Treatment of heart failure

Abstract

The present invention provides a composition for the treatment or prevention of heart failure in patients with lung disease. [Solution] A composition is provided comprising isolated mitochondria or a combined mitochondrial agent, for use in the treatment or prevention of heart failure in patients with lung disease, wherein the composition is administered to the patient in a therapeutically effective dose.

Description

Technical Field

[0001] Claim of Priority This application claims the benefit of U.S. Provisional Application No. 62 / 806,473, filed Feb. 15, 2019. The entire content of the foregoing is incorporated herein by reference.

[0002] Field The present disclosure relates to the therapeutic use of mitochondria and combined mitochondrial agents.

Background Art

[0003] Mitochondria are double-membrane-bound organelles found in the cytoplasm of nucleated eukaryotic cells. They are found in almost all cells of the human body, except red blood cells. They are the major sites of the cell's energy metabolism and produce adenosine triphosphate (ATP) for various cell functions. Usually, more than 90% of the cell's demand for ATP is supplied by the cell's own mitochondria.

[0004] Mitochondria are composed of two concentric membranes with specialized functions. The inner mitochondrial membrane contains the protein ATP synthase. The outer mitochondrial membrane, which contains a number of integral membrane proteins, surrounds the entire organelle.

[0005] The structure of mitochondria is strikingly similar to that of some modern prokaryotes. In fact, mitochondria are thought to be derived from an ancient symbiosis when nucleated cells engulfed aerobic prokaryotes. In the symbiotic relationship, the host cell became dependent on the engulfed prokaryote for energy production, and the prokaryotic cell began to depend on the protective environment provided by the host cell.

[0006] [[ID=3l]] Due to the major functions of mitochondria in cell metabolism, mitochondria may be used in the treatment of various disorders, and there is also a need to utilize mitochondria for drug delivery and several other therapeutic and diagnostic purposes.

Summary of the Invention

[0007] This disclosure provides pharmaceutical compositions comprising mitochondria and methods for treating disorders using such pharmaceutical compositions. This specification further provides diagnostic and imaging methods using such pharmaceutical compositions. The described methods are at least in part based on the discovery that isolated mitochondria themselves, and isolated mitochondria bound to therapeutic agents, diagnostic agents, and / or contrast agents, can be delivered to a patient's tissues by injecting them into the patient's blood vessels. That is, direct injection or application of mitochondria to target tissue is intended by the specific methods described herein, but is not necessarily required. Rather, in some cases, the methods described herein take advantage of the discovery that mitochondria, after being injected or infused, for example into an artery, can traverse the arterial wall and be taken up by cells in the patient's tissue. The methods described herein can provide local and general distributions of mitochondria, or mitochondria together with therapeutic agents, diagnostic agents, and / or contrast agents, to tissues or cells for various therapeutic, diagnostic, and / or imaging purposes using relatively simple medical procedures.

[0008] Provided herein, among other things, are methods for treating or preventing heart failure in a subject, comprising administering a therapeutically effective amount of a composition comprising isolated mitochondria or a combined mitochondrial agent to the subject. In some embodiments, the composition is administered to the subject by intramyocardial injection. In some embodiments, the subject has or is at risk of developing heart failure—right ventricular hypertrophy (RVH), left ventricular hypertrophy (LVH), right ventricular failure (RVF), or left ventricular failure (LVF). In some embodiments, the subject has a lung disease. In some embodiments, the lung disease affects right ventricular function. In some embodiments, the composition is administered to the subject by injecting the composition into the subject's blood vessels. In some embodiments, the mitochondria are autologous. In some embodiments, the mitochondria are allogeneic. In some embodiments, the mitochondria are heterogeneous.

[0009] Provided herein, among other things, are methods for maintaining right ventricular (RV) contractility, maintaining RV capillary density, preventing RV dilation, or delaying the onset of RVF in a subject, the methods comprising administering a therapeutically effective amount of a composition comprising isolated mitochondria or a combined mitochondrial agent to the subject. In some embodiments, the composition is administered to the subject by intramyocardial injection. In some embodiments, the subject has or is at risk of developing heart failure—right ventricular hypertrophy (RVH), left ventricular hypertrophy (LVH), right ventricular failure (RVF), or left ventricular failure (LVF). In some embodiments, the subject has a lung disease. In some embodiments, the lung disease affects right ventricular function. In some embodiments, the composition is administered to the subject by injecting the composition into the subject's blood vessels. In some embodiments, the mitochondria are autologous. In some embodiments, the mitochondria are allogeneic. In some embodiments, the mitochondria are heterogeneous.

[0010] Provided herein, among other things, are methods for maintaining left ventricular (LV) contractility, maintaining LV capillary density, preventing LV dilation, or delaying the onset of left ventricular failure (LVF) in a subject, the method comprising administering to the subject a therapeutically effective amount of a composition comprising isolated mitochondria or a combined mitochondrial agent. In some embodiments, the composition is administered to the subject by intramyocardial injection. In some embodiments, the subject has or is at risk of developing heart failure—right ventricular hypertrophy (RVH), left ventricular hypertrophy (LVH), right ventricular failure (RVF), or left ventricular failure (LVF). In some embodiments, the subject has a lung disease. In some embodiments, the lung disease affects left ventricular function. In some embodiments, the composition is administered to the subject by injecting the composition into the subject's blood vessels. In some embodiments, the mitochondria are autologous. In some embodiments, the mitochondria are allogeneic. In some embodiments, the mitochondria are heterogeneous.

[0011] Provided herein, among other things, is a method for maintaining the ventricular contractility of a subject, and this method is, Identifying those who need it, and Administer a therapeutically effective dose of a composition containing isolated mitochondria or combined mitochondrial agents to the target. This includes. In some embodiments, subjects are identified by measuring end-systolic pressure volume (ESPV).

[0012] Provided herein, among other things, is a method for maintaining the density of ventricular capillaries in a given area, and this method is, Identifying those who need it, and Administer a therapeutically effective dose of a composition containing isolated mitochondria or combined mitochondrial agents to the target. Includes.

[0013] Provided herein, among other things, is a method for reducing the risk of ventricular dilation in subjects, and this method is, Identifying those who need it, and Administer a therapeutically effective dose of a composition containing isolated mitochondria or combined mitochondrial agents to the target. Includes.

[0014] In some embodiments, subjects are identified as having diabetes, obesity, hypertension, alcohol abuse, cocaine use and abuse, bacterial infection, viral infection, fungal infection, parasitic infection, exposure to toxins (e.g., lead, mercury, or cobalt), arrhythmia, or late pregnancy complications.

[0015] Provided herein, in particular, is a method for delaying the onset of heart failure in subjects, and this method is, Identifying those who need it, and Administer a therapeutically effective dose of a composition containing isolated mitochondria or combined mitochondrial agents to the target. This includes. In some embodiments, subjects are identified as having right ventricular hypertrophy or left ventricular hypertrophy.

[0016] Provided herein, among other things, are methods for treating heart failure, delaying the onset of heart failure, and reducing the risk of developing heart failure in a subject, the method comprising administering a therapeutically effective amount of a composition comprising isolated mitochondria or a combined mitochondrial agent to the subject. In some embodiments, the composition is administered to the subject by intramyocardial injection. In some embodiments, the method comprises identifying a subject as being at risk of developing heart failure. In some embodiments, the subject has a lung disease. In some embodiments, the composition is administered to the subject by injecting the composition into the blood vessels leading to the heart.

[0017] Provided herein, among other things, are methods for treating cardiac hypertrophy, delaying the onset of cardiac hypertrophy, and reducing the risk of developing cardiac hypertrophy in a subject, the method comprising administering a therapeutically effective amount of a composition comprising isolated mitochondria or a combined mitochondrial agent to the subject. In some embodiments, the composition is administered to the subject by intramyocardial injection. In some embodiments, the method comprises identifying the subject as being at risk of developing cardiac hypertrophy. In some embodiments, the subject has a lung disease. In some embodiments, the composition is administered to the subject by injecting the composition into the blood vessels leading to the heart.

[0018] In certain embodiments, the blood vessels are blood vessels or parts of the vascular system that carry blood to a target site, target organ, or target region, such as the coronary arteries of the target, the hepatic portal vein of the target, the pancreatic arteries of the target, or the prostatic arteries of the target.

[0019] In certain embodiments, mitochondria may have different sources; for example, they may be autologous, allogeneic, or heterologous. In certain embodiments, autologous mitochondria may have exogenous mtDNA. In some embodiments, mitochondria originate from a first-degree relative of the subject.

[0020] In some embodiments, the described method includes the step of collecting mitochondria isolated from cells before administration. The isolated mitochondria or combined mitochondrial agent can be administered to the subject immediately after the isolated mitochondria have been collected from the cells.

[0021] In one embodiment, the present disclosure provides a composition comprising isolated mitochondria and / or combined mitochondrial agents and a carrier. In some embodiments, the composition is a pharmaceutical composition. The carrier may be any suitable carrier, such as respiratory buffer, mitochondrial buffer, sterile mitochondrial buffer, University of Wisconsin (UW) solution, blood, serum, or contrast agent.

[0022] In all methods and / or compositions described herein, the combined mitochondrial agent may include a pharmaceutical agent. The pharmaceutical agent may be a therapeutic agent, a contrast agent, a diagnostic agent, or any combination thereof. The contrast agent may be radioactive. In some embodiments, the contrast agent is 18 The drug is either F-rhodamine 6G or iron oxide nanoparticles. In some embodiments, the drug is covalently bound to mitochondria. Alternatively, the drug is embedded in mitochondria. The combined mitochondrial agent may contain an antibody or antigen-binding fragment. Furthermore, in all methods and / or compositions described herein, the mitochondria may be autologous, allogeneic, or heterologous. In some embodiments, the mitochondria have exogenous DNA (e.g., mtDNA).

[0023] As used herein, the term “isolated mitochondria” means functional, intact mitochondria that do not contain external eukaryotic cellular material.

[0024] A “combined mitochondrial agent” is an isolated mitochondria artificially combined with a drug, diagnostic agent, contrast agent, or other agent. The agent is combined with mitochondria in any way, as long as the mitochondria and the agent are in physical contact with each other, for example, by being bound to the mitochondria (e.g., chemically or electrostatically), attached to the mitochondria, embedded in the mitochondrial membrane, substantially encapsulated within the mitochondria, or completely encapsulated by the mitochondria. The combined mitochondrial agent is designed so that the mitochondria act as a “carrier” that can deliver the agent to the patient’s tissues after injection.

[0025] The terms “subject” and “patient” are used throughout this specification to describe human or non-human animals to which treatment by the methods of this disclosure is offered. Veterinary applications are expressly anticipated by this disclosure. These terms include, but are not limited to, birds, reptiles, amphibians, and mammals, such as humans, other primates, rodents such as pigs, mice and rats, rabbits, guinea pigs, hamsters, cattle, horses, cats, dogs, sheep and goats. Preferred subjects are humans, livestock, and domesticated pets such as cats and dogs.

[0026] The term “to treat” is used herein to indicate delaying the onset of a condition, such as a disease described herein, inhibiting its effects, mitigating its effects, or extending the lifespan of a patient suffering from the condition.

[0027] "Ischemia-related diseases" are diseases involving ischemia. As used herein, ischemia is a reduction in blood flow to organs and / or tissues. This reduction in blood flow may be caused by any appropriate mechanism, including, among other things, partial or complete occlusion (obstruction), stenosis (constriction), and / or leakage / rupture of one or more blood vessels supplying blood to organs and / or tissues.

[0028] "Immediately after mitochondria are collected from the cell" means the time immediately after mitochondria are collected from the cell, before a substantial decrease in mitochondrial viability can occur.

[0029] Where used herein, the term “transplantation” is used throughout this specification as a general term to describe the process of transferring an organ, tissue, cell mass, individual cell, or organelle to a recipient. The term “cell transplantation” is used throughout this specification as a general term to describe the process of transferring at least one cell, for example, islet cells or stem cells, to a recipient. For example, such a transplant may be performed by removing beta cells (or intact islets) from a donor’s pancreas and placing them in a recipient patient whose pancreas is unable to produce sufficient insulin. These terms encompass all categories of transplantation known in the art, with the exception of blood transfusions. Transplantation is classified by site and genetic relationship between the donor and recipient. This term includes, for example, autologous transplantation (removal and transfer of cells or tissue from one site in a patient to the same or another site in the same subject), allogeneic transplantation (transplantation between members of the same species), and xenotransplantation (transplantation between members of different species).

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art. Similar or equivalent methods and materials may be used in carrying out or testing the present invention, but suitable methods and materials are described below. All publications, patent applications, patents, and other references referenced herein are incorporated by reference in their entirety. In case of any conflict, this specification, including definitions, shall prevail. Furthermore, materials, methods, and examples are illustrative and not intended to limit the scope.

