were cut into serial sections of approximately 1 cm from base to apex. Each section was weighed and photographed. Heart sections were stained with TTC for 30 min at 37 °C and photographed a second time. Infarct size was calculated as a percentage of the AAR. Images (including a ruler) were analyzed and the ratios of infarct / AAR, infarct / LV area, and AAR / LV were calculated. Heart sections were stored frozen in 10% neutral buffered formalin (NBF) at -60 °C to -90 °C for possible future analysis. Treatment in in vivo bovine myocardial I / R After 60 min of occlusion, TAT-WVELHFFN (0.075 mg kg-1 BW) or sodium chloride was administered by intravenous bolus injection 5 min before reperfusion. Transthoracic echocardiography was used to measure LV contractile function in wild-type mice in response to TAT-WVELHFFN treatment. Echocardiography was performed before and 5 h after injection of TAT-WVELHFFN (267 nmol kg-1) and at desired time points before and after I / R using Vevo 2100 and Vevo 3100 imaging systems (FUJIFILM VisualSonics, The Netherlands). The investigator was blinded with respect to untreated and treated groups. After sedation with 2% isoflurane by volume and removal of hair, mice were placed on a heating plate with constant heart rate, respiratory rate and temperature monitoring via a rectal probe. Left ventricular fractional shortening was determined by M-mode in the mid-ventricular parasternal short axis. LV ejection fraction was calculated using Simpson's method. Cytosol isolation and mitochondrial fractionation. Hearts were washed with ice-cold homogenization buffer (250 mM sucrose, 10 mM HEPES and 1 mM EGTA, pH 7.4), homogenized in 4 ml buffer containing 0.5% bovine serum albumin, and centrifuged at 700×g for 10 min at 4° C. to remove unbroken tissue and nuclei. For general fractionation, the supernatant was centrifuged at 15,000×g for 10 min at 4° C. to obtain the cytosolic fraction in the supernatant and mitochondria in the pellet. The mitochondrial pellet was further cleared of contaminants by washing twice in isolation buffer. The cytosolic and mitochondrial fractions were further processed for Western blotting. Subfractionation of MIM and MOM.Hearts were washed with ice-cold homogenization buffer (250 mM sucrose, 10 mM HEPES and 1 mM EGTA, pH 7.4), homogenized in 4 ml buffer containing 0.5% bovine serum albumin, and centrifuged at 700 × g for 10 min at 4 °C to remove unbroken tissue and nuclei. The supernatant was centrifuged at 15,000 × g for 10 min at 4 °C to pellet mitochondria. The supernatant was then centrifuged at 105,000 × g for 30 min at 4 °C to obtain cytoplasm. The pellet containing mitochondria was carefully washed twice with homogenization buffer. Mitochondrial protein concentration was determined by the Bradford method. For preparation of inner and outer membranes, isolated mitochondria were suspended in 0.25 M sucrose solution, incubated in 0.3% digitonin for 20 min on ice, and centrifuged at 9,500 × g for 15 min. The supernatant was centrifuged at 105,000×g for 1 h at 4°C to obtain MIM in the supernatant. Pellet II was suspended in buffer A (0.25 M sucrose, 10 mM Tris-HCl, pH 7.4) and incubated overnight at 4°C. Pellet I was suspended in 0.1 M Na2CO3 solution, incubated on ice for 20 min, and centrifuged at 105,000×g for 30 min. Pellet III was suspended in buffer A and incubated overnight at 4°C. The next day, pellets II and III were loaded onto a discontinuous sucrose gradient (0.5 ml of 51.3% sucrose, 0.5 ml of 44.3% sucrose, 1 ml of 37.4% sucrose, and 1 ml of 23.2% sucrose in 0.1 M phosphate buffer (pH 7.4)) and centrifuged at 105,000×g for 2 h at 4°C. Fractions (200 μL) were collected and analyzed by Western blotting with the indicated antibodies. Histopathological analysis Infarct area Mouse hearts were perfused with PBS before collection, fixation and preparation of paraffin-embedded sections for histopathological analysis. Standard Gomori trichrome staining was performed to visualize the infarct area. Slices were incubated in Weigert's iron hematoxylin working solution (Merck, Darmstadt, Germany) for 10 min at room temperature and then washed 10 times in H2O.Slices were then incubated in Gomori's trichrome solution (Morphisto, Cologne, Germany) for 15 min at room temperature and washed 10 times in H2O, 2 times in 0.5% acetic acid, and 10 times in H2O. To detect apoptotic nuclei, TUNEL staining was performed using the ApopTag Red In situ Apoptosis Detection Kit (Millipore, S7165, Darmstadt, Germany) according to the manufacturer's instructions. The total number of TUNEL-positive nuclei was counted in five planar sections of the heart to calculate the number of TUNEL-positive nuclei per heart and per section for spatial distribution analysis. Images were captured under an inverted epifluorescence microscope (Zeiss Axio Observer Z1, Oberkochen, Germany) and processed with ImageJ software 1.52a (NIH). Necrosis Troponin staining was performed by deparaffinizing (I / R) mouse heart sections, washing twice in HO and twice in TBS-T (0.1% Tween® 20), and blocking with 5% normal goat serum in PBS for 1 h. Sections were incubated with anti-troponin I antibody (Invitrogen, 1:100 dilution) at 4°C overnight and stained with secondary goat anti-rabbit Alexa Fluor 594 antibody (1:200 dilution). After washing in TBS-T and staining with DAPI (Invitrogen, Carlsbad, CA, USA; 1:5,000 dilution), sections were mounted in ProLong Gold Antifade (ThermoFisher Scientific, Waltham, MA, USA). Images were captured under an inverted epifluorescence microscope (Zeiss Axio Observer Z1, Oberkochen, Germany). Troponin in plasma To examine necrotic cell death, blood was collected from the abdominal aorta after 30 min of ischemia followed by 24 h of reperfusion, blood was centrifuged at 3,000 × g for 10 min at 4 °C, and plasma was collected. Troponin I levels were measured using a Troponin I ELISA (Life Diagnostics, CTNI-1-US, West Chester, PA, USA). Real-time quantitative RT-PCR (real-time qRT-PCR).Total RNA was isolated from hearts using the RNeasy Mini Kit (Qiagen, Hilden, Germany, 74104) according to the manufacturer's instructions. Purified RNA was quantified using a NanoDrop instrument (ThermoFisher Scientific, Waltham, MA, USA). cDNA was generated using a High Capacity RNA-to-cDNA Kit (Life Technologies, Carlsbad, CA, USA). Real-time qRT-PCR was performed using TaqMan Gene Expression Assays (ThermoFisher Scientific, Waltham, MA, USA, Mm00833810) in a 7900HT Fast Real-Time PCR System (ABI PRISM® 7900HT, Applied Biosystems, Foster City, CA, USA). Reactions were normalized to 18S rRNA levels and relative transcript abundance was calculated using the comparative Ct method. Size Exclusion Chromatography One mg of cardiac cytosol lysate was applied to a Superdex® 200 Increase 10 / 300 GL (GE Healthcare, Chicago, IL, USA) equilibrated with SEC buffer (10 mM Tris (pH 7.5), 1 mM EGTA, 200 mM sucrose, protease phosphatase inhibitors (Halt, Thermo Fisher Scientific, Waltham, MA, USA) and run at 4°C. Fractions of 1000 μl were collected, of which 500 μl were concentrated to 40 μl using Amicon® Ultra 0.5 ml Centrifugal Filters (Merck Millipore, Burlington, MA, USA). The collected samples were directly processed for subsequent SDS-PAGE and Western blotting. To estimate the molecular weight of BNIP3 oligomers, gel filtration molecular weight markers (#1511901, BioRad, Hercules, CA, USA) were applied to the column to obtain a standard curve.Fluorescence measurements of Cy5.5-labeled TAT-WVELHFFN were performed in triplicate on an Infinite® 200 PRO microplate reader (Tecan Group Ltd., Switzerland) equipped with black flat-bottom 96-well plates using 150 μl of unprocessed SEC fraction / well. Fluorescence intensity was measured at 630 nm (± 9 nm) excitation / 680 nm (± 20 nm) emission. Western blotting SDS-PAGE Frozen tissues, HL-1 cells and human CM were lysed in ice-cold RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 0.5 mM EDTA, 1% NP-40 and protease and phosphatase inhibitors, pH 7.4). Isolated mitochondria were lysed in Mito-lysis buffer (200 mM sucrose, 10 mM HEPES, 1 mM EGTA, 1% Triton® X-100, protease and phosphatase inhibitors, pH 7.4) and centrifuged at 20,000×g for 15 min at 4° C. to clear the lysate. Protein concentrations in the supernatants were measured using the DC protein assay (Bio-Rad Laboratories Inc., Hercules, CA, USA). Samples were then diluted with 4x LDS sample buffer and 10x reducing agent (Invitrogen, Carlsbad, CA, USA), boiled at 95°C