[0031] Other features and advantages of the present invention will become apparent from the following detailed description and claims. [Brief explanation of the drawing]

[0032] [Figure 1] Figure 1 is a schematic diagram illustrating the method for isolating mitochondria. [Figure 2] Figure 2 is a schematic diagram showing the outcomes of diseases related to ventricular overload. [Figure 3] Figure 3 is a schematic diagram illustrating the outline of the mitochondrial transplantation method in the subjects. [Figure 4] Figure 4 is a schematic diagram illustrating an animal model study utilizing pulmonary artery banding (PAB). [Figure 5] Figure 5 is a schematic diagram showing the timeline of measurements and analyses for the experiment. [Figure 6] Figure 6 is a line graph showing the percentage changes in functional area (FAC) at baseline, 1 month after PAB, and at euthanasia for the control (C, also called the "fake"), PAB-V (vehicle), and PAB-M (mitochondrial) groups. [Figure 7] Figure 7 is a line graph showing the tricuspid annular planar contractile range of motion (TAPSE) in mm at baseline, 1 month after PAB, and at euthanasia for the control (C, also called the "fake"), PAB-V (vehicle), and PAB-M (mitochondrial) groups. [Figure 8] Figure 8 is a line graph showing the right ventricular (RV) wall thickness in cm at baseline, 1 month after PAB, and at euthanasia for the control (C, also called the "fake"), PAB-V (vehicle), and PAB-M (mitochondrial) groups. [Figure 9] Figure 9 is a box plot showing the dP / dt max (mmHg / sec) at the time of euthanasia for the control group (C, also called the "fake" group), the PAB-V (vehicle) group, and the PAB-M (mitochondrial) group. [Figure 10] Figure 10 is a line graph showing the baseline and euthanasia dP / dt max in mmHg / sec for the control (C, also called the "fake"), PAB-V (vehicle), and PAB-M (mitochondrial) groups. [Figure 11A] Figure 11A is an immunofluorescence image showing TUNEL staining to illuminate apoptotic cells (white arrows) in the TUNEL-positive control group. Cardiomyocytes are stained with desmin, and nuclei are stained with DAPI. [Figure 11B] Figure 11B is an immunofluorescence image showing TUNEL staining to illuminate apoptotic cells (white arrows) in the control / sham group. Cardiomyocytes are stained with desmin, and nuclei are stained with DAPI. [Figure 11C] Figure 11C is an immunofluorescence image showing TUNEL staining to illuminate apoptotic cells (white arrows) in the PAB-V group. Cardiomyocytes are stained with desmin, and the nuclei are stained with DAPI. [Figure 11D] Figure 11D is an immunofluorescence image showing TUNEL staining to illuminate apoptotic cells (white arrows) of the PAB-M group. Cardiomyocytes are stained with desmin, and the nuclei are stained with DAPI. [Figure 11E] Figure 11E is a bar graph showing the ratio of desmin per field of view to the number of nuclei per field of view (P<0.01**). [Figure 12A]Figure 12A is an immunofluorescence image showing CD31 staining to illuminate capillary density (white arrows) in the control / sham group. Cardiomyocytes are stained with desmin, and nuclei are stained with DAPI. [Figure 12B] Figure 12B is an immunofluorescence image showing CD31 staining to illuminate the capillary density (white arrows) of the PAB-V group. Cardiomyocytes are stained with desmin, and nuclei are stained with DAPI. [Figure 12C] Figure 12C is an immunofluorescence image showing CD31 staining to illuminate the capillary density (white arrows) of the PAB-M group. Cardiomyocytes are stained with desmin, and nuclei are stained with DAPI. [Figure 13A] Figure 13A shows electron microscopy images of the number and shape of mitochondria in the control / false group. [Figure 13B] Figure 13B is an electron microscopy image showing the number and shape of mitochondria in the PAB-V group. [Figure 13C] Figure 13C is an electron microscopy image showing the number and shape of mitochondria in the PAB-C group. [Figure 14] Figure 14 is a schematic diagram showing the disease outcomes and results associated with mitochondrial transplantation therapy. [Figure 15] Figure 15 shows immunofluorescence images of a control, RV hypertrophy (RVH), and RVH with mitochondrial transplantation. [Figure 16] Figure 16 is a box plot showing ATP levels in control cardiomyocytes, untreated hypertrophic cardiomyocytes (without H1 mitometabolism), and mitochondrial-treated hypertrophic cardiomyocytes (with H1 cardiac mitometabolism, H1 gastrocnemius mitometabolism, and H1 soleus mitometabolism). *p=0.05 vs control, #p=0.001 vs untreated hypertrophic cardiomyocytes (without H1 mitometabolism). [Figure 17A] Figure 17A is an immunofluorescence image showing TUNEL staining to illuminate apoptotic cells (white arrows) in the control / sham, PAB-V, and PAB-M groups. Cardiomyocytes are stained with desmin, and nuclei are stained with DAPI. [Figure 17B]Figure 17B is a box plot showing TUNEL-positive nuclei per 1000 nuclei (*p=0.01 vs control and #p=0.05 PAB-V vs PAB-M). [Figure 17C] Figure 17C shows microscopic examinations of representative histological sections for detecting fibrosis in the control / sham, PAB-V, and PAB-M groups. [Figure 17D] Figure 17D is a box plot showing the percentage of fibrosis per visual field at the end of the study (*p=0.01 vs. control and #p=0.05 PAB-V vs. PAB-M). [Figure 18A] Figure 18A is a box plot showing the baseline RV wall thickness (cm) for the control (C, also called "fake"), PAB-V, and PAB-M groups. [Figure 18B] Figure 18B is a box plot showing the RV wall thickness (cm) one month after PAB for the control (C, also called "false"), PAB-V, and PAB-M groups. [Figure 18C] Figure 18C is a box plot showing the RV wall thickness (cm) at the time of euthanasia for the control (C, also called "fake"), PAB-V, and PAB-M groups. [Figure 19A] Figure 19A is a box plot showing the baseline functional area change (FAC) as a percentage for the control (C, also called "fake"), PAB-V, and PAB-M groups. [Figure 19B] Figure 19B is a box plot showing the percentage of functional area change (FAC) one month after PAB for the control (C, also called "fake"), PAB-V, and PAB-M groups. [Figure 19C] Figure 19C is a box plot showing the percentage of functional area changes (FAC) in euthanasia for the control (C, also called "fake"), PAB-V, and PAB-M groups. [Figure 20A] Figure 20A is a box plot showing the baseline tricuspid annular plane contractile range of motion (TAPSE) in mm for the control (C, also called "fake"), PAB-V, and PAB-M groups. [Figure 20B] Figure 20B is a box plot showing the planar contractile range of motion (TAPSE) of the tricuspid annular valve one month after PAB for the control (C, also called "fake"), PAB-V, and PAB-M groups in mm. [Figure 20C] Figure 20C is a box plot showing the tricuspid annular planar contractile range of motion (TAPSE) in mm during euthanasia in the control (C, also called "fake"), PAB-V, and PAB-M groups. [Figure 21A] Figure 21A is a box plot showing the baseline dP / dt max (mmHg / sec) for the control (C, also called "fake"), PAB-V, and PAB-M groups. [Figure 21B] Figure 21B is a box plot showing the dP / dt max (mmHg / sec) in euthanasia for the control (C, also called "fake"), PAB-V, and PAB-M groups. [Figure 22] Figure 22 is a schematic diagram summarizing animal model studies using pulmonary artery banding (PAB) and some clinical findings. [Modes for carrying out the invention]

[0033] Detailed explanation Right ventricular hypertrophy (RVH) and failure (RVF) are major causes of cardiac morbidity and mortality affecting long-term outcomes in patients with pulmonary hypertension, outflow tract obstruction causing abnormal load on the right ventricle (RV), or where the right ventricle functions as a systemic ventricle. As an initial compensatory step, the RV adapts to these hemodynamic changes by increasing wall thickness, providing greater contractility to overcome the increased afterload. Ultimately, these mechanisms are insufficient, and the hypertrophy progresses to diastolic and systolic failure. Clinical observations indicate that these compensatory changes more effectively maintain systolic function and are preserved for longer periods on the left side than on the right side, where failure occurs more rapidly. Mitochondrial function directly affects cardiac function and contractility. The lack of adaptation of the RV to increased long-term pressure load is associated with a lack of ability of mitochondrial and calcium processing mechanisms to keep up with the demands from myocardial tissue thickening. Wasted calcium circulation, accompanied by adenosine triphosphate (ATP) consumption and electron transport chain (ETC) dysfunction, further restricts ATP synthesis and leads to bioenergy impairment (McCully JD, Rousou AJ, Parker RA, Levitsky S. Age and gender differences in mitochondrial oxygen consumption and free matrix calcium during ischemia / reperfusion and with cardioplegia and diazoxide. Ann Thorac Surg. 2007;83:1102-1109). These events eventually overwhelm cellular regulatory mechanisms, leading to a more rapid deterioration of cardiac function in RVH.

[0034] Mitochondrial enzyme activity and mitochondrial DNA (mtDNA) content play a crucial role in RV dysfunction, as they gradually decrease from hypertrophy to failure. In response to these findings, this disclosure, using a combination of matched sample microarray and proteomic analysis, demonstrates that mitochondrial function, in relation to mitochondrial quantity / mass, is as important as myocardial tissue development in the adaptation of thin-walled RVs to pathological loading. Furthermore, activation of pro-apoptotic pathways and downregulation of calcium signaling pathways, particularly those related to mitochondria, are associated with progression to failure. Initial upregulation of oxidative phosphorylation and associated calcium processing for mitochondrial stabilization corresponds to meeting the energy demands of cardiac muscle growth that adapt thin-walled RVs to pressure overload. However, prolonged exposure to increased pressure loads renders mitochondria unable to adapt, leading to rapid deterioration accompanied by reduced contractile function. Progression to heart failure is associated with a decrease in energy storage capacity, as compensatory mechanisms can no longer support the imbalance between reduced energy supply and increased demands for adaptive RV wall thickening.

[0035] The central role of mitochondria in the progression from hypertrophy to heart failure is recognized. Current treatments for patients with right heart failure are limited to therapies that primarily target lung function, rather than directly addressing the significant myocardial energy deficit resulting from mitochondrial dysfunction. Previous studies have demonstrated a successful and safe technique of therapeutic transplantation of autogenic mitochondria. This involves replacing and / or supplementing the native pool of damaged mitochondria with viable mitochondria isolated from healthy tissue. However, the success of the therapy has mainly been demonstrated in established models of acute ischemia-reperfusion injury where the beneficial effects of mitochondrial transplantation are established (McCully JD, Cowan DB, Pacak CA, Toumpoulis IK, Dayalan H, Levitsky S. Injection of isolated mitochondria during early reperfusion for cardioprotection. Am J Physiol Heart Circ Physiol. 2009;296(1):H94-105). Beyond the adaptive maintenance of mitochondrial function due to progressive pressure-overload hypertrophy, it is not well known whether long-term mitochondrial dysfunction can be achieved through mitochondrial transplantation. Furthermore, the combination of metabolic adaptive changes and mitochondrial dysfunction may indicate that the source of mitochondria for transplantation plays a crucial role. As reported, mitochondria adapt to the roles required by the tissue supplying them (Fernandez-Vizarra E, Enriquez JA, Perez-Martos A, Montoya J, Fernandez-Silva P. Tissue-specific differences in mitochondrial activity and biogenesis. Mitochondrion. 2011;11(1):207-213). Mitochondria in fast-twitch skeletal muscle are accustomed to high-glucose metabolic cells that can rapidly adapt to increasing energy demands compared to slow-twitch skeletal muscle.It is established that hypertrophied and dysfunctional cardiomyocytes switch to using glucose as a metabolic substrate (Doenst T, Nguyen TD, Abel ED. Cardiac metabolism in heart failure: Implications beyond ATP production. Circ Res. 2013;113:709-724). Therefore, the source of mitochondria used for transplantation may be relevant, as the goal is to restore the defective energy production and mitochondrial dynamics of the damaged heart. Since cardiac mitochondria are already impaired in a damaged heart, mitochondria must be harvested from other cell sources for transplantation.

[0036] This disclosure is based in part on the remarkable discovery that mitochondria can be used to prevent, treat, and / or mitigate one or more symptoms of heart failure, even before heart failure occurs. Thus, in one aspect, this disclosure provides a method for minimizing heart failure, reducing the risk of heart failure, improving at least one symptom of heart failure, and preventing or treating cellular, tissue, and / or organ damage associated with heart failure in subjects at risk of heart failure.

[0037] In some embodiments, the methods herein for treating or preventing heart failure in a subject include administering a therapeutically effective amount of a composition comprising isolated mitochondria or a combined mitochondrial agent to the subject. In some embodiments, the composition is administered to the subject by direct injection, intramyocardial injection, or infusion. In some embodiments, the subject has or is at risk of developing right ventricular hypertrophy (RVH), left ventricular hypertrophy (LVH), right ventricular failure (RVF), or left ventricular failure (LVF).

[0038] This disclosure is also based at least in part on the discovery that isolated mitochondria, and isolated mitochondria bound to therapeutic agents, diagnostic agents, and / or contrast agents, can be delivered to a patient's tissues by injecting them into the patient's blood vessels. Those skilled in the art can distribute mitochondria locally and / or generally to a patient's tissues and / or cells for various purposes using relatively simple medical procedures. Furthermore, mitochondria can be used, for example, as carriers for delivering therapeutic agents, diagnostic agents, and / or contrast agents to a patient's tissues. It should be further noted that, compared to some conventional therapeutic regimens including nanoparticles, mitochondria are non-toxic and do not cause substantially harmful immune or autoimmune responses.

[0039] While not intended to be bound by any particular theory, it is thought that injected mitochondria initially attach to the endothelium and then spill out through the capillary wall. After being injected or infused into the arteries, the mitochondria may pass through the vascular endothelium and be taken up by tissue cells through the actin-dependent internalization process of endosomes.

[0040] Combined mitochondrial agents Combined mitochondrial agents include mitochondria that are physically bound to drugs such as therapeutic agents, diagnostic agents, and / or contrast agents.