for 5 min, and loaded onto NuPAGE™ 4-12% Bis-Tris Protein Gels (Invitrogen, Carlsbad, CA, USA). The following antibodies were incubated overnight at 4°C: anti-ANT1 (Abcam, Cambridge, UK, ab110322, 1:1,000 dilution), anti-AIF (Santa Cruz Biotechnology, Dallas, TX, USA, sc9416, 1:200 dilution), anti-BNIP3 (Abcam, Cambridge, UK, ab38621, 1:1,000 dilution), anti-BAX (Abcam, Cambridge, UK, ab7977, ab32503, 1:500 dilution), anti-cytochrome c (Abcam, Cambridge, UK, ab13575, 1:1,000 dilution), anti-TOMM22 (Abcam, Cambridge, UK, ab57523, 1:500 dilution), anti-tubulin (Abcam, Cambridge, UK, ab15246, 1:5,000 dilution) and anti-active BAX (Enzo Life Western blot analysis was performed on nitrocellulose membranes after blocking with TBS-T containing 5% milk using 1:1,000 dilution of 1000 mM NaCl (Bio-Rad Laboratories Inc., Hercules, CA, USA). Membranes were incubated with HRP-conjugated secondary from the appropriate species for 1 h at room temperature with subsequent detection and exposed to SuperSignal West Pico Plus Substrate (ThermoFisher Scientific, Waltham, MA, USA) as directed by the manufacturer. Protein concentrations of BN-PAGE isolated cytosolic fractions were measured using the DC protein assay (Bio-Rad Laboratories Inc., Hercules, CA, USA). Samples were then diluted with 4× NativePAGE™ sample buffer (Invitrogen, Carlsbad, CA, USA) and loaded onto NativePAGE™ 4–16% Bis-Tris protein gels (Invitrogen, Carlsbad, CA, USA).Gel runs were performed at 4°C using the NativePAGE™ Running Buffer Kit (Invitrogen, Carlsbad, CA, USA). Western blot analysis was performed on PVDF membranes using the iBind™ Flex system (Invitrogen, Carlsbad, CA, USA) with anti-BNIP3 (Abcam, Cambridge, UK, ab38621, 1:1,000 dilution) and anti-BAX (Abcam, Cambridge, USA, ab32503, 1:500 dilution). Co-immunoprecipitation experiments Cytosolic lysates were cleared by centrifugation at 20,000×g for 15 min at 4°C. Protein concentrations in the cytosolic fractions were measured using the DC protein assay (Bio-Rad Laboratories Inc., Hercules, CA, USA). Co-immunoprecipitation was performed using Protein G-coupled Dynabeads (Invitrogen, Carlsbad, CA, USA) covalently coupled to anti-BNIP3 (Abcam, Cambridge, UK, 10 μg / sample) or anti-BAX (Abcam, Cambridge, UK, 0.011 μg / sample). After washing off unbound antibodies, 500 μg of protein lysate was subjected to coupled bead antibodies overnight at 4 °C with rotation. Precipitates bound to bead antibodies were removed from unbound lysate and resuspended in 4 × LDS sample buffer (Invitrogen, Carlsbad, CA, USA), 10 × reducing agent (Invitrogen, Carlsbad, CA, USA), and boiled at 70 °C for 10 min to separate precipitates from bead antibodies. The precipitates were then analyzed by Western blot on nitrocellulose membranes using anti-BNIP3 (Abcam, Cambridge, UK, ab38621, 1:1,000) and anti-BAX (Abcam, Cambridge, UK, ab32503, 1:500). For fluorescence measurements, the precipitates were resuspended in 10x reducing agent, boiled at 70°C for 10 min, excited at 485 nm, and emission measured at 520 nm. 4x LDS was then added and Western blotting was performed as described.Immunostaining of BNIP3 in tissues Wild-type and Bnip3- / - mouse hearts were washed in vivo with NaCl blood-free, then removed and incubated overnight at 4°C in 40% sucrose solution. After fixation with 4% paraformaldehyde for 15 min, 8 μm slices were cut and washed three times for 5 min with PBS containing 0.5% Triton® X-100 for cell permeabilization. Slices were stained with Mito-Tracker orange (Invitrogen, Carlsbad, CA, USA) and incubated with anti-BNIP3 antibody overnight at 4°C. Staining was performed with AlexaFluor 488-conjugated goat anti-rabbit secondary antibody and DAPI (Invitrogen, Carlsbad, CA, USA, 1:5,000 dilution). Confocal sections were visualized under a confocal laser scanning microscope using a 63× / 1.4 Oil DIC M27 objective (Zeiss Elyra PS; Zeiss, Oberkochen, Germany). Caspase-3 activity Caspase-3 activity was measured in the AAR after 1 h of reperfusion using a Caspase-3 Assay Kit (Abcam, Cambridge, UK, ab39401) according to the manufacturer's instructions. Bioenergetics For bioenergetic studies, ATP concentration in the AAR was analyzed after 30 min of reperfusion using an ATP Bioluminescent Assay Kit (Sigma-Aldrich, St. Louis, MO, USA, FLAA). Tissue Clearing Hearts were fixed in 4% paraformaldehyde (w / v PFA) for 4 h at 4°C. Samples need to be protected from light in all the following steps. The hearts were then dehydrated using an ascending series of ethanol (50%, 70%, 100% (v / v) EtOH in ddH2O) with shaking: overnight in 50% EtOH at 4 °C, 30 min in 70% EtOH at room temperature, and 30 min in 70% EtOH at room temperature. To increase the clarity of the samples, the samples were bleached using freshly prepared bleaching solution (5% (v / v) hydrogen peroxide, 5% (v / v) dimethyl sulfoxide in 100% ethanol) for 4 h at 4 °C with shaking. Afterwards, the samples were washed three times in 100% ethanol for 30 min at 4 °C with shaking.For the final clearing step, the hearts were warmed to room temperature for 5 min and subsequently transferred to a glass vial containing pure ethyl cinnamate (ECi, Sigma Aldrich). Samples were cleared for at least 4 h before imaging and kept at room temperature and protected from light before and after imaging. Light sheet fluorescence microscopy and image processing Samples were imaged using an Ultramicroscope II and ImSpector software (both from LaVision BioTec). Cleared hearts were immersed in pure ECi in a quartz cuvette. As TATWVELHFFN was coupled to the Cy 5.5 fluorophore, the excitation wavelength of the light sheet was set to 639 nm with the corresponding 680 / 30 bandpass emission filter. Additionally, autofluorescence was imaged in the FITC channel (488 nm excitation, 525 / 50 bandpass emission filter). To prevent deformation, the specimen was clamped in a specimen holder, apex and aorta aligned horizontally between two blocks of ECi-clarified phytagel (1% phytagel in H2O). The ligation knot used to induce myocardial I / R needs to remain in situ and point downwards during imaging to reduce blockage of excitation or emission light. A whole-heart data set was acquired at a total magnification of 1.2x with a z-spacing of 10 μm between the optical planes. The sheet width was 4200 and the numerical aperture was 0.148. The longest wavelength was imaged first to prevent photobleaching. 16-bit OME.TIF stacks were converted to Imaris files (.ims) (ImarisFileConverterx64, BitPlane). 3D reconstructions and subsequent analyses were performed using Imaris software (BitPlane). Mitochondrial Experiments MIM Permeabilization To analyze MIM potential, 5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) was used by performing the MitoProbe™ JC-1 Assay Kit for Flow Cytometry (ThermoFisher Scientific, Waltham, MA, USA 34152). Each probe contained 250 μg of isolated mitochondria incubated with 1.5 μg JC-1 / mg.Changes in mitochondrial MIM potential were analyzed by flow cytometry using a FACS Verse (Becton Dickinson, Franklin Lakes, NJ, USA) with an excitation wavelength of 514 nm and emission wavelengths of 529 nm and 590 nm. To assess MIM permeabilization due to mPTP opening, mitochondria were isolated from the AAR after 10 min of reperfusion. Mitochondrial swelling was measured by changes in light scattering at 540 nm. The final buffer volume was 200 μl (250 mM sucrose, 10 mM HEPES and 1 mM EGTA, pH 7.4) containing 0.5 mg / mL mitochondria. To distinguish between cytochrome c release induced by mPTP opening or MOMP formation, isolated mitochondria (1 mg / mL) were incubated in buffer (150 mM KCl, 5 mM Tris-HCl, pH 7.4) and energized with 5 mM succinate and 2 μM rotenone. rBNIP3 (1 μg), rBAX (1 μg) and 150 mM Ca2+ were added per mg protein. 5 mM Pi was used to induce mPTP opening and swelling. Optical density at 540 nm was measured over 40 min. Cytochrome c content in the supernatant was analyzed by Western blotting. MOM permeabilization To demonstrate the requirement of BNIP3 for BAX pore formation, isolated mitochondria were incubated with cytoplasm. To inhibit VDAC, BAX and BNIP3, the inhibitors DIDS (1 μg), anti-BAX antibody (1 μg) and BNIP3ΔTM (1 μg) were added. Cytosolic concentrations of cytochrome c, AIF and active BAX were quantified by Western blotting. Cell culture Isolated adult cardiomyocytes To isolate adult cardiomyocytes, wild-type mice were anesthetized with ketamine (100 mg kg-1 ip) and xylazine (10 mg kg-1 ip) and hearts were rapidly excised. The explanted hearts were perfused in a retrograde manner and digested with HEPES buffer (110 mM NaCl, 2.5 mM KCl, 1.2 mM KH2PO4, 2.5 mM MgSO4, 25 mM HEPES, 10 mM glucose monohydrate, 4 μM collagenase type II and 28 μM Ca2+, pH 7.4) for 25 min at 37 °C.The cell suspension was filtered through a 250 μm mesh collector and centrifuged at 18 × g for 1 min. The cell pellet was resuspended in 100 μM HEPES buffer with stepwise increasing Ca2+ concentrations. Cells were seeded on cell culture dishes pre-coated with laminin and washed after 4 h to remove dead and non-adherent cells. Cells were cultured in modified medium 199 containing Earl's salts, 2 mM carnitine, 5 mM creatine and 5 mM taurine supplemented with 100 IU / mL penicillin and 100 μg / ml streptomycin. Cytosine-D-arabinofuranoside (10 μM. ) was added to inhibit proliferation of non-myocytes. Hypoxia / reoxygenation (H / R) For hypoxia experiments, cells were incubated in HEPES buffer (113 mM NaCl, 4.7 mM KCl, 12 mM HEPES, 1.2 mM MgSO4, 30 mM taurine and 1.3 mM CaCl2, pH 7.4) under 1% O2 at 37°C for 2 h. For reoxygenation, cells were incubated in HEPES buffer supplemented with 5.5 mM glucose under 21% O2 at 37°C for 2 h. Phosphorylation experiments To evaluate BNIP3 serine phosphorylation under AKT inhibition, HL-1 cells were treated with 10 μM AKT1 / 2 inhibitor (Sigma-Aldrich, St. Louis, MO, USA, A6730). The levels of BNIP3 serine phosphorylation and active BAX were analyzed by Western blotting. Peptide effects in human CM To evaluate the in vitro effects of TAT-WVELHFFN, human CM were treated with TATWVELHFFN (2 nmol in 500 μl HEPES buffer) and TAT-WVELAASN (2 nmol in 500 μl HEPES) during H / R. To visualize apoptotic and necrotic human CM after H / R, cells were stained with 7-AAD (red), Apoxin Green (green) and CytoCalcein (blue) using the Apoptosis / Necrosis Detection Kit (Abcam, Cambridge, UK, ab176749) according to the manufacturer's instructions. Images were captured under an EVOS FL microscope (ThermoFisher Scientific, Waltham, MA, USA). To analyze MIM potential, human CM were stained with JC-1. Cells were incubated with 6 μM JC-1 in medium for 30 min at 37 °C, washed with PBS buffer at 37 °C, and fixed with 4% paraformaldehyde for 15 min at room temperature. DAPI staining was performed, and cells were analyzed using an EVOS FL microscope (ThermoFisher Scientific, Waltham, MA, USA). Transmission electron microscopy (TEM) Electron microscopic analysis of I / R injury in cardiac tissue specimens and isolated mitochondria was performed as previously described with minor modifications (Hendgen-Cotta et al., 2018). Specifically, cacodylate buffer was used for the fixation and washing steps. Staining procedures were performed with potassium ferrocyanide (II), thiocarbohydrazide, and lead nitrate aspartate solutions. Samples were infiltrated and embedded in epoxy resin (Durcupan, Sigma-Aldrich, St. Louis, MO, USA). Peptide synthesis. L-amino acid peptides were synthesized by JPT Peptide Technologies (Berlin, Germany) using a solid-phase resin-based methodology and purified to >90% by HPLC (confirmed by mass spectrometry; Figure S6). The N-terminus of the peptide was capped with an acetyl group and the C-terminus with an amide group.The TAT-BNIP3-20A sequence, GRKKRRQRRRPQMSQSGEENLQGSWVELHFSN, consisted of 12 amino acids from the HIV-1 PTD at the N-terminus and 20 amino acids from BNIP3 1-20 at the C-terminus. The TAT-BNIP3-20C sequence, GRKKRRQRRRPQLDAQHESGRSSSKSSHCDSP, consisted of 12 amino acids from the HIV-1 PTD at the N-terminus and 20 amino acids from BNIP3 42-61 at the C-terminus. The TAT-octapeptide sequence, GRKKRRQRRRPQWVELHFFN, consisted of 12 amino acids from the HIV-1 PTD at the N-terminus and 8 amino acids from BNIP3 13-20 containing the substitution S19F at the C-terminus. The control TAT-octapeptide sequence, GRKKRRQRRRPQWVELAASN, consisted of 12 amino acids from the HIV-1 PTD at the N-terminus and 8 amino acids from BNIP3 13-20 with two substitutions H17A and F18A at the C-terminus. For in vivo and cell culture experiments, peptides were dissolved in HO and diluted with 0.9% NaCl. For protein-peptide interaction studies, peptide libraries were synthesized and immobilized on microarray slides. Peptides were synthesized and immobilized for (i) BNIP3 / BAX interaction studies (provided in Table S1), (ii) BNIP3 / BNIP3 interaction studies in which C-, N- or C / N-terminal truncations of the wild-type sequence of BNIP3 1-20 were performed (provided in Table S2A); and (iii) BNIP3 / BNIP3 interaction studies in which a single residue of the wild-type sequence of BNIP3 1-20 was replaced with 18 neutral amino acids (provided in Table S2B). Interaction studies with peptide microarrays. For protein-peptide binding studies, including rBNIP3-BAX peptide interaction analysis, rBNIP3-BNIP3 peptide alanine scan, truncation analysis, and substitution analysis, rBNIP3 was used at concentrations of 5 μg / mL and 1 μg / mL. The studies were performed by JPT Peptide Technologies (Berlin, Germany).For fluorescent labeling, the DyLight Microscale Antibody Labeling Kit with label DyLight 650 (ThermoFisher Scientific, Waltham, MA, USA) was used. The assay was performed using an automated Tecan HS 4800 microarray processing station. The microarrays were incubated for 2 h at 30 °C with customer-provided samples diluted in blocking buffer. Before each step, the microarrays were washed with washing buffer. The microarrays were scanned using a high-resolution fluorescent scanner. The laser settings and the applied resolution were identical for all measurements performed. The obtained images were analyzed and quantified using GenePix spot recognition software (Molecular Devices, San Jose, CA, USA). The mean signal intensity was extracted for each spot (between 0 and 65535 arbitrary units). For further data evaluation, the so-called MMC2 value was determined. The MMC2 is equal to the mean value of all three instances on the microarray, except when the coefficient of variation (CV) (standard deviation divided by the mean) is >0.5. In this case, the average of the two closest values ​​(MC2) was assigned to MMC2. Overlay assays For protein-protein / peptide overlay assays, 300 ng of rBNIP3 or rBAX was spotted onto a nitrocellulose membrane. After 5 min of incubation followed by 5 min of washing with TBS-T, the membrane was incubated for 1.5 h with 2.5 μg of rBNIP3 or rBAX protein labeled with Dy650, or 50 μg of TAT-WVELHFFN labeled with Lys(5(6))-Fam in 200 μl of TBS-T. After two 5 min washes with TBS-T, fluorescence was detected using an ImageQuant system (Amersham, Little Chalfont, UK). Docking simulation experiments Docking simulation experiments were performed using Autodock Vina and HADDOCK. The structure of BAX (pdb-ID: 4S0O) was obtained from the Protein Data Bank, while the structures of BNIP3 and peptide TAT-WVELHFFN were modeled using Modeller 9.15.The template structure corresponded to PDB codes 2K7W and 2KA1. The generated model was energy minimized using NAMD2.9 and CHARMM36 force field. Proximity ligation assay To evaluate the BNIP3-BAX interaction, proximity ligation assays were performed in human CM after 1 h hypoxia / 1 h reoxygenation using Duolink In Situ Red Starter Kit mice (Sigma-Aldrich, St. Louis, MO, USA, DUO 92101-1 KT) using antibodies against BNIP3 (ab10433, 1:1,000 dilution) and BAX (ab7977, 1:1,000) according to the manufacturer's protocol, followed by visualization by confocal laser scanning microscopy (Zeiss Elyra PS; Zeiss, Oberkochen, Germany). Circular dichroism spectroscopy. Circular dichroism spectra of BNIP3 protein and TAT-WVELHFFN peptide were recorded in 1× PBS, pH 7.4 at 37°C on a Jasco J-715 spectropolarimeter (Jasco, Pfungstadt, Germany). Peptide uptake, distribution and stability For uptake and distribution analysis, mice were injected with TAT-WVELHFFN-Lys(5(6)-FAM) (267 nmol kg-1 in 50 μL of 0.9% NaCl). Sham-operated mice and baseline values ​​served as controls. For pharmacokinetic studies, TAT-WVELHFFN was incubated in human whole blood, serum and plasma for 0, 10, 20, 30 and 60 min at 37°C. Treatment with proteinase K (0.3 mg / ml) served as control. Human samples Left ventricular tissue samples were obtained from three patients with suspected myocarditis who underwent endomyocardial biopsy (ethical approval from the ethical committee, University of Duisburg-Essen, No. 17-7392-BO). The diagnosis was not confirmed histologically. The median age of the three donors was 47 years.