[0041] A therapeutic agent can be any drug having a therapeutic or prophylactic use. Exemplary therapeutic agents include, for example, drugs for treating ischemia-related disorders, cytotoxic drugs for treating cancer, among many others. In some cases, mitochondria can deliver therapeutic agents to specific cells, such as tumor cells. A therapeutic agent may be, for example, an intracellular inhibitor, an inactivator, a toxin, a stopping agent, and / or a cell proliferation inhibitor / cytotoxic substance that, upon entering the cell, inhibits, destroys, stops, denatures, and / or modifies the cell so that it can no longer function normally and / or survive. A therapeutic agent may be a drug for restoring proper cell function, such as a DNA vector for gene therapy. A therapeutic agent may be, for example, an inorganic or organic compound, a small molecule (less than 500 daltons) or a large molecule; a proteinaceous molecule such as a peptide, polypeptide, protein, post-translational modified protein, or antibody; or a nucleic acid molecule such as double-stranded DNA, single-stranded DNA, double-stranded RNA, single-stranded RNA, or a triple-helical nucleic acid molecule. In some embodiments, the therapeutic agent may be derived from a library of natural or synthetic molecules from any known organism (e.g., animals, plants, bacteria, fungi, protists, or viruses). In some embodiments, the therapeutic agent may be a monomer or a macromolecule. Some exemplary therapeutic agents include cytotoxic agents, DNA vectors, small interfering RNA (siRNA), microRNA (miRNA), reactive peptides, nanoparticles, microspheres, and fluorescent molecules.

[0042] Diagnostic agents are agents used for diagnosis. When mitochondria deliver the diagnostic agent to a cell, in some embodiments, the diagnostic agent can be designed to measure intracellular conditions, such as intracellular pH and oxidative stress.

[0043] Contrast agents are drugs used for contrast-enhanced imaging techniques. Techniques or modalities include, but are not limited to, X-ray, computed tomography (CT), magnetic resonance imaging (MRI), scintigraphy, fluorescence, and ultrasound. Contrast agents may be fluorescent and / or radioactive. In some embodiments, contrast agents may also be diagnostic agents. Exemplary contrast agents include MitoTracker fluorophores (Thermo Fisher Scientific Inc.), CellLight® RFP, BacMam 2.0 (Thermo Fisher Scientific Inc.), and pH-sensitive pHrodo fluorescent dyes (Thermo Fisher Scientific Inc.). 18 F-Rhodamine 6G, 18 This includes, but is not limited to, F-labeled rhodamine B, magnetic iron oxide nanoparticles, and gold-based and platinum-based nanoparticles.

[0044] As described above, a combined mitochondrial agent includes mitochondria and drugs that are in direct and / or indirect physical contact with each other. For example, a drug may bind to mitochondria, adhere to mitochondria, be embedded in the mitochondrial membrane, or be completely or partially encapsulated in mitochondria. In some cases, a drug can be covalently bound to mitochondria. In some cases, a drug is bound to components of the mitochondrial membrane directly via covalent bonds (e.g., carboxamide bonds and disulfide bonds) or indirectly via a linker (e.g., a peptide linker) or another covalently bound drug. In other examples, a drug may be noncovalently bound to mitochondria via, for example, hydrophobic interactions, van der Waals interactions, and / or electrostatic interactions.

[0045] In some embodiments, the combined mitochondrial agent may include two or more different types of agents, such as two different types of therapeutic agents, three different types of contrast agents, one therapeutic agent and one contrast agent, a therapeutic agent and a diagnostic agent, etc. Those skilled in the art will understand that any variation is possible.

[0046] One particularly useful linker for binding mitochondria to drugs is one that provides sustained drug release upon injection. This can be achieved, for example, using a hydrazone functional group. For instance, a hydrazone is formed to covalently bond a drug to components on the mitochondrial membrane. Once this combined mitochondrial agent is taken up by a cell, a change in pH hydrolyzes the hydrazone, releasing the bound drug into the cell.

[0047] In some embodiments, therapeutic agents, diagnostic agents, and / or contrast agents can be bound to the mitochondrial outer membrane using functionalized surface chemistry. In some cases, heterobifunctional chemistry can bind therapeutic agents, diagnostic agents, and / or contrast agents to the mitochondrial surface, and once internalized, these agents can be released through interactions with intercellular esterases (e.g., through interactions with acetoxymethyl esters) or through UV photoactivation or near-infrared photoactivation strategies. UV light activation and near-infrared light activation strategies are discussed, for example, in Zhou, Fang, Hanjie Wang, and Jin Chang, "Progress in the Field of Constructing Near-Infrared Light-Responsive Drug Delivery Platforms," ​​Journal of Nanoscience and Nanotechnology 16.3 (2016): 2111-2125; Bansal, Akshaya, and Yong Zhang, "Photocontrolled nanoparticle delivery systems for biomedical applications," Accounts of chemical research 47.10 (2014): 3052-3060; and Barhoumi, Aoune, Qian Liu, and Daniel S. Kohane, "Ultraviolet light-mediated drug delivery: Principles, applications, and challenges," Journal of Controlled Release 219 (2015): 31-42 ("Drug Delivery via Ultraviolet Radiation: Principles, Applications, and Challenges"). Each of these is incorporated in whole by reference.

[0048] Pharmaceuticals and other compositions This disclosure provides compositions comprising isolated mitochondria, compositions comprising combined mitochondrial agents, compositions comprising both isolated mitochondria and combined mitochondrial agents, and methods for using such compositions.

[0049] The pharmaceutical compositions described herein may comprise mitochondrial and / or combined mitochondrial agents, as well as pharmaceutically acceptable carriers. As used herein, the term “pharmaceutically acceptable carrier” includes saline, solvents, dispersion media, coatings, antimicrobial and antifungal agents, isotonic agents and absorption retarders, etc., that are compatible with pharmaceutical administration. In some embodiments, the pharmaceutically acceptable carrier is phosphate-buffered saline, saline, Krebs buffer, Tyrode's solution, contrast media, or OmniPak, or a mixture thereof. In some embodiments, a pharmaceutically acceptable carrier is sterile mitochondrial buffer (300 mM sucrose; 10 mM K+-HEPES (potassium-buffered (4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid, pH 7.2); 1 mM K+-EGTA (potassium-buffered ethylene glycol tetraacetic acid, pH 8.0)). In some embodiments, a pharmaceutically acceptable carrier is respiratory buffer (250 mM sucrose, 2 mM KH2PO4, 10 mM MgCl2, 20 mM K-HEPES buffer (pH 7.2), and 0.5 mM K-EGTA (pH 8.0)).

[0050] Pharmaceutical compositions are typically formulated to be compatible with their intended route of administration. Examples of routes of administration include parenteral administration, such as intravenous, intradermal, subcutaneous, oral (e.g., inhalation), sublingual, transdermal (e.g., topical), transmucosal, and rectal administration.

[0051] Pharmaceutical compositions can be formulated for a variety of clinical uses, such as imaging, wound treatment, injury treatment, organ preservation, improvement of mitochondrial function of organs or tissues, and skin care. In some cases, a pharmaceutically acceptable carrier is a contrast agent for imaging purposes. In some embodiments, pharmaceutical compositions may include disinfectants, antibacterial agents (e.g., antibiotics), antifungal agents, bactericides, analgesics, anesthetics, steroids, nutritional supplements, essential oils, and the like. Anesthetics are agents that can prevent pain during surgery or treatment. Exemplary analgesics include, but are not limited to, paracetamol, nonsteroidal anti-inflammatory drugs, salicylates, ibuprofen, and lidocaine. Exemplary antibacterial agents include, but are not limited to, dichlorobenzyl alcohol, amylmetacresol, and antibiotics. Exemplary antibiotics include penicillin carbapenems, cephalosporin aminoglycosides, bacitracin, gramicidin, mupirocin, chloramphenicol, thiamphenicol, lincomycin, clindamycin, macrolides, novobiocin, polymyxin, rifamycin, spectinomycin, tetracycline, vancomycin, teicoplanin, streptogramin, antifolic acid agents, sulfonamides, trimethoprim, pyrimethamine, nitrofuran, methenamine mandelate, methenamine hiprate, nitroimidazole, quinolones, fluoroquinolones, isoniazid, ethambutol, pyrazinamide, para-aminosalicylic acid, cycloserine, capreomycin, ethionamide, prothionamide, thiasetazone, and biomycin. Disinfectants are antimicrobial substances that can be applied to living tissues / skin to reduce the likelihood of infection, sepsis, or putrefaction. Exemplary disinfectants include, but are not limited to, chlorhexidine and its salts, benzalkonium and its salts, triclosan, and cetylpyridium chloride. Exemplary antifungal agents include, but are not limited to, tolnaftate, miconazole, fluconazole, clotrimazole, econazole, ketoconazole, itraconazole, terbinafine, amphotericin, nystatin, and natamycin.Exemplary steroids include, but are not limited to, prednisone acetate, prednisone valerate, prednisolone, alclomethasone dipropionate, fluocinolone acetonide, dexamethasone, methylprednisolone, desonide, pivolate, crocoltron pivolate, triamcinolone acetonide, prednicarbate, fluticasone propionate, flullandrenolide, mometasone florate, desoxymethasone, betamethasone, betamethasone dipropionate, betamethasone valerate, betamethasone propionate, betamethasone benzoate, diflorasone diacetate, fluocinonide, halcinonide, amcinonide, halobetasol propionate, and clobetasol propionate. Exemplary dietary supplements include, but are not limited to, vitamins, minerals, herbal products, and amino acids. Vitamins include, but are not limited to, vitamin A, the B vitamin family, vitamin C, the D vitamin family, vitamin E, and vitamin K. Ether oils include, but are not limited to, mint, sage, fir, lavender, basil, lemon, juniper, rosemary, eucalyptus, marigold, chamomile, and orange. Many of these agents are described, for example, in WO2008152626, which is incorporated in its entirety by reference. Compositions containing mitochondria and / or combined mitochondrial agents can be formulated in any form, e.g., liquid, semi-solid, or solid. Exemplary compositions include, among others, liquids, creams, ointments (salves), oils, emulsions, and liposomal formulations.

[0052] Method for preparing a composition containing mitochondria and / or a combined mitochondrial agent. Isolation of mitochondria Mitochondria for use in the methods described herein may be isolated or provided from any source, e.g., cultured cells or tissues. Exemplary cells include, but are not limited to, muscle tissue cells, cardiac fibroblasts, cultured cells, HeLa cells, prostate cancer cells, yeast, and any mixture thereof. Exemplary tissues include, but are not limited to, liver tissue, skeletal muscle, heart, brain, and adipose tissue, among others. Mitochondria may be isolated from autologous, allogeneic, and / or heterologous cells. In some cases, mitochondria may be isolated from genetically modified cells, e.g., cells having modified mtDNA or modified nuclear DNA.

[0053] Mitochondria can be isolated from cells or tissues by any means known to those skilled in the art. In one example, a tissue or cell sample is collected and then homogenized. Following homogenization, mitochondria are isolated by repeated centrifugation. Alternatively, the cell homogenate can be filtered through a nylon mesh filter. Typical methods for isolating mitochondria include, for example, McCully JD, Cowan DB, Pacak CA, Toumpoulis IK, Dayalan H and Levitsky S, Injection of isolated mitochondria during early reperfusion for cardioprotection, Am J Physiol 296, H94-H105. PMC2637784 (2009) (Injection of isolated mitochondria during early reperfusion for cardioprotection); Frezza, C., Cipolat, S., & Scorrano, L, Organelle isolation: functional mitochondria from mouse liver, muscle and cultured filroblasts. Nature protocols, 2(2), 287-295. This is described in (2007) (Isolation of Organelles: Functional Mitochondria from Mouse Liver, Muscle, and Cultured Fibroblasts); and in the PCT application (PCT / US2015 / 035584;WO2015192020) entitled “Products and Methods for Isolating Mitochondria,” which are incorporated by reference, respectively.

[0054] How to create a combined mitochondrial agent Those skilled in the art will understand that drugs can be conjugated to mitochondria in any number of ways, such as by attaching to mitochondria, partially or completely embedding in the mitochondrial membrane, encapsulating in mitochondria, or encapsulating within mitochondria.

[0055] While not intended to be constrained by theory or a specific approach, the mitochondrial outer membrane is considered adhesive and therefore particularly well-suited for combination with various drugs. In some embodiments, drugs can be attached to the mitochondrial outer membrane simply by incubation. For example, an effective amount of drug can be thoroughly mixed with isolated mitochondria in a buffer, such as a respiratory buffer, at a temperature favorable to the isolated mitochondria, e.g., 0°C to 26°C, 0°C to 4°C, or approximately 0°C, 4°C, 26°C. This procedure helps to attach an effective amount of drug (such as nanoparticles, DNA vectors, or RNA vectors) to mitochondria.

[0056] In some embodiments, organic cations (e.g., rhodamine and tetramethylrosamine) are readily sequestered by functioning mitochondria due to the potential on the mitochondrial membrane. A healthy mitochondrial membrane maintains a potential difference between the inside and outside of the organelle; this is called the membrane potential. This membrane potential is a direct result of mitochondrial functional processes and can be lost if the mitochondria are not functioning properly. Lipid-soluble cations are sequestered by mitochondria as a result of their positive charge and their solubility in both the inner membrane lipids and the matrix aqueous space. Similarly, in some other embodiments, anions can adhere to the outer membrane of mitochondria due to their negative charge. To bind mitochondria to these pharmaceuticals, an effective amount of the pharmaceutical should be thoroughly mixed with the isolated mitochondria in a buffer, e.g., respiratory buffer, at a temperature favorable to the isolated mitochondria, e.g., about 0°C or 4°C.

[0057] Therapeutic agents, diagnostic agents, and / or contrast agents can bind to phospholipids, peptides, or proteins on the mitochondrial membrane via chemical bonds. For example, molecules containing fluorophores (pHrodo Red (Thermo Fisher Scientific, Inc.)) or metal particles (e.g., 30 nm magnetic iron oxide nanoparticles (Sigma)) can covalently bond to exposed amine groups of proteins or peptides exposed on the intact mitochondrial outer membrane using succinimidyl ester conjugates. These reactive reagents react with unprotonated aliphatic amine groups, such as the amine terminus of proteins or the ε-amino group of lysine residues, to form stable carboxamide bonds. In another example, when a pharmaceutical product, such as MitoTracker® OrangeCMTMRos (Invitrogen, Carlsbad, California, now Thermo-Fisher Scientific, Cambridge, Massachusetts), is mixed with functional mitochondria, they are oxidized and then react with thiols on proteins and peptides on the mitochondria to form conjugates.