[0267] Human Cardiomyocytes (HCM) HCM were obtained from PromoCell and cultured according to the manufacturer's instructions. Cells were incubated with 0, 1 and 10 μM doxorubicin and simultaneously treated with 0 and 1.5 pM TAT-WVELHFFN for 4 and 22 hours, respectively. Mitochondrial membrane potential was measured using TMRM-Kit (Abcam ab228569), calcium overload using Rhod-2AM, and cell viability using a cytotoxicity kit (Molecular Probes L3224), all according to the manufacturer's instructions.

[0268] In vivo DOX treatment Wild-type mice aged 12±3 weeks received 8 ip injections of 0.9% saline (vehicle control) or DOX (3 mg / kg body weight) every 2 days. TAT-WVELHFFN was administered ip at the same time. Serial echocardiography was performed on all mice at baseline and on days 16 and 42. Left ventricular ejection fraction, fractional shortening and isovolumic relaxation time were assessed. Cardiac troponin I (cTNI-ELISA life Diagnostic CTNI-1-US), BNP (BNP-ELISA EIAM-BNP) and LDH (activity assay Sigma MAK066) were measured according to the manufacturer's instructions. Mitochondrial OCR was assessed on a Seahorse XF24 Analyzer using the Mitochondrial Stress Test (Agilent).

[0269] Quantitative and statistical analysis Statistical tests were performed and graphs were generated using GraphPad Prism 7 software (GraphPad, San Diego, CA, USA). Data are presented as mean ± SEM, and p values ​​were calculated as detailed in the corresponding legend. Sample size was determined based on previous experimental experience or based on common practice in the field. All replicates constitute biological replicates. For in vivo studies, mice were randomly assigned to groups. For all histopathological and immunostaining analyses, experimenters were blinded to the experimental conditions. Comparisons between characteristics of subject groups were analyzed with a two-tailed Student's t test. For comparisons between three or more groups, one-way or two-way ANOVA with Bonferroni's post hoc test was used. Availability of data and software ImageJ software 1.52a and Fiji / ImageJ 1.52e are available at the website http: / / imagej.net / Fiji.

Claims

1. A composition for ameliorating infarction damage in a subject at risk for an infarction damage risk event, the composition comprising a mitochondrial membrane transport inhibitor.

2. The composition of claim 1 , wherein the mitochondrial membrane translocation inhibitor inhibits BAX translocation to the mitochondrial membrane.

3. The composition of claim 1 , wherein the mitochondrial membrane transport inhibitor inhibits BNIP3 transport to the mitochondrial membrane.

4. The method of claim 1 , wherein the infarct injury risk event comprises a heart attack.

5. The composition of claim 1 , wherein the infarction injury risk event comprises a stroke.

6. The composition of claim 1 , wherein the infarction injury risk event comprises renal failure.

7. The composition of claim 1 , wherein the infarction injury risk event comprises acute circulatory failure.

8. The composition of claim 1 , wherein the infarction injury risk event comprises organ transplantation.

9. The method of claim 1 , wherein the infarction injury risk event comprises surgery.

10. The composition of claim 1 , wherein the migration inhibitor is administered intravenously.

11. The composition of claim 1, wherein the migration inhibitor is administered before an infarct injury risk event.

12. The composition of claim 9, wherein the migration inhibitor is administered before an infarction injury risk event.

13. The method of claim 1 , wherein the infarction injury risk event comprises surgery.

14. The composition of claim 1, wherein the migration inhibitor is administered after an infarct injury risk event.

15. The composition of claim 1, wherein the migration inhibitor is administered prior to reperfusion.

16. The composition of claim 14 , wherein the infarction injury risk event comprises a contusion injury.

17. The composition of claim 14 , wherein the infarction injury risk event comprises heart failure.

18. The composition of claim 14 , wherein the infarction injury risk event comprises a physiological response to stress.

19. The composition of claim 14 , wherein the infarct injury risk event comprises a physiological response to diabetes.

20. 15. The composition of claim 14, wherein the infarct injury risk event comprises a physiological response to hypertension.

21. The composition of claim 14 , wherein the infarction injury risk event comprises a physiological response to hyperlipidemia.

22. 15. The composition of claim 14, wherein the infarct injury risk event comprises a physiological response to obesity.

23. 15. The composition of claim 14, wherein the infarct injury risk event comprises a physiological response to a genetic disorder.

24. The composition of claim 14 , wherein the infarct injury risk event comprises a physiological response to lung injury.

25. The composition of claim 14 , wherein the infarction injury risk event comprises an inflammatory response.

26. The composition of claim 14 , wherein the infarction injury risk event comprises an autoinflammatory response.

27. 10. The composition of claim 1, wherein the BAX translocation inhibitor is administered in multiple doses.

28. 28. The composition of claim 27, wherein the multiple doses are administered at regular intervals.