[0058] There are numerous reactive chemical moieties (e.g., carboxylic acid, amine functionalization, etc.) that can be used to attach therapeutic agents, diagnostic agents, and / or contrast agents to the surface of mitochondria.

[0059] Drugs can be attached to either the outer or inner mitochondrial membrane via protein binding, amine binding, or other attachment methods. Alternatively, drugs can be attached to the mitochondrial membrane via hydrophobic interactions, van der Waals interactions, and / or electrostatic interactions.

[0060] In many cases, therapeutic agents, diagnostic agents, and contrast agents can be simply mixed with isolated mitochondria and incubated in a buffer (e.g., respiratory buffer) for a sufficient amount of time (e.g., several minutes, 5 minutes, 10 minutes, or 1 hour) under favorable conditions (e.g., 0°C to 26°C, 0°C to 4°C, or approximately 0°C, 4°C, 26°C, pH 7.2–8.0).

[0061] Exemplary methods for preparing combined mitochondrial agents are described in McCully et al, Injection of isolated mitochondria during early reperfusion for cardioprotection, Am J Physiol 296, H94-H105. PMC2637784 (2009) (Injection of isolated mitochondria during early reperfusion for cardioprotection); and Masuzawa et al, Transplantation of autologously derived mitochondria protects the heart from ischemia-reperfusion injury, Am J Physiol 304, H966-982. PMC3625892 (2013) (Transplantation of autologously derived mitochondria protects the heart from ischemia-reperfusion injury). Each of the aforementioned is incorporated in its entirety by reference.

[0062] Method for preparing a composition containing mitochondria and / or a combined mitochondrial agent. Isolated mitochondria and combined mitochondrial agents can be mixed with pharmaceutically acceptable carriers to prepare pharmaceutical compositions. Pharmaceutically acceptable carriers include any compounds or compositions useful for promoting the storage, stability, administration, cell targeting, and / or delivery of mitochondria and / or combined mitochondrial agents, including, but not limited to, suitable vehicles, diluents, solvents, excipients, pH modifiers, salts, colorants, rheological modifiers, lubricants, coatings, fillers, defoamers, polymers, hydrogels, surfactants, emulsifiers, adjuvants, preservatives, phospholipids, fatty acids, monoglycerides, diglycerides and triglycerides and their derivatives, waxes, oils, and water. In some embodiments, isolated mitochondria and / or combined mitochondrial agents are suspended in water, saline, buffer, respiratory buffer, or sterile mitochondrial buffer for in vivo delivery. The compositions described herein may include, but are not limited to, physiological saline, phosphate buffer, phosphate-buffered saline (PBS), or respiratory buffer, as pharmaceutically acceptable salts, buffers, or buffer systems. Vehicles capable of facilitating in vivo delivery to cells, such as liposomes, can be used to facilitate the delivery of the combined mitochondrial agents to target cells.

[0063] Methods for preparing compositions, such as liquid, semi-solid, and solid compositions (e.g., liquids, creams, lotions, ointments, oils, etc.), are well known in the art. Those skilled in the art will understand that such known methods can be modified to add mitochondria and / or combined mitochondrial agents, thereby adding one or more steps to form the compositions described herein. Those skilled in the art will understand that, in some cases, the compositions described herein may include multiple types of combined mitochondrial agents. For example, this includes compositions containing mitochondria, where each mitochondria is essentially associated with multiple types of agents. It also includes compositions containing mitochondria, where each mitochondria is paired with only one type of agent, but the composition contains a mixture of mitochondrial / agent pairs.

[0064] Treatment of cardiovascular disease The heart is a highly energetic organ that requires a continuous supply of oxygen to maintain normal function. Under aerobic conditions, the heart primarily draws energy from mitochondria, which make up 30% of the volume of cardiomyocytes. Following the onset of ischemia, high-energy phosphate levels rapidly decrease, accompanied by changes in mitochondrial structure, volume, oxygen consumption, and ATP synthesis.

[0065] This disclosure provides methods for treating or preventing cardiovascular diseases (e.g., heart failure). Cardiovascular diseases refer to a class of diseases involving the heart or blood vessels. Cardiovascular diseases include, for example, coronary artery disease (CAD), such as angina and myocardial infarction (commonly known as heart attack), stroke, heart failure, hypertensive heart disease, rheumatic heart disease, cardiomyopathy, cardiac arrhythmias, congenital heart disease, valvular heart disease, carditis, aortic aneurysm, peripheral artery disease, thromboembolic disease, and venous thrombosis.

[0066] Heart failure, also known as chronic heart failure, refers to a condition in which the heart is unable to maintain sufficient blood flow to meet the body's needs. Signs and symptoms of heart failure typically include shortness of breath, excessive fatigue, and swelling of the lower extremities. Limited exercise capacity is also common in patients with heart failure. In this context, "to treat" means to improve at least one symptom of the disorder associated with the disease. Often, treatment improves blood supply and alleviates one or more symptoms (such as shortness of breath, excessive fatigue, and swelling of the lower extremities).

[0067] Generally, this method involves administering the compositions described herein (for example, compositions comprising isolated mitochondria or compositions comprising combined mitochondrial agents) to subjects who are in need of, or who have been determined to need, such treatment.

[0068] In some aspects, the methods described herein can also be used to maintain ventricular contractility (e.g., right ventricular (RV) contractility), maintain capillary density (e.g., RV capillary density), prevent ventricular dilation (e.g., RV dilation), delay the onset of heart failure (e.g., RVF), or reduce the risk of developing cardiovascular disease (e.g., heart failure).

[0069] This disclosure provides methods for minimizing heart failure, reducing the risk of heart failure, improving at least one symptom of heart failure, and preventing or treating cellular damage, tissue damage, and / or organ damage associated with heart failure in subjects at risk of heart failure.

[0070] As used herein, the term “at risk of heart failure” means having a higher risk of heart failure compared to the average person in the population (e.g., within the same age group). In some embodiments, the risk is about or at least 50%, 60%, 70%, 80%, 90%, or 100% higher than the average person in the population. In some embodiments, the risk is about or at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 times higher than the average person in the population. In some embodiments, the age group is at least 40, 50, 60, 70, or 80 years old.

[0071] This increased risk of heart failure may be due to a variety of factors, such as genetic factors (e.g., genetic mutation), environmental factors (e.g., occupational risk, pollution), various diseases, medical procedures (e.g., surgery, organ / tissue transplantation), and behaviors (e.g., smoking, inactivity). Once a subject is identified as being at risk of heart failure, a therapeutically effective dose of the composition described herein may be administered to the subject to reduce the risk of heart failure. Risk may also arise from potential medical procedures. As used herein, the term “medical procedure” refers to a set of actions aimed at achieving an outcome in the provision of healthcare. Medical procedures may include, for example, diagnostic procedures, therapeutic procedures, and surgical procedures. Some medical procedures include, for example, extracorporeal membrane oxygenation (ECMO), chemotherapy, radiotherapy, tracheal intubation, gene therapy, anesthesia, resection, amputation, cardiopulmonary resuscitation (CPR), cryosurgery, endoscopic surgery, unilateral laminectomy, image-guided surgery, knee cartilage replacement therapy, laminectomy, laparoscopic surgery, lithotomy, lobotomy, neovaginoplasty, radiosurgery, stereotactic surgery, vaginoplasty, transplantation (e.g., tissue or organ transplantation), and xenograft. Healthcare providers can determine whether medical procedures and behaviors (e.g., smoking) may increase the risk of heart failure. Many risk factors are known in the art, including hypertension, myocardial infarction, abnormal heart valves, cardiomyopathy, family history of heart disease, and diabetes. In these cases, to minimize risk, therapeutically effective doses of the compositions described herein may be administered to the target prior to these procedures.

[0072] In some embodiments, the methods described herein can be used to treat or prevent heart failure in a subject. In some embodiments, the subject has or is at risk of developing right ventricular hypertrophy (RVH), left ventricular hypertrophy (LVH), right ventricular failure (RVF), or left ventricular failure (LVF).

[0073] Right ventricular hypertrophy (RVH) is a condition defined by abnormal hypertrophy of the myocardium surrounding the right ventricle. RVH usually results from chronic lung disease or a structural defect in the heart. One of the most common causes of RVH is pulmonary hypertension (PH). Pulmonary hypertension is characterized by increased blood pressure in the blood vessels that supply blood to the lungs. Pulmonary hypertension can lead to increased pulmonary artery pressure. The right ventricle attempts to compensate for this increased pressure by changing its shape and size. Hypertrophy of individual muscle cells results in an increase in the thickness of the right ventricular wall. Common causes of pulmonary hypertension include chronic obstructive pulmonary disease (COPD), pulmonary embolism, and other restrictive lung diseases. RVH often develops as a result of these disorders. RVH also develops in response to structural defects in the heart. One common cause is tricuspid regurgitation. Tricuspid regurgitation is a disorder in which the tricuspid valve does not close properly, allowing blood to flow backward. Other structural defects that can lead to RVH include Tetralogy of Fallot, ventricular septal defect, pulmonary valve stenosis, and atrial septal defect. RVH is also associated with abdominal obesity, elevated fasting blood glucose levels, elevated systolic blood pressure, and partial shortening of the left ventricular wall. Other risk factors for RVH include smoking, sleep apnea, and strenuous activity.

[0074] Accordingly, in one embodiment, the present disclosure provides a method for reducing the risk of developing right ventricular hypertrophy. The method involves identifying a subject as being at risk of developing right ventricular hypertrophy and administering the composition described herein to the subject. In some embodiments, the method involves identifying a subject as having, for example, pulmonary hypertension, COPD, pulmonary embolism, restrictive lung disease, tricuspid regurgitation, Tetralogy of Fallot, ventricular septal defect, pulmonary stenosis, atrial septal defect, abdominal obesity, elevated fasting blood glucose levels, elevated systolic blood pressure, and / or partial shortening of the left ventricular wall.

[0075] Left ventricular hypertrophy (LVH) is a thickening of the myocardium in the left ventricle of the heart. LVH itself is not a disease, but it is usually a marker of heart-related disorders. Disease processes that can cause LVH include any disease that increases the afterload that the heart must contract, as well as several major diseases of the heart muscle. Causes of increased afterload that can lead to LVH include aortic stenosis, aortic regurgitation, and hypertension. The primary heart muscle disease that causes LVH is known as hypertrophic cardiomyopathy, which can lead to heart failure. Long-standing mitral regurgitation can also lead to LVH as a compensatory mechanism.

[0076] Accordingly, in one embodiment, the present disclosure provides a method for reducing the risk of developing left ventricular hypertrophy. This method involves identifying a subject as being at risk of developing left ventricular hypertrophy and administering the composition described herein to the subject. In some embodiments, the method includes identifying a subject as having, for example, aortic stenosis, aortic regurgitation, hypertension, hypertrophic cardiomyopathy, and / or mitral regurgitation.

[0077] Heart failure (HF), also known as congestive heart failure, is a condition in which the heart is unable to pump enough blood to maintain blood flow and meet the body's needs. Signs and symptoms of heart failure typically include shortness of breath, excessive fatigue, and swelling of the lower extremities. Limited exercise capacity is also a common feature. Common causes of heart failure include a previous myocardial infarction (heart attack), hypertension, atrial fibrillation, valvular heart disease, excessive alcohol use, infections, and coronary artery disease such as cardiomyopathy. The left side of the heart receives oxygen-rich blood from the lungs and pumps it into the systemic circulation (the rest of the body excluding the pulmonary circulation). When the left side of the heart stops functioning, blood flows back into the lungs (congestion), causing respiratory symptoms and fatigue due to insufficient oxygenated blood supply. Right-sided heart failure is often caused by cor pulmonal disease, which is usually due to difficulties in the pulmonary circulation, such as pulmonary hypertension or pulmonary valve stenosis.

[0078] Accordingly, in one aspect, the present disclosure provides a method for reducing the risk of developing heart failure. This method includes identifying a subject as being at risk of developing heart failure and administering the composition described herein to the subject. In some embodiments, the subject has LVH or RVH and is therefore at high risk of developing heart failure. In another aspect, the method described herein may also be used to treat or reduce the risk of developing pulmonary heart disease or lung disorders (e.g., chronic obstructive pulmonary disease, chronic bronchitis, emphysema, cystic fibrosis, pleural effusion, or bronchiectasis).

[0079] Methods for diagnosing cardiovascular disorders are known in the art. One primary method for diagnosing cardiovascular disorders is echocardiography, which can measure the thickness of the heart muscle. For example, electrocardiograms (ECGs) are often used to show signs of increased voltage from the heart in patients with LVH.

[0080] This disclosure also provides methods for treating ischemic heart disease and other ischemia-related diseases. Attempts to reduce myocardial tissue necrosis and improve post-ischemic function using pharmacological and / or exogenous substrate interventions alone or in combination with procedural techniques have provided only limited cardioprotection. Despite these interventions, mitochondrial damage and dysfunction continue to represent a major problem after myocardial ischemia and remain a significant cause of morbidity and mortality. Mitochondrial damage occurs primarily during ischemia rather than during reperfusion, and maintaining mitochondrial respiratory function promotes recovery of contractility and reduces the size of myocardial infarctions.

[0081] The methods described herein can be used to treat ischemic heart disease. For example, an effective amount of isolated mitochondria can be injected into the target blood vessel, e.g., the target coronary vascular system. For example, about 1 × 10⁻⁶ 7Mitochondria can be administered to the target coronary vascular system. The injected mitochondria are internalized by cardiomyocytes after transplantation, increasing oxygen consumption, upregulating chemokines that enhance post-infarction cardiac function, and upregulating the expression of protein pathways crucial for maintaining myocardial energy. Alternatively, an effective dose of mitochondria can be directly injected into the area at risk (local ischemic area). The injection can be repeated several times in different locations within the heart.

[0082] Reperfusion injury is tissue damage caused by the blood supply when blood returns to tissues after a period of ischemia or oxygen deprivation. When oxygen and nutrients are deficient during the ischemic period, inflammation and oxidative damage occur when blood flow is restored. The inflammatory response further leads to tissue reperfusion injury. Therefore, in some cases, treatment may include administering immunosuppressants to the patient. Immunosuppressants can be administered separately, for example, or as concurrent therapy with mitochondrial agents. Alternatively, immunosuppressants can be bound to mitochondria to form combined mitochondrial agents that can be used for treatment. Bisphosphonates are particularly useful immunosuppressants.

[0083] Ischemia / reperfusion injury in several other organs is often associated with mitochondrial damage or dysfunction. These organs include, but are not limited to, the lungs, kidneys, liver, skeletal muscle, and brain. These injuries or diseases include, but are not limited to, ischemic colitis, mesenteric ischemia, cerebral ischemia, stroke, acute limb ischemia, cyanide, and gangrenous malformations. The methods described may also be used to treat ischemic injury in these organs / tissues. For these treatments, isolated mitochondria and / or combined mitochondrial agents may be injected directly into organ tissue or into blood vessels carrying blood to the target organ / tissue or site of injury.

[0084] Heart surgery Isolated mitochondria and / or combined mitochondrial agents can be delivered to the heart to reduce the threat, allow the heart to escape surgical intervention (e.g., cardiac arrest), and enable cardiac recovery without increasing cardiac rate or oxygen demand. In some embodiments, the method involves direct injection of isolated mitochondria and / or combined mitochondrial agents into the heart. In some methods, isolated mitochondria and / or combined mitochondrial agents are injected into the coronary arteries.

[0085] Image creation In many cases, mitochondria can be made to adhere to a contrast agent by co-incubating them with the contrast agent. Examples of such contrast agents include ThermoFisher Scientific Inc.'s MitoTracker and pHrodo fluorophores. 18 This includes, but is not limited to, F-rhodamine 6G and iron oxide nanoparticles.

[0086] Combined mitochondrial agents containing contrast agents can be injected into tissues, such as cardiac tissue, or perfused through blood vessels. Tissues containing labeled mitochondria can be examined using imaging techniques such as positron emission tomography (PET), microcomputed tomography (μCT), magnetic resonance imaging (MRI), bright-field microscopy, and 3D super-resolution microscopy. Those skilled in the art will understand that other imaging techniques or modalities may be used, including, but not limited to, X-ray, scintigraphy, fluorescence, and ultrasound.

[0087] Administration Isolated mitochondria and combined mitochondrial agents can be administered to patients by intravenous, intra-arterial, intraperitoneal, intramuscular injection, and / or intraosseous injection. In some embodiments, isolated mitochondria and combined mitochondrial agents may be delivered by direct injection or intravascular injection.

[0088] When mitochondria are injected into tissue, they are taken up by cells around the injection site. Therefore, in some embodiments, the injection site is the target site. In some other embodiments, mitochondria are injected into blood vessels that carry blood to the target site, e.g., an organ, tissue, or injury site. While not intended to be bound by any theory, evidence suggests that mitochondria delivered by direct injection are internalized by cells via actin-dependent endocytosis. However, mitochondrial uptake by vascular delivery appears to be more complex. The rapid and widespread uptake of mitochondria when delivered by vascular injection suggests the involvement of mechanisms that allow mitochondria to rapidly pass through the blood vessel wall. Some studies support the idea that cells can routinely escape circulation. Certain cardiac and mesenchymal stem cells appear to be actively expelled from the vascular system through processes distinct from leakage (Cheng, K., Shen, D., Xie, Y., Cingolani, E., Malliaras, K., Marban, E., 2012, Brief report: Mechanism of extravasation of infused stem cells. Stem Cells. 30, 2835-2842; Allen, TA, Gracieux, D., Talib, M., Tokarz, DA, Hensley, MT, Cores, J., Vandergriff, A., Tang, J., de Andrade, JB, Dinh, PU, ​​Yoder, JA, Cheng, K., 2017. Angiopellosis as an Alternative Mechanism of Cell Extravasation. Stem Cells.). 35,170-180 (Angioperosis as an alternative mechanism to cell overflow). Stem cell migration across the vascular wall requires large-scale endothelial remodeling.Mitochondria may use similar remodeling mechanisms to pass through blood vessel walls. Another possible mechanism for mitochondrial uptake might be something like leakage. Some cells routinely escape from circulation. For example, leukocyte spillage (i.e., leakage) between venous endothelial cells is a well-understood process involving cell adhesion proteins. Furthermore, injected mitochondria may spill through the spaces between endothelial cells and up the capillary wall. After mitochondria have passed through the vascular endothelium, they are taken up by tissue cells through an actin-dependent internalization process of endosomes.

[0089] Mitochondria or combined mitochondrial agents may be administered to a patient as a single treatment, or as a series of treatments, for example, approximately 1, 2, 5, 8, 10, 20, 30, 50, or 60 days, 1 year, indefinitely, or intermittently or continuously until the physician determines that administration of mitochondria or combined mitochondrial agents is no longer necessary.

[0090] In one method of administration, mitochondria or combined mitochondrial agents are injected directly into organ tissue, such as heart tissue. The injection may, in some cases, be repeated several times at different sites within the organ. In such a method, a sterile 1 ml insulin syringe with a small needle (e.g., 28 gauge) can be used for injection, with each injection site being, for example, approximately 1.2 × 10⁻⁶. 6 It can receive mitochondria.

[0091] Those skilled in the art will understand that the amount of mitochondria and / or combined mitochondrial agents to be administered to a patient, such as a composition containing mitochondria and / or combined mitochondrial agents, varies depending, for example, the type of disorder being treated, in particular the route of administration, the duration of treatment, the size of the area being treated, and / or the location of the treatment site in the patent. Those skilled in the art can determine the dosage to be administered depending on these and other variables. For example, a total of about 1 × 10 7The mitochondria can be administered to the blood vessels of a subject, for example, to treat focal ischemia of the myocardium. As another example, in the case of a larger organ or affected area, more mitochondria, for example, from 1×10 10 to 1×10 14 mitochondria can be injected into the blood vessels. Conversely, in the case of a small focal lesion, from 1x10 3 to 1x10 6 mitochondria can be injected into the patient. Thus, the effective amount of mitochondria or a combined mitochondrial agent (or a composition containing the same) is the total amount of mitochondria or a combined mitochondrial agent sufficient to bring about the desired therapeutic effect. The effective amount is, for example, at least or about 1×10 2 mitochondria, or a combined mitochondrial agent, for example, from about 1×10 3 to about 1×10 14 、about 1×10 4 to about 1×10 13 、about 1×10 5 to about 1x10 12 、about 1x10 6 to about 1x10 11 、about 1x10 7 to about 1x10 10 、about 1x10 3 to about 1x10 7 、about 1x10 4 to about 1x10 6 、about 1x10 7 to about 1x10 14 、or about 1x10 8 to about 1x10 13 、about 1x10 9 [[ID=​​​​​​​​​​​​​​​​​​​​​​​​​, 1x10 13 , or at least or about 1x10 14 , or for example, 1 × 10 14 The amount may exceed this. As used herein, the term “total amount” in the context of administration to a patient may refer to the total amount of mitochondria or combined mitochondrial agents in a single dose (e.g., one injection, one dose administered in an infusion) or multiple doses (e.g., multiple injections), depending on the dosing plan being implemented.

[0092] Isolated mitochondria and / or combined mitochondrial agents can be administered to subjects every 12 to 24 hours via various routes, e.g., direct injection, intravenous delivery. In some embodiments, isolated mitochondria or combined mitochondrial agents can be administered to subjects every 5 to 10 minutes (e.g., every 5 minutes, every 10 minutes) via various routes, e.g., direct injection, intravenous infusion.

[0093] For the treatment of cardiovascular or pulmonary diseases, isolated mitochondria and / or combined mitochondrial agents can be administered to various blood vessels, including, for example, the aorta, vena cava (e.g., superior or inferior vena cava), coronary veins, circumflex arteries, left coronary artery, left anterior descending branch, pulmonary veins, right coronary artery, pulmonary veins, or pulmonary arteries.

[0094] Isolated mitochondria and / or combined mitochondrial agents may be administered to subjects at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, or 30 days or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months, or at least about 1, 2, 3, 4, or 5 years prior to the onset of right ventricular hypertrophy (RVH), left ventricular hypertrophy (LVH), right ventricular failure (RVF), or left ventricular failure (LVF). In some embodiments, isolated mitochondria and / or combined mitochondrial agents may be administered to subjects within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, or 30 days, or within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 24 months, or within approximately 1, 2, 3, 4, or 5 years, after the subject is identified as being at risk of developing a cardiovascular disease that may lead to heart failure (e.g., obesity, right ventricular hypertrophy, etc.).

[0095] In some embodiments, isolated mitochondria and / or combined mitochondrial agents may be administered to a subject within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, or 30 days, or within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 24 months, or within approximately 1, 2, 3, 4, or 5 years, after the subject is identified as having some other disorder that may lead to cardiovascular disease or heart failure (e.g., obesity, right ventricular hypertrophy, etc.).

[0096] In some embodiments, isolated mitochondria and / or combined mitochondrial agents may be administered to subjects at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, or 30 days, or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months, or at least about 1, 2, 3, 4, or 5 years, after the onset and / or diagnosis of right ventricular hypertrophy (RVH), left ventricular hypertrophy (LVH), right ventricular failure (RVF), or left ventricular failure (LVF).

[0097] In some embodiments, isolated mitochondria or combined mitochondrial agents may be injected directly into tissues or organs by needles with gauges 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, and 34. In some other embodiments, isolated mitochondria or combined mitochondrial agents may be delivered to a target site by catheter.

[0098] In some cases, mitochondria are newly isolated and viable. Mitochondria or combined mitochondrial agents may be administered to the subject within approximately 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, or 120 minutes after mitochondria are isolated. In some cases, mitochondria or combined mitochondrial agents are administered to the subject within approximately 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, or 120 minutes after the start of the mitochondrial isolation process. Mitochondria and / or combined mitochondrial agents may, in some cases, be stored for a short period before use (e.g., about or at least 10 minutes, about or at least 20 minutes, about or at least 30 minutes, about or at least 40 minutes, about or at least 50 minutes, about or at least 60 minutes, about or at least 1 hour, about or at least 2 hours, about or at least 3 hours, about or at least 4 hours, or about or at least 24 hours).

[0099] Mitochondria for therapeutic purposes can be isolated from cells or tissues of autologous, allogeneic, and heterogeneous sources. In some cases, mitochondria are collected from cultured cells or tissues of a target, and these mitochondria are administered to the same target. In some other cases, mitochondria are collected from cultured cells or tissues of a second target, and these mitochondria are administered to the first target. In some cases, mitochondria are collected from cultured cells or tissues of different species (e.g., mouse, pig, yeast). [Examples]

[0100] The present invention is further described in the following examples, which do not limit the scope of the invention as described in the claims.

[0101] Example 1: Exemplary method for isolating mitochondria from tissue samples or cultured cells preparation The following solutions can be prepared to isolate intact, viable, and respiratoryly capable mitochondria. To successfully isolate mitochondria using the method of the present invention, the solutions and tissue samples are kept on ice to maintain mitochondrial viability. Even when maintained on ice, isolated mitochondria will show a decline in functional activity over time (Olson et al., J Biol Chem 242:325-332, 1967). These solutions are prepared in advance, if possible.

[0102] 1M K-HEPES stock solution (pH adjusted to 7.2 with KOH). 0.5 MK-EGTA stock solution (pH adjusted to 8.0 with KOH). 1M KH2PO4 stock solution. 1M MgCl2 stock solution.

[0103] Homogenized buffer (pH 7.2): 300 mM sucrose, 10 mM K-HEPES, and 1 mM K-EGTA. The buffer can be stored at 4°C. Respiratory buffer: 250 mM sucrose, 2 mM KH2PO4, 10 mM MgCl2, 20 mM K-HEPES buffer (pH 7.2), and 0.5 mM K-EGTA (pH 8.0). The buffer can be stored at 4°C. 10XPBS stock solution: Dissolve 80g of NaCl, 2g of KCl, 14.4g of Na2HPO4, and 2.4g of KH2PO4 in 1L of redistilled H2O (pH 7.4). 1XPBS is prepared by pipetting 100 mL of 10XPBS into 1 L of redistilled H2O. Subtilisin A stock is prepared by weighing 4 mg of subtilisin A into a 1.5 mL microfuse tube. The stock can be stored at -20°C until use. BSA stock is prepared by weighing 20 mg of BSA into a 1.5 mL microfuse tube. The stock can be stored at -20°C until use.