29. The composition of claim 1 , wherein the BAX translocation inhibitor comprises a chimeric peptide.

30. 30. The composition of claim 29, wherein the chimeric peptide comprises a segment having at least 75% identity to at least 8 consecutive residues of BNIP3.

31. 31. The composition of claim 30, wherein the at least eight consecutive residues of BNIP3 include a phenylalanine residue at position 7 of the at least eight consecutive residues of BNIP3.

32. 32. The composition of claim 31, wherein the 8 contiguous residues comprise residues having at least 75% identity to residues corresponding to residues 13-20 of BNIP3.

33. 33. The composition of claim 32, wherein the eight contiguous residues comprise residues having at least 87.5% identity to residues corresponding to residues 13-20 of BNIP3.

34. 30. The composition of claim 29, wherein the chimeric protein comprises a segment having 75% identity to no more than 50 residues of BNIP3.

35. 30. The composition of claim 29, wherein the chimeric protein comprises a segment having 87.5% identity to no more than 50 residues of BNIP3.

36. 30. The composition of claim 29, wherein the chimeric protein does not contain a BH3 motif.

37. 30. The composition of claim 29, wherein the chimeric protein does not contain a PESTQ motif.

38. The composition of any one of claims 29 to 37, wherein the chimeric protein comprises a BAX binding motif.

39. 30. The composition of claim 29, wherein the migration inhibitor reduces damage from a subsequent infarct event by at least 10%.

40. 30. The composition of claim 29, wherein the migration inhibitor reduces damage from a subsequent infarct event by at least 20%.

41. 30. The composition of claim 29, wherein the migration inhibitor reduces damage from a subsequent infarct event by at least 30%.

42. 30. The composition of claim 29, wherein the migration inhibitor reduces damage from a subsequent infarct event by at least 40%.

43. 30. The composition of claim 29, wherein the migration inhibitor reduces damage from a subsequent infarct event by at least 50%.

44. 30. The composition of claim 29, for monitoring recovery from infarction injury.

45. 1. A composition for reducing damage from an infarct event to a subject suffering from an infarct event, the composition comprising a mitochondrial membrane transport inhibitor.

46. 46. ​​The composition of claim 45, wherein the migration inhibitor is administered prior to reoxygenation following treatment of the infarct event.

47. 47. The composition of claim 46, wherein the peptide is administered at least 5 minutes before reperfusion.

48. 47. The composition of claim 46, wherein the peptide is administered up to 5 minutes before reperfusion.

49. 47. The composition of claim 46, wherein the peptide is administered at least one day before an infarction risk event.

50. 47. The composition of claim 46, wherein the peptide is administered at least two days before an infarction risk event.

51. 47. The composition of claim 46, wherein the peptide is administered at least three days before an infarction risk event.

52. 47. The composition of claim 46, wherein the peptide is administered at least 7 days before the infarction risk event.

53. 46. ​​The composition of claim 45, wherein the migration inhibitor is administered simultaneously with reoxygenation following treatment of the infarct event.

54. 46. ​​The composition of claim 45, wherein the treatment reduces BAX mitochondrial levels by at least 30% compared to an untreated baseline.

55. 46. ​​The composition of claim 45, wherein the treatment reduces BNIP3 mitochondrial levels by at least 30% compared to an untreated baseline.

56. 46. ​​The composition of claim 45, wherein the treatment reduces mitochondrial swelling by at least 30% compared to an untreated baseline.

57. 46. ​​The composition of claim 45, wherein the treatment reduces BAX activity levels by at least 75% compared to an untreated baseline.

58. 46. ​​The composition of claim 45, wherein the treatment reduces cytochrome c release by at least 75% compared to an untreated baseline.

59. 46. ​​The composition of claim 45, wherein the treatment improves the Inf / AAR ratio by at least 50% compared to an untreated baseline.

60. 46. ​​The composition of claim 45, wherein the treatment reduces caspase activity compared to an untreated baseline.

61. 46. ​​The composition of claim 45, wherein the treatment reduces membrane depolarization compared to an untreated baseline.

62. 46. ​​The composition of claim 45, wherein the infarct injury risk event comprises a heart attack.

63. 46. ​​The composition of claim 45, wherein the infarct injury risk event comprises a stroke.

64. 46. ​​The composition of claim 45, wherein the migration inhibitor is administered intravenously.

65. 46. ​​The composition of claim 45, wherein the migration inhibitor is administered via a catheter.

66. 46. ​​The composition of claim 45, wherein the migration inhibitor comprises a chimeric peptide.

67. 67. The composition of claim 66, wherein the chimeric protein comprises a segment having at least 75% identity to at least 8 consecutive residues of BNIP3.

68. 68. The composition of claim 67, wherein the at least eight consecutive residues of BNIP3 include a phenylalanine residue at position seven of the at least eight consecutive residues of BNIP3.

69. 69. The composition of claim 68, wherein the eight consecutive residues comprise residues corresponding to residues 13-20 of BNIP3.

70. 68. The composition of claim 67, wherein the chimeric protein comprises a segment having 75% identity to 50 residues or less of BNIP3.

71. 71. The composition of claim 70, wherein the chimeric protein does not contain a BH3 motif.

72. 71. The composition of claim 70, wherein the chimeric protein does not contain a PESTQ motif.

73. The composition of any one of claims 70 to 72, wherein the chimeric protein comprises a BAX binding motif.

74. 46. ​​The method of claim 45, further comprising monitoring recovery from infarction injury.

75. 10. A method of evaluating a molecule for ameliorating infarction, said method comprising assaying for binding of said molecule to a BAX / BNIP3 oligomeric complex.

76. 76. The method of claim 75, wherein binding of said molecule to said BAX / BNIP3 oligomeric complex indicates efficacy in ameliorating infarction.

77. 76. The method of claim 75, wherein said assaying comprises assaying for co-localization of the molecule and a BAX / BNIP3 oligomeric complex.

78. 78. The method of claim 77, wherein the colocalization is assayed by fluorescence microscopy.

79. 76. The method of claim 75, wherein said assaying comprises assaying for co-migration of said molecule and a BAX / BNIP3 oligomeric complex.

80. 80. The method of claim 79, wherein said co-migration is performed under conditions that maintain the integrity of oligomeric BAX / BNIP3 oligomeric complexes.

81. 80. The method of claim 79, wherein the co-migration is assayed using gel electrophoresis.

82. 82. The method of claim 81, wherein the gel electrophoresis comprises SDS-PAGE.