[0104] Isolating mitochondria from tissue Figure 1 shows an overview of the procedure for isolating mitochondria using tissue dissociation and differential filtration. Two 6 mm biopsy sample punches are transferred to 5 mL of homogenization buffer in a dissociation C tube, and the samples are homogenized using the 1-minute homogenization program of the tissue dissociation apparatus (A). Subtilisin A stock solution (250 μL) is added to the homogenate in the dissociation C tube, and incubated on ice for 10 minutes (B). The homogenized material is centrifuged at 750 x G for 4 minutes (as an optional step). The homogenate is filtered on ice in a 50 mL conical centrifuge tube through a pre-moistened 40 μm mesh filter, and then 250 μL of BSA stock solution is added to the filtrate (C). The filtrate is re-filtered on ice in a 50 mL conical centrifuge through a new pre-moistened 40 μm mesh filter (D). The filtrate is refiltered on ice through a new, pre-moistened 10 μm mesh filter in a 50 mL conical centrifuge tube (E). The filtrate is refiltered on ice through a new, pre-moistened 6 μm mesh filter in a 50 mL conical centrifuge tube. The resulting filtrate can be used immediately or concentrated by centrifugation. For concentration, the filtrate is transferred to a 1.5 mL microfuse tube and centrifuged at 9000 x g for 10 minutes at 4°C (F). The supernatant is removed, the pellet containing mitochondria is resuspended and placed in 1 mL of respiratory buffer (G).

[0105] Immediately before isolation, dissolve subtilisin A in 1 mL of homogenization buffer. Immediately before isolation, dissolve BSA in 1 mL of homogenization buffer. Collect two fresh tissue samples using a 6 mm biopsy sample punch and store them on ice in 1 XPBS in a 50 mL conical centrifuge tube. Transfer the two 6 mm punches of tissue to a dissociation C tube containing 5 mL of ice-cold homogenization buffer. Homogenize the tissue by mounting the dissociation C tube in a tissue dissociation device and selecting a pre-configured mitochondrial isolation cycle (60 seconds of homogenization).

[0106] Remove the dissociation C tube and transfer it to an ice bucket. Add subtilisin A stock solution (250 μL) to the homogenate, mix by inverting, and incubate the homogenate on ice for 10 minutes. Place a 40 μm mesh filter on ice in a 50 mL conical centrifuge tube, pre-moisten the filter with homogenization buffer, and filter the homogenate into the 50 mL conical centrifuge tube on ice.

[0107] Add the freshly prepared BSA stock solution (250 μL) to the filtrate and mix by inversion. (This step is omitted if mitochondrial protein measurement is required.) Place a 40 μm mesh filter in a 50 mL conical centrifuge tube on ice, pre-moisten the filter with homogenizing buffer, and filter the homogenate into a 50 mL conical centrifuge tube on ice. Place a 10 μm filter in a 50 mL conical centrifuge tube on ice, pre-moisten the filter with homogenizing buffer, and filter the homogenate into a 50 mL conical centrifuge tube on ice. Transfer the filtrate to two pre-cooled 1.5 mL microfuse tubes and centrifuge at 9000 xg for 10 minutes at 4°C. Remove the supernatant, resuspend the pellet, and add it to 1 mL of ice-cold breathing buffer. Mitochondria isolated from tissue are either used immediately for injection or to prepare combined mitochondrial agents.

[0108] Isolating mitochondria from cultured cells Mitochondria can be isolated from cultured cells. The procedure is essentially the same as that for isolating mitochondria from tissue samples, except that cultured cells, such as human fibroblasts, are used instead of biopsy samples.

[0109] Number of mitochondria The number of viable mitochondria is measured by labeling aliquots (10 μl) of isolated mitochondria with MitoTracker Orange CMTMRos or MitoTracker Red CMXRos (5 μmol / l; Invitrogen, Carlsbad, California, now Thermo-Fisher Scientific, Cambridge, Massachusetts). Aliquots of labeled mitochondria are spotted onto a slide and counted using a rotating disk confocal microscope with a 63x C-apochromatic objective lens (1.2 W Korr / 0.17 NA, Zeiss). Mitochondria are counterstained with the mitochondrial-specific dye MitoFluor Green or MitoTrackerDeep Red FM (Invitrogen, Carlsbad, California, now Thermo-Fisher Scientific, Cambridge, Massachusetts). Unstained cells or tissues are used to select appropriate wavelengths for measuring autofluorescence and background fluorescence. Briefly, 1 μl of labeled mitochondria is placed on a microscope slide and covered. Mitochondrial counts are measured using MetaMorph Imaging Analysis software at low magnification (×10) covering the entire sample area.

[0110] Example 2: Exemplary method for preparing combined mitochondrial agents Mitochondria and 18 Combine F-rhodamine 6G using electrical potential. 18F-Rhodamine 6G (40-100 μCi in a 20 μl volume) was diluted to 1.0 mL at 4°C with mitochondrial isolation solution A (homogenization buffer: 300 mM sucrose, 10 mM K-HEPES, and 1 mM K-EGTA, pH 7.2), and then the mitochondria isolated with mitochondrial isolation solution A (0.5 ml, 1 x 10⁶) were collected. 7 -1x10 8 Mix thoroughly with (including). In the mixture, 18 F-rhodamine 6G is electrophoretically distributed into the mitochondrial matrix via the inner mitochondrial membrane in response to electrical potential and thus sequestered by functional mitochondria. The mixture is incubated on ice for 10–30 minutes. The mixture is washed three times by centrifugation at 9,000 rpm (10,000 g) for 10 minutes, and the pellet is resuspended in mitochondrial isolation solution A each time. After the final wash, the pellet is resuspended in respiratory buffer.

[0111] The mitochondrial outer membrane combines mitochondria with iron oxide nanoparticles. Iron oxide nanoparticles containing succinimidyl ester (10 mg) were suspended in a respiratory buffer at 4°C, and isolated mitochondria (1 x 10⁶) were extracted. 7 ~1x10 8 Mix thoroughly with 1.0 ml of [the substance]. Iron oxide binds to the mitochondrial amine groups of the outer mitochondrial membrane by the succinimidyl esteramine reaction. Incubate the mixture on ice for 10-30 minutes. Wash the mixture three times by centrifugation at 9,000 rpm (10,000 g) for 10 minutes, and resuspend the pellet in mitochondrial isolation solution A each time. After the final wash, resuspend the pellet in respiratory buffer.

[0112] Combining mitochondria with two pharmaceuticals 18 F-Rhodamine 6G (40-100 μCi in a 20 μl volume) and iron oxide nanoparticles containing succinimimidyl ester (10 mg) were combined and diluted to a volume of 1.0 mL at 4°C with mitochondrial isolation solution A. Then, isolated mitochondria (0.5 ml, 1 x 10⁶) were added to the mitochondrial isolation solution.7 -1x10 8 Mix thoroughly with (including). Incubate the mixture on ice for 10-30 minutes. Wash the mixture three times by centrifugation at 9,000 rpm (10,000 g) for 10 minutes, and resuspend the pellet in mitochondrial isolation solution A each time. After the final wash, resuspend the pellet in respiratory buffer.

[0113] Combining mitochondria via thiols MitoTracker® fluorophore (5 μmol / l; Invitrogen, Carlsbad, California, now Thermo-Fisher Scientific, Cambridge, Massachusetts) is mixed with isolated mitochondria (1.0 mL) in respiratory buffer. When the probe is mixed with functional mitochondria, they are oxidized and then react with thiols on the proteins and peptides on the mitochondria to form conjugates. The mixture is incubated on ice for 10 minutes and then in the dark at 4°C. The mixture is washed three times by centrifugation at 9,000 rpm (10,000 g) for 10 minutes, and the pellet is resuspended in mitochondrial isolation solution A each time. After the final wash, the pellet is resuspended in respiratory buffer.

[0114] Example 3: Prevention of heart failure in hypertrophy due to pressure overload by transplantation of autologous mitochondria. the purpose: A key event in the progression from right ventricular hypertrophy (RVH) to right ventricular failure (RVF) is apoptosis of cardiomyocytes due to mitochondrial dysfunction. Since transplantation of respiratoryable mitochondria is available, the aim of these experiments was to determine whether injection of autologous mitochondria could prevent heart failure.

[0115] method: The RVH / RVF model was created by ligating (banding) the pulmonary artery at 50% (gradient = 15–20 mmHg) in immature piglets (n=6 / group). Sham surgery animals served as controls (Ctr). Animals were followed for 8 weeks by echocardiography (M-mode, RV free wall thickness measured by TAPSE). Four weeks after surgery, ligated animals were treated with either mitochondria isolated from the piglet's own calf muscle (PAB-mito) or a vehicle (PAB-V) injected into the free wall of the RV. At euthanasia, tissue was histologically analyzed to measure cardiomyocyte hypertrophy, fibrosis (H&E, Masson's trichrome, desmin), and apoptosis by TUNEL. Invasive PV loop measurements (Ved, Dp / Dt max, Pdev) were obtained at baseline and at euthanasia.

[0116] result: All animals survived to the end of the study. One month after surgery, ligated animals showed signs of hypertrophy with a significantly thicker RV free wall compared to Ctr (0.27±0.03 cm vs. 0.4±0.02 cm; P<0.01; Figure 8). RV wall thickness further increased to the end of the study in PAB-mites, although the PAB-V heart was already severely dilated (0.5±0.04 cm vs. 0.28±0.08 cm; P<0.01; Figure 8). Total cardiac weight (Ctr: 100.6±11.4g, PAB-V: 132.4±31.9g, PAB-Mito: 141.5±31.4g; P<0.05) and histological hypertrophy calculations (desmin / nucleus ratio: Ctr: 0.17±0.02, PAB-V: 0.45±0.01, PAB-Mito: 0.42±0.; P<0.05; Figures 11A-11E) corresponded to these findings. There was no loss of apoptotic cardiomyocytes in Ctr and PAB-Mito hearts, but in PAB-V hearts it was 3±1 / total nucleus (Figures 12A-12C). Dp / Dtmax significantly increased from 831.9±170.5 in all baseline groups to 1006±178.2 in PAB-Mito compared to the decrease in PAB-V (501.2±158.9), and remained unchanged in Ctr (843.5±27.6) hearts at euthanasia (P<0.05) (Figures 9-10). Baseline TAPSE (10.3±1.7 mm) significantly decreased in PAB-V hearts (6.5±0.6 mm) compared to the significant improvement in PAB-Mito (12.3±1.1 mm) hearts (P<0.01) (Figure 7).

[0117] Conclusion: Mitochondrial transplantation maintained hypertrophic adaptations and preserved contractile function in RVs. Directly addressing myocardial dysfunction by targeting mitochondrial dysfunction can be used to treat patients with lung disease affecting right heart function.

[0118] Example 4: Autologous mitochondrial transplantation for the treatment of right heart failure Right ventricular hypertrophy (RVH) and failure (RVF) are major causes of cardiac morbidity and mortality. A key event in progression to RVF is apoptosis of cardiomyocytes due to mitochondrial dysfunction. Since transplantation of respiratoryable mitochondria is available, the aim of this study was to determine whether local intramyocardial injection of autologous mitochondria could treat heart failure.

[0119] The beneficial effects of mitochondria transplanted from different sources were measured in cultured hypertrophic cardiomyocytes. An RVH / RVF model using pulmonary artery ligation in immature piglets with sham controls (n=6 / group) was used for treatment (procedure) with autologous mitochondria isolated from the piglets' own calf muscles (PAB-M) and vehicle injection into the free wall of RV (PAB-V). Animals were followed for 8 weeks with echocardiography (free wall thickness, systolic function), and Dp / Dtmax was measured at the end of the study, which included histological analysis of cardiomyocyte hypertrophy, fibrosis, and apoptosis. Neither internalization nor ATP levels showed significant differences regardless of the mitochondrial source used. At 4 weeks, ligated animals showed RVH (C: 0.27±0.03 cm vs. PAB: 0.4±0.02 cm wall thickness; P=0.01), which further increased in PAB-M, while PAB-V was already severely dilated (0.5±0.04 cm vs. 0.28±0.08 cm; P=0.01). There was no difference in systolic function at baseline, but it was significantly decreased in PAB-V hearts compared to a significant improvement in PAB-M. This was also reflected in Dp / Dtmax at the end of the study. There was very little apoptotic cardiomyocyte loss and fibrosis in C, but significant loss and fibrosis were observed in the PAB-V hearts, which had the highest number of hypertrophied hearts (C: 1±0.4 vs. PAB-V: 13±1.6; p=0.001 and vs. PAB-M: 8±1.9; p=0.01; PAB-V vs. PAB-M p=0.05). By directly addressing myocardial dysfunction through mitochondrial transplantation, we maintain the hypertrophic adaptation of RV and preserve its contractile function.

[0120] Methods and results: Animal models Immature male Yorkshire piglets weighing 5-10 kg (N=18) underwent pulmonary artery banding (PAB) or sham surgery. The piglets were sedated with terazole (4.5 mg / kg im), xylazine (2 mg / kg im), and atropine (0.04 mg / kg im). After tracheal intubation, ventilation was initiated with isoflurane (1-3%) and air. EKG, blood oxygen saturation (maintained >97%), body temperature, and end-tidal carbon dioxide were monitored. Femoral and venous lines were placed. The piglets were positioned on the right side, draped, and a left thoracotomy was performed in the fourth intercostal space. Lidocaine (1%, iv) was administered before thoracotomy to prevent ventricular fibrillation. The pulmonary artery (PA) was incised from the ascending aorta and ligated by needle puncture while monitoring the pressure of the RV and distal PA. The PA was ligated to 50% of its original diameter. A 4F angiography catheter (Merit Medical Systems, South Jordan, Utah) was inserted into the PA and connected to a PowerLab data acquisition system (DAQ, AD Instruments, Series 16 / 35) to acquire data for baseline pressure calculation. Baseline echocardiography was obtained on the epicardium before and after PA banding. The thoracotomy was closed in three layers, and pleural air was drained onto the thoracic tube. Bupivacaine (0.25%; <0.03 mg / kg) was administered as a local analgesic, and postoperative systemic analgesia was provided for the first 72 hours via benamin / grunixin meglumine (1–2 mg / kg im) and fentanyl patch (1–4 ug / kg perdermal). The piglets were recovered in a 37°C incubator and then immediately returned to the enclosure. Sham surgery (C, N=6) involved opening and closing the chest by local manipulation at the site of the PA. Following intraoperative echocardiography, the progression of RV hypertrophy was measured every other week. Throughout these procedures, the animals were maintained under isoflurane (1-3%) sedation.

[0121] Four weeks after PAB, animals were treated by direct injection of vehicle (PAB-V, N=6) or autologous mitochondria (PAB-M, N=6) into the free wall of the RV, following the same anesthesia protocol as described above. Under sterile conditions, muscle biopsies were performed from the calf muscles of piglets, and mitochondria were isolated as described above. Under direct visualization via a subxiphoid approach, 1 ml of buffer (300 mM sucrose, 10 mM K-HEPES, and 1 mM K-EGTA, pH 7.2 and 4°C) containing 10 × 10⁶ / ml of autologous mitochondria (PAB-M, N=6) or buffer alone (PAB-V, N=6) was injected into 10 sites in the free wall of the RV using a 30G needle. Echocardiography was performed before injection and immediately after mitochondrial / vehicle injection. The incisions were closed in layers, and the animals recovered.

[0122] All animals survived for a further 4 weeks (8 weeks after the first PAB) and were monitored by echocardiography every other week. At the end of the study, the piglets were anesthetized in the same manner as above, except that anesthesia was maintained via a face mask instead of intubation. A median sternotomy was performed, and PA (proximal and distal PA bands) and aortic pressure were invasively measured using a 4F angiography catheter (Merit Medical Systems, South Jordan, Utah). In addition, PA and RV pressure-volume curves were calculated from data obtained using a 7F Scisense pressure-volume (PV) loop catheter (Transonic, Ithaca, New York) inserted into the RVOT. The PV loop catheter was connected to a Powerlab, and the signal was automatically calibrated using an ADVantage pressure-volume system (ADV500, Transonic, Ithaca, New York). After all measurements were performed, Fetal Plus® was administered for euthanasia, the heart was resected, and the body was flushed with phosphate-buffered saline (PBS) on ice. RV free wall biopsies were obtained, rapidly frozen, and stored in liquid nitrogen until further use. Individual RV free wall tissues were embedded in an optimal cutting temperature (OCT) compound, rapidly frozen, and stored at -80°C until sectioning was performed. Fresh RV free walls were used for wet / dry weight calculations.

[0123] Isolated cardiomyocyte culture model We measured mitochondrial internalization in hypertrophied cardiomyocytes using a cell culture model of neonatal rat cardiomyocytes. Furthermore, we tested the functional advantages of various mitochondrial sources in this model. Pharmacologically induced hypertrophied samples were compared to non-hypertrophied control samples. All isolated cell experiments were performed redundantly.

[0124] In short, neonatal rat cardiomyocytes were isolated using a commercially available isolation kit (Worthington) and cultured as previously described in detail (Choi YH, Stamm C, Hammer PE, et al. Cardiac conduction through engineered tissue. Am J Pathol. 2006;169:72-85). Two days after culturing, cells were assigned to either control or hypertrophy. To stimulate cardiac hypertrophy in vitro, cardiomyocytes were serum-starved for 24 hours and then treated with angiotensin II (100 nM; Sigma-Aldrich) for 24 hours.

[0125] Isolation of mitochondria from muscle tissue To determine whether the source of mitochondria plays a role in the benefit of restoring mitochondrial function, mitochondria from two different skeletal muscle sources, fast-twitch and slow-twitch muscle fibers, were collected by punch biopsy from gastrocnemius muscle and soleus muscle obtained from mother rats and compared to RV cardiac mitochondria. This method yields over 99.5% viable mitochondria from 100 mg of tissue sample. After homogenizing the muscle tissue with a commercially available tissue dissociator in homogenization buffer (300 mM sucrose, 10 mM K-HEPES, and 1 mM K-EGTA, pH 7.2 and 4°C), 250 μl of buffer solution containing subtilisin A was added to 1 ml of homogenization buffer. The homogenate was inverted and mixed, incubated on ice for 10 minutes, and then fractionally filtered. The filtrate was transferred to two pre-cooled microfuse tubes and centrifuged at 9,000 xg for 10 minutes at 4°C. The supernatant was removed, and the combined pellet was resuspended in 1 ml of ice-cold respiratory buffer (250 mmol / l sucrose, 2 mmol / l KH2PO4, 10 mmol / l MgCl2, 20 mmol / l K+-HEPES buffer, pH 7.2, 0.5 mmol / l K+-EGTA, pH 8.0, 5 mmol / l glutamate, 5 mmol / l malate, 8 mmol / l succinate, 1 mmol / l ADP). Mitochondria were counted using a Coulter counter (Beckman Coulter Life Sciences, Indianapolis, Indiana).

[0126] Measurement of internalization of transplanted mitochondria Mitochondria were pre-labeled with pHrodo red particle labels (ThermoFisher, Waltham, Massachusetts) at 4°C for 10 minutes and washed four times with respiratory buffer. PHrodo fluorescence provides a positive indicator of internalization because fluorescence follows only uptake by viable mitochondria. Labeled mitochondria were resuspended in fresh respiratory buffer, and the final wash supernatant was saved. This wash supernatant was used to determine nonspecific labeling by incubation of control cells with this supernatant (data not shown). Labeled mitochondria (1 x 100 / well) were co-cultured with cardiomyocytes (~50,000 / well). After 24 hours, the medium was removed, cells were washed four times with 1x PBS, and 200 μl of fresh medium was added to each well. For staining, cells were permeabilized with 0.1% Triton X-100 in PBS for 3 minutes and incubated for 1 hour with primary antibody diluted 1:1000 in 10% fetal bovine serum (FBS) in PBS. As the primary antibody, desmin of cardiomyocytes was used, along with a species-appropriate Alexa Fluor 488-conjugated secondary antibody (ThermoFisher, Waltham, Massachusetts). The nuclei were simultaneously stained with 4',6-diamidino-2-phenylindole (DAPI) (ThermoFisher). Internalization was detected based on red fluorescent mitochondria in green desmin-stained cardiomyocytes. Internalization was evaluated by Zeiss fluorescence microscopy.

[0127] Measurement of mitochondrial function ATP content was measured using the ATPlite luminescent ATP detection assay system (Perkin Elmer, Waltham, Massachusetts). A different cell set was used for these experiments because the fluorescent dye used to label mitochondria may interfere with mitochondrial function.

[0128] Echocardiography Echocardiographic measurements were acquired at baseline, pre-treatment (4 weeks post-ligation), and at the end of the study (8 weeks post-ligation). All studies were performed using a Philips iE33 device with an S8-3 transducer (Philips Healthcare, Amsterdam, Netherlands), and all cycles were saved with concurrent ECG recordings. RV function was assessed from four-chamber views by fractional area change (FAC) and tricuspid annular plane contractile range of motion (TAPSE). Free wall thickness of the RV was measured in M-mode recordings acquired from parasternal long axis (PLAX) and parasternal short axis (PSA) views at end-diastole.

[0129] Invasive pressure-volume (PV) measurement The pressure-volume curves for PA and RV were calculated from data obtained using a 7F Scisense pressure-volume (PV) loop catheter (Transonic Systems Inc., Ithaca, New York). This catheter was inserted through the right adnexa and secured with 4-0 Prolen sutures (Ethicon Inc., Somerville, New Jersey). The PV loop catheter was connected and automatically calibrated using an ADVantage pressure-volume system (ADV500, Transonic, Ithaca, New York). Measurements were obtained at baseline and at euthanasia. Data were acquired using a Powerlab data acquisition system (DAQ, AD Instruments, Series 16 / 35) and analyzed with the provided LabChart 7 Acquisition software (AD Instruments, Sydney, Australia). RV peak pressure (Pdev, mmHg), RV end-diastolic pressure (Ped, mmHg), and maximum change in RV pressure over time (dp / dt max, mmHg / s) were measured. Maximum volume (Vmax) and end-diastolic volume (Ved) were calculated using a PV loop catheter.

[0130] RV histological analysis RV tissue was embedded in an optimal cutting temperature (OCT) compound, rapidly frozen, and stored at -80°C until use. Sections were collected, and the frozen slides were stored at -80°C until use for staining. All slides were visualized using a ZeissObserver.Z1 fluorescence microscope with a Nikon 20x objective lens (NA=20x / 0.45). Ten randomly selected fields from each slide were photographed with a Leica digital color camera and analyzed in ImageJ (version 2.0.0-rc-43, obtained from the National Institutes of Health, Bethesda, Maryland).

[0131] Measurement of cardiomyocyte hypertrophy In addition to echocardiography, RV hypertrophy was assessed using immunofluorescence staining of desmin to visualize cardiomyocytes (1:50, Santa Cruz Biotechnology Inc., Dallas, Texas) and DAPI (1:1000, Dako, Carpinteria, California), and the number of nuclei per field of view was measured. The ratio of desmin to the number of nuclei per field of view was calculated using ImageJ.

[0132] Measurement of myocardial apoptosis Without being constrained by theory, since cardiomyocyte apoptosis has been shown to be primarily a result of mitochondrial dysfunction, we measured cardiomyocyte apoptosis by TUNEL staining using the ApopTagPlus fluorescein InSitu Apoptosis Detection Kit (MilliporeSigma, Burlington, Massachusetts). Cardiomyocytes were counterstained with desmin (1:50, Santa Cruz Biotechnology Inc., Delas, Texas) and the nuclei with DAPI (1:1000, Dako, Carpinteria, California). TUNEL-positive nuclei were manually counted. The total number of nuclei per field of view was measured using ImageJ. Data were expressed as the ratio of apoptotic nuclei per 1000 cardiomyocyte nuclei.

[0133] Measurement of cardiomyopathy A separate set of tissue sections was stained with Masson's trichrome, resulting in fibrous (collagen-rich) areas appearing blue and cardiac muscle appearing red. The blue and red areas (fibrosis and cardiac muscle) were measured, and their ratio served as an estimate of fibrosis. Slides were visualized using a Nikon 10x objective lens. Ten randomly selected fields of view were acquired for each tissue sample and analyzed in ImageJ. Results are expressed as the ratio of blue to red areas.

[0134] statistical analysis All results are reported as mean ± standard error of the mean (SEM). After confirming the normal distribution of the data, ANOVA and Bonferroni post-hoc analyses were performed for comparison of multiple groups, and statistical significance was calculated (SPSS 23, IBM Corporation, Armonk, New York). Probability values ​​less than or equal to 0.05 were considered statistically significant.

[0135] Cardiomyocyte culture model Cardiomyocyte size was measured as an indicator of hypertrophy (H) following angiotensin II exposure. Post-treatment, cardiomyocyte size significantly increased compared to control cardiomyocytes, expressed as the ratio of nuclei per field of view (C: 2.4 ± 0.2 vs. H: 4.2 ± 0.5; p = 0.01). Mitochondria were internalized in hypertrophied RV cardiomyocytes at the same scale as in control cardiomyocytes (Figure 15).

[0136] ATP levels, expressed per cardiomyocyte, were decreased in hypertrophied cardiomyocytes compared to controls (C: 404±28 vs. no H-mitos: 256±23; p=0.01), but normalized to above-normal levels after mitochondrial transplantation. No statistical differences were observed based on the mitochondrial source used (H-cardiac mitos: 541±36 vs. H-gastrocnemius mitos: 527±98 vs. H-soleus mitos: 531±19; ns). Skeletal muscle mitochondria responded similarly to mitochondria isolated from cardiomyocytes (Figure 16).

[0137] Animal models All 18 piglets survived until the end of the study. The mean gradient (mmHg, mean ± SEM) measured across the entire PA band at the end of the study did not differ significantly between the PAB-V (12.1 ± 1.6) group and the PAB-M (9.7 ± 1.9) group (P=0.8). There was no significant difference in body weight (kg) between the three groups, either before the intervention (C: 12.2 ± 1.5, PAB-V: 11.4 ± 1.5, PAB-M: 11.9 ± 0.9; P=0.9) or at the end of the study (C: 17.3 ± 3.1, PAB-V: 16.6 ± 1.2, PAB-M: 17.8 ± 0.9; P=0.4). However, total cardiac weight (grams) at the end of the study was significantly higher in PA-banded animals compared to sham surgery controls (C: 100.6 ± 4.7 vs. PAB-V: 132.4 ± 13 and PAB-M: 141.6 ± 12.8; P<0.05). There were no significant differences in the RV wet weight / dry weight ratio (wet-dry weight / dry weight) among the three groups (C: 4.1 ± 0.05, PAB-V: 5.3 ± 0.09, PAB-M: 4.9 ± 0.3; P=0.5).

[0138] Histological analysis Hypertrophy was histologically assessed by calculating the ratio of myocardial area to the number of nuclei per field of view. Both PAB groups showed a significant increase in muscle mass compared to sham surgery controls at the study endpoint (C: 0.2 ± 0.02 vs. PAB-V: 0.36 ± 0.02 and PAB-M: 0.36 ± 0.3; P = 0.001). This finding correlated with the presence of cardiomyocyte apoptosis, with the PAB-V heart showing the highest number of apoptosis-positive cardiomyocyte nuclei (C: 1 ± 0.4 vs. PAB-V: 13 ± 1.7 vs. PAB-M: 8 ± 1.9; p ≤ 0.05; Figures 11A-11E and 17A-17B). PAB-V mitochondria were also swollen and cristae were reduced (Figures 13A-13C). These findings were also supported by a significant increase in myocardial fibrosis in hypertrophic hearts treated with vehicles compared to hypertrophic hearts treated with mitochondria (C: 4 ± 0.55 vs. PAB-V: 15 ± 1.3 vs. PAB-M: 10 ± 1.1; p ≤ 0.05; Figures 17C-17D).