83. 82. The method of claim 81, wherein said gel electrophoresis comprises Western blot analysis.

84. 81. The method of claim 80, wherein the co-migration is performed under non-denaturing conditions.

85. 76. The method of claim 75, wherein said assaying comprises assaying for co-precipitation of said molecule and a BAX / BNIP3 oligomeric complex.

86. 86. The method of claim 85, wherein said co-precipitation comprises immunoprecipitation.

87. 76. The method of claim 75, wherein the assay comprises contacting the molecule with cells that have been subjected to oxygen deprivation.

88. 76. The method of claim 75, wherein the assay comprises contacting the molecule with the cells within 10 minutes of subjecting the cells to oxygen deprivation.

89. A method for evaluating a molecule for ameliorating infarction, said method comprising contacting said molecule with cells subjected to oxygen deprivation and assaying for localization of the BAX / BNIP3 complex.

90. 90. The method of claim 89, wherein retention of BAX / BNIP3 outside the mitochondria of the cell indicates efficacy in ameliorating infarction.

91. 90. The method of claim 89, wherein inhibition of BAX / BNIP3 translocation to the mitochondria of the cell indicates efficacy in ameliorating infarction.

92. 90. The method of claim 89, wherein inhibition of BAX translocation into the mitochondria of the cell demonstrates efficacy in ameliorating infarction.

93. 90. The method of claim 89, wherein inhibition of BNIP3 translocation to the mitochondria of the cell indicates efficacy in ameliorating infarction.

94. 90. The method of claim 89, wherein assaying for localization of the BAX / BNIP3 complex comprises immunofluorescence.

95. 90. The method of claim 89, wherein assaying for localization of the BAX / BNIP3 complex comprises assaying for mitochondrial integrity.

96. 90. The method of claim 89, wherein assaying for localization of the BAX / BNIP3 complex comprises assaying for mitochondrial swelling.

97. 90. The method of claim 89, wherein assaying for localization of the BAX / BNIP3 complex comprises assaying for cytochrome c release.

98. 90. The method of claim 89, wherein assaying for localization of the BAX / BNIP3 complex comprises assaying caspase-3 activity.

99. 99. The method of any one of claims 89 to 98, wherein the assay comprises contacting the molecule with the cells within 10 minutes of subjecting the cells to oxygen deprivation.

100. A composition for ameliorating a side effect of treatment, the composition comprising a mitochondrial stabilizing agent.

101. 101. The composition of claim 100, wherein the treatment comprises administering a chemotherapeutic agent.

102. 101. The composition of claim 100, wherein the mitochondrial stabilizing agent comprises a mitochondrial import inhibitor.

103. 102. The composition of claim 101, wherein the chemotherapeutic agent targets cancerous cells.

104. 102. The composition of claim 101, wherein the chemotherapeutic agent targets a tumor.

105. 102. The composition of claim 101, wherein the chemotherapeutic agent causes myocardial cell death.

106. The chemotherapeutic agent is an alkylating agent such as altretamine, busulfan, carboplatin, carmustine, cisplatin, cyclophosphamide, dacarbazine, ifosfamide, lomustine, melphalan, temozolomide, or trabectedin; 5-fluorouracil, 6-mercaptopurine, azacitidine, capecitabine, clofarabine, cytarabine, floxuridine, fludarabine, gemcitabine, methotrexate, pemetrexed, or pentostaphylococcus aureus; 102. The composition of claim 101, comprising a compound selected from the list consisting of: antimetabolites such as vincristine, vinblastine, vinorelbine, paclitaxel, docetaxel, etoposide, teniposide, irinotecan, topotecan; plant alkaloids such as daunorubicin, doxorubicin, liposomal doxorubicin, epirubicin, idarubicin, and valrubicin.

107. 102. The composition of claim 101, wherein the chemotherapeutic agent comprises a compound selected from the list consisting of daunorubicin, doxorubicin, liposomal doxorubicin, epirubicin, idarubicin, and valrubicin.

108. 102. The composition of claim 101, wherein the chemotherapeutic agent comprises an anthracycline.

109. 107. The composition of claim 106, wherein the anthracycline comprises doxorubicin.

110. 102. The composition of claim 101, wherein the inhibitor affects BNIP3 activity.

111. The composition of claim 101, wherein the inhibitor affects at least one indication selected from the list including DOX cell death induction, mitochondrial membrane potential stabilization, prevention of mitochondrial pore opening, prevention of mitochondrial calcium ion overload, prevention of mitochondrial ROS or accumulation.

112. 102. The composition of claim 101, wherein the inhibitor attenuates cardiotoxicity.

113. The composition of claim 101, wherein the inhibitor attenuates cardiomyocyte death.

114. 102. The composition of claim 101, wherein the inhibitor preserves cardiac function.

115. The composition of claim 101, wherein the inhibitor increases mitochondrial fitness.

116. The composition of claim 101, wherein the inhibitor preserves autophagic flux.

117. 102. The composition of claim 101, wherein the inhibitor comprises a polypeptide.

118. 102. The composition of claim 101, wherein the inhibitor comprises a BNIP3 fragment.

119. 102. The composition of claim 101, wherein the inhibitor comprises a BAX fragment.

120. A composition described in any one of claims 101 to 119, characterized in that administration of the composition includes injection.

121. 1. A composition for use in ameliorating the adverse effects of treatment, the composition comprising a chemotherapeutic agent and a mitochondrial import inhibitor.

122. 122. The composition for use of claim 121, wherein the chemotherapeutic agent targets cancerous cells.

123. 122. The composition for use of claim 121, wherein the chemotherapeutic agent targets a tumor.

124. 122. The composition for use of claim 121, wherein the chemotherapeutic agent causes myocardial cell death.

125. The chemotherapeutic agent is selected from the group consisting of alkylating agents such as altretamine, busulfan, carboplatin, carmustine, cisplatin, cyclophosphamide, dacarbazine, ifosfamide, lomustine, melphalan, temozolomide, and trabectedin; antimetabolites such as 5-fluorouracil, 6-mercaptopurine, azacitidine, capecitabine, clofarabine, cytarabine, floxuridine, fludarabine, gemcitabine, methotrexate, pemetrexed, pentostatin, pralatrexate, trifluridine, and tipiracil; and HER2 inhibitors, such as trastuzumab and pertuzumab.

122. The composition for use of claim 121, comprising a compound selected from the list consisting of antibodies such as nivolumab (anti-PD-1), avelumab (anti-PD-L1), ipilimumab (anti-CTLA-4), leratolimab (anti-LAG-3), and the like; plant alkaloids such as vincristine, vinblastine, vinorelbine, paclitaxel, docetaxel, etoposide, teniposide, irinotecan, and topotecan; and antitumor agents such as daunorubicin, doxorubicin, liposomal doxorubicin, epirubicin, idarubicin, and valrubicin.

126. 122. The composition for use of claim 121, wherein the chemotherapeutic agent comprises a compound selected from the list consisting of daunorubicin, doxorubicin, liposomal doxorubicin, epirubicin, idarubicin, and valrubicin.

127. 122. The composition for use of claim 121, wherein the chemotherapeutic agent comprises an anthracycline.

128. 128. The composition for use of claim 127, wherein the anthracycline comprises doxorubicin.

129. 122. The composition for use of claim 121, wherein the inhibitor affects BNIP3 activity.

130. 122. The composition for use of claim 121, wherein the inhibitor affects at least one indication selected from the list including DOX cell death induction, mitochondrial membrane potential stabilization, prevention of mitochondrial pore opening, prevention of mitochondrial calcium ion overload, prevention of mitochondrial ROS or accumulation.

131. 122. The composition for use of claim 121, wherein the inhibitor attenuates cardiotoxicity.

132. 122. The composition for use of claim 121, wherein the inhibitor attenuates cardiomyocyte death.

133. 122. The composition for use of claim 121, wherein the inhibitor preserves cardiac function.

134. 122. The composition for use of claim 121, wherein the inhibitor increases mitochondrial fitness.

135. 122. The composition for use of claim 121, wherein the inhibitor preserves autophagic flux.

136. 122. The composition for use of claim 121, wherein the inhibitor comprises a polypeptide.

137. A composition for use as described in claim 121, wherein the inhibitor comprises a BNIP3 fragment.

138. 122. The composition for use of claim 121, wherein the inhibitor comprises a BAX fragment.

139. The composition for use according to claim 121, wherein administration of the composition comprises injection.

140. A composition comprising a mitochondrial import inhibitor for ameliorating harm to cardiac function, wherein the composition is administered to a patient at risk of harm associated with mitochondrial disruption.

141. The composition of claim 140, wherein the harm associated with mitochondrial disruption is risk of harm to cardiac function.

142. 142. The composition of claim 141, wherein the patient at risk of harm to cardiac function comprises a patient who has suffered a myocardial infarction.

143. 142. The composition of claim 141, wherein the patient at risk of harm to cardiac function includes a patient suffering from cardiac arrest.

144. 142. The composition of claim 141, wherein patients at risk of harm to cardiac function include patients expected to undergo surgery.

145. 142. The composition of claim 141, wherein the patient at risk of harm to cardiac function includes a patient undergoing surgery.

146. 142. The composition of claim 141, wherein the patient at risk of harm to cardiac function includes a patient who has undergone surgery.

147. 142. The composition of claim 141, wherein the patient at risk of harm to cardiac function includes a patient receiving a chemotherapy agent.

148. 148. The composition of claim 147, wherein the chemotherapeutic agent targets cancer.

149. The composition of claim 147, wherein the chemotherapeutic agent targets a tumor.

150. The composition of claim 147, wherein the chemotherapeutic agent and the mitochondrial import inhibitor are administered simultaneously.

151. The composition of claim 147, wherein the chemotherapeutic agent and the mitochondrial import inhibitor are administered in a common composition.

152. The composition of claim 147, wherein the chemotherapeutic agent and the mitochondrial import inhibitor are not administered simultaneously.

153. 148. The composition of claim 147, wherein the chemotherapeutic agent targets cancerous cells.

154. The composition of claim 147, wherein the chemotherapeutic agent targets a tumor.

155. The composition of claim 147, wherein the chemotherapeutic agent causes myocardial cell death.

156. The chemotherapeutic agent may be an alkylating agent such as altretamine, busulfan, carboplatin, carmustine, cisplatin, cyclophosphamide, dacarbazine, ifosfamide, lomustine, melphalan, temozolomide, or trabectedin; an antimetabolite such as 5-fluorouracil, 6-mercaptopurine, azacitidine, capecitabine, clofarabine, cytarabine, floxuridine, fludarabine, gemcitabine, methotrexate, pemetrexed, pentostatin, pralatrexate, trifluridine, or tipiracil; a HER2 inhibitor such as trastuzumab, pelamocalcin, or the like; The composition of claim 147, comprising a compound selected from the list consisting of: antibodies such as tuzumab, margetuximab, immune checkpoint inhibitors, e.g., nivolumab (anti-PD-1), avelumab (anti-PD-L1), ipilimumab (anti-CTLA-4), and leratolimab (anti-LAG-3); plant alkaloids such as vincristine, vinblastine, vinorelbine, paclitaxel, docetaxel, etoposide, teniposide, irinotecan, and topotecan; and antitumor agents such as daunorubicin, doxorubicin, liposomal doxorubicin, epirubicin, idarubicin, and valrubicin.

157. 148. The composition of claim 147, wherein the chemotherapeutic agent comprises a compound selected from the list consisting of daunorubicin, doxorubicin, liposomal doxorubicin, epirubicin, idarubicin, and valrubicin.

158. 148. The composition of claim 147, wherein the chemotherapeutic agent comprises an anthracycline.

159. 157. The composition of claim 156, wherein the anthracycline comprises doxorubicin.

160. 148. The composition of claim 147, wherein the inhibitor affects BNIP3 activity.

161. The composition of claim 147, wherein the inhibitor affects at least one indication selected from the list including DOX cell death induction, mitochondrial membrane potential stabilization, prevention of mitochondrial pore opening, prevention of mitochondrial calcium ion overload, prevention of mitochondrial ROS or accumulation.

162. The composition of claim 147, wherein the inhibitor attenuates cardiotoxicity.

163. The composition of claim 147, wherein the inhibitor attenuates cardiomyocyte death.

164. The composition of claim 147, wherein the inhibitor preserves cardiac function.

165. The composition described in claim 147, wherein the inhibitor increases mitochondrial fitness.

166. The composition described in claim 147, wherein the inhibitor maintains autophagic flux.

167. The composition of claim 147, wherein the inhibitor comprises a polypeptide.

168. 148. The composition of claim 147, wherein the inhibitor comprises a BNIP3 fragment.

169. The composition described in claim 147, wherein the inhibitor comprises a BAX fragment.

170. A composition described in any one of claims 147 to 169, characterized in that administration of the composition comprises injection.

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