[0139] Echocardiography In addition to histological evaluation of myocardial hypertrophy, right ventricular wall thickness in centimeters was measured using end-diastolic M-mode recordings. Baseline wall thickness did not differ significantly between the pre-intervention groups (C: 0.25 ± 0.01, PAB-V: 0.24 ± 0.01, PAB-M: 0.25 ± 0.01; P=0.48), but it significantly increased in the ligated group within 4 weeks after ligation (C: 0.28 ± 0.01 vs. PAB-V: 0.4 ± 0.02 and PAB-M: 0.38 ± 0.02; P<0.001). At the end of the study, the mitochondria-treated hearts maintained wall thickness, while the vehicle-treated hearts returned to a baseline showing dilation (C: 0.28 ± 0.01 vs. PAB-V: 0.34 ± 0.03; P=0.15; vs. PAB-M: 0.47 ± 0.02; P=0.05). (Figures 8 and 18A–18C)

[0140] Baseline tricuspid annular plane contraction range of motion (TAPSE, mm) did not differ between groups (C: 10.6 ± 0.2, PAB-V: 10 ± 0.4, PAB-M: 9.8 ± 0.2; P=0.2), but was significantly lower in the ligated group compared to the sham control 4 weeks after PAB (C: 12.3 ± 0.6 vs. PAB-V: 8.2 ± 0.3 and PAB-M: 8 ± 0.3, P<0.001). There was no difference between the two hypertrophy groups before mitochondrial treatment (PAB-V vs. PAB-M; P=0.9). Four weeks after mitochondrial transplantation, the treated hypertrophied hearts were functionally significantly superior to those treated with the vehicle (PAB-V: 6.7±0.2 vs. PAB-M: 12.2±0.4; P<0.001), and there was no difference between the mitochondrial-treated hearts and the sham-operated controls (C: 13±0.5 vs. PAB-M: 12.2±0.4; P=0.42). (Figures 6 and 19A-19C)

[0141] Changes in functional domain (FAC, %) did not differ among the three groups at baseline (C: 38.2±1.4, PAB-S: 41.2±3.4, PAB-M: 41.3±2.1; P=0.60), but were significantly different in the hypertrophy group 4 weeks after ligation compared to the sham surgery control (C: 43±1.6, PAB-V: 23.7±1.5, PAB-M: 25±2.5, P<0.001). Both hypertrophy groups did not differ from each other before mitochondrial therapy, but at the end of the study, contractile function of vehicle-treated hearts was significantly reduced compared to mitochondrial therapy and the sham surgery control (C: 46.3±1.9 and PAB-M: 45.7±0.9 vs. PAB-V: 21.5±1.9; P<0.001. (Figures 7 and 20A-20C)).

[0142] Invasive pressure-volume (PV) measurement Vmax (ml / min) and Ved (ml / min) were higher in group PAB-V compared to groups C and PAB-M at the end of the study, but the difference was not statistically significant (C: 83.7±11.8, PAB-V: 122.1±30.3, PAB-M: 94.5±13.8; P=0.42 and C: 73.8±8.3, PAB-V: 99.9±25.3, PAB-M: 84.8±13.6; P=0.57). Pdev (mmHg) was higher in ligated animals at the end of the study compared to sham surgical controls, but the difference was not statistically significant (C: 10±0.9, PAB-V: 18.6±6.2, PAB-M: 20.5±1.9, P=0.16).

[0143] Prior to intervention, all animals started with a mean dP / dt max (mmHg / sec) of 831.9 ± 56.8. Animals were not significantly different from each other at baseline (P>0.05). However, DP / dt max (mmHg / sec) was significantly higher at the study endpoint in hypertrophic hearts treated with mitochondria compared to hearts treated with the vehicle (PAB-V: 506.6 ± 88.1 vs. PAB-M: 894.9 ± 119.23; P<0.05). Conversely, PAB-M hearts were not significantly different from sham-operated control hearts (C: 777 ± 29.4; P=0.9). (Figures 9-10 and 21A-21B)

[0144] Conclusion: Mitochondrial transplantation maintained hypertrophic adaptations and preserved contractile function in RVs. Directly addressing myocardial dysfunction by targeting mitochondrial dysfunction can be used to treat patients with lung disease affecting right heart function.

[0145] The objective of this study was to delay the onset of heart failure by targeting defects affecting mitochondrial energy. Without being constrained by theory, cardiac mitochondrial dysfunction is critical in heart failure, partly due to the increased energy demands of hypertrophied RVs. Our intervention aimed to improve mitochondrial function to maximize energy production through the transplantation of respiratoryable mitochondria. We established that autologous exogenous mitochondria obtained from skeletal muscle sources internalize in hypertrophied cardiomyocytes and increase mitochondrial function to a comparable degree to mitochondria obtained from RV cardiomyocytes. Autologous mitochondrial transplantation in a large animal model of pulmonary artery banding established that cardiomyocytes are protected from apoptotic cell loss. Furthermore, maintaining hypertrophic growth led to the preservation of systolic function compared to untreated hypertrophied hearts showing signs of diastolic and systolic dysfunction. Therefore, in some embodiments, methods for preventing or reducing apoptotic cell loss in cardiomyocytes and preserving or improving cardiac systolic function, comprising administering a mitochondrial-containing composition to a patient, are described herein.

[0146] Without being bound by theory, the right ventricle is the primary determinant of the prognosis in pulmonary disease-related heart failure. The indications and maladjustments of the RV are crucial throughout the disease course. Initially, RV contractility increases due to changes in muscle properties and compensatory muscle hypertrophy until a certain uncoupling point occurs where afterload exceeds contractility. This indicates maladjustment, which is characteristic of ventricular dilation. In right heart failure, treatment is primarily limited to targeting lung function rather than directly intervening in the significant energy deficit of the RV myocardium resulting from mitochondrial dysfunction (Hoeper MM, Kramer T, Pan Z, et al. Mortality in pulmonary arterial hypertension: prediction by the 2015 European pulmonary hypertension guidelines risk stratification model. Eur Respir J. 2017;50(2):pii:1700740 (Mortality in pulmonary arterial hypertension: prediction by the 2015 European pulmonary hypertension guidelines risk stratification model); Galie N, Humbert M, Vachiery JL, et al. 2015 ESC / ERS Guidelines for the diagnosis and treatment of pulmonary hypertension. Eur Resp J. 2015;46:903-975 (2015 ESC / ERS Guidelines for the diagnosis and treatment of pulmonary hypertension); Tonelli AR, Arelli V, Minai OA, et al. Causes and circumstances of death in pulmonary arterial hypertension. Am J Respir Crit Care Med. 2013;188(3):365-369 (Causes and circumstances of death in pulmonary arterial hypertension).

[0147] Without being constrained by theory, mitochondrial dysfunction, which results in a reduced ability to produce ATP, is known to affect cardiac function because approximately 90% of a cell's ATP is used for contraction and relaxation and calcium regulation (Doenst T, Nguyen TD, Abel ED. Cardiac metabolism in heart failure: Implications beyond ATP production. Circ Res. 2013;113:709-724). Mitochondrial dysfunction may be due to a lack of mitochondrial quality control, leading to defects in metabolic signaling, bioenergy, calcium transport, reactive oxygen species (ROS) generation, and activation of cell death pathways. This results in a vicious feedforward cycle that leads to apoptotic cardiomyocyte death (Brown DA, Perry JB, Allen ME, et al. Expert consensus document: mitochondrial function as a therapeutic target in heart failure. Nat Rev Cardiol. 2016;14:238-250). Altered mitochondrial function is recognized as a cause of pressure overload hypertrophy and impairment. Human studies of end-stage heart failure have shown that markers of energy metabolism decrease in heart failure, leading to the idea that heart failure is like an engine running out of fuel (Doenst T, Nguyen TD, Abel ED. Cardiac metabolism in heart failure: Implications beyond ATP production. Circ Res. 2013;113:709-724; Neubauer S. The failing heart-an engine out of fuel. N Engl J Med. 2007;356:1140-1151).However, mitochondria play a more complex role in regulating metabolism and cell death than simply starving the energy of dysfunctional myocardium. A piglet model of right heart failure has highlighted the importance of mitochondrial function and structural quality in right heart failure caused by pressure overload, with mitochondria showing impaired oxidative phosphorylation and significant structural damage (Noly PE, Piquereau J, Coblence M, et al. Right ventricular mitochondrial respiratory function in a piglet model of chronic pulmonary hypertension. J Thorac Cardiovasc Surg 2020;159(1):129-140). The data herein show that cardiomyocytes were better preserved from apoptotic cell loss after mitochondrial treatment, while hypertrophied hearts treated with vehicle showed more apoptotic cell death.

[0148] Without being constrained by theory, the metabolic demands of hypertrophied RVs increase significantly, and since lean RVs need to increase muscle mass to compensate for the increased pressure load, it is necessary to immediately strengthen the required mitochondrial support. However, mitochondria cannot keep up with the rapidly increasing muscle growth (Friehs I, Cowan DB, Choi YH, et al. Pressure-overload hypertrophy of the developing heart reveals activation of divergent gene and protein pathways in the left and right ventricular myocardium. Am J Physiol Circ Physiol. 2013;304(5):H697-708 (Pressure-overload hypertrophy of the developing heart reveals activation of divergent gene and protein pathways in the left and right ventricular myocardium); Phillips D, Aponte AM, Covian R, Neufeld E, Yu ZX, Balaban RS. Homogenous protein programming in the mammalian left and right ventricle free walls. Physiol Genomics. 2011;43(21):1198-1206 (Homogeneous protein programming in the mammalian left and right ventricle free walls)). In our animal model, pressure overload does not reverse, and mitochondrial adaptation to the thickened RV muscle is necessary to maintain function. Based on our results, this compensatory adaptation to cope with increased pressure load does not occur in the long term, as indicated by the reduction in wall thickness of hypertrophied hearts treated in vehicles. In contrast, mitochondrial transplantation maintains the hypertrophic adaptive growth of RV. Furthermore, the data showed that the source of mitochondria for transplantation is not a determinant of their benefits. There was no difference in which mitochondrial source was used. Skeletal muscle mitochondria responded similarly to mitochondria isolated from cardiomyocytes. Therefore, cardiomyocyte mitochondria are not necessary for treating mitochondrial dysfunction via RVH / RVF.Exogenous autologous skeletal muscle mitochondria maintain the contractile function of dysfunctional RVs.

[0149] Mitochondrial transplantation as a therapeutic intervention targets all aspects of mitochondrial function and structure. Because mitochondrial structural integrity must also be addressed, mitochondrial metabolic manipulation alone is insufficient to treat a dysfunctional right ventricle. Furthermore, mitochondrial transplantation targets the impaired mitochondrial dynamics in a hypertrophied / dysfunctional right heart. Potential mechanisms under consideration include the disruption of mitochondrial biosynthesis and the generation of new mitochondria as early events in the pathophysiology of heart failure. In the early stages of compensatory hypertrophy, mitochondrial biosynthetic signaling is maintained. In contrast, as decompensated heart failure becomes apparent, mitochondrial biosynthetic signaling decreases.

[0150] In conclusion, this study deepened our understanding of the benefits of mitochondrial transplantation. In particular, the results show that exogenous autologous skeletal muscle mitochondria maintain the contractile function of a dysfunctional heart.

[0151] Example 5: Autologous mitochondrial transplantation via intracoronary injection for myocardial protection We conducted experiments to investigate pre-ischemic autologous mitochondrial transplantation (MT) in the coronary arteries as a therapeutic strategy for prophylactic cardioprotection in a porcine model.

[0152] method: A cannula was inserted into the left coronary artery of Yorkshire pigs (n=26). Mitochondria (1×10 9 ) or buffer (vehicle [Veh]) is a single bolus (MT S ) or consecutive (10 injections in 60 minutes; MT SS It was delivered as a single injection. It was delivered as a bolus antegrade into the left major coronary artery (1 × 10⁶ in 6 mL). 9 ). Continuous injection (1 x 10⁶ doses in 6 mL of respiratory buffer per injection). 9Ten injections of the drug were administered every five minutes. Fifteen minutes after the injection, the heart received transient local ischemia (RI) by snagging the left anterior descending artery. Thirty minutes after the RI, the snare was released, and the heart was reperfused for 120 minutes.

[0153] result: Coronary artery blood flow (CBF) and myocardial function temporarily increased during the period prior to radioisotopes (RI). 30 minutes after RI, MT S and MT SS The heart significantly increased CBF, which persisted throughout the reperfusion (Veh vs MT). S and MT SS (P=0.04). MT S and MT SS This is due to a significant improvement in ejection fraction (Veh vs MT). S (P<0.001; Veh vs MTSS, P=0.04) and generated pressure (Veh vs MTSS) S , P<0.001; Veh vs MT SS It showed segmental shortening (Veh vs MT). S P=0.03; Veh vs MT SS (P<0.001), reduction rate (Veh vs MT) S , P<0.001; Veh vs MT SS (P=0.04), and strain analysis (Veh vs. MT) S P=0.002; Veh vs MT SS Local function, as assessed by (P=0.003), also improved significantly. Although there was no difference in risk areas between the treatment groups, infarct size was significantly reduced in both MT groups (Veh vs. MT). S and MT SS (P<0.001).

[0154] Conclusion: Pre-ischemic mitral therapy (MT) administered by single or sequential intracoronary injections provides prophylactic myocardial protection, significantly reducing infarct size and enhancing overall and local cardiac function.

[0155] Example 6: Myocardial protection by intracoronary delivery of mitochondria Autologous mitochondrial transplantation involves supplying ischemic tissue with viable, respiratoryable mitochondria isolated from the patient's own body to mitigate the effects of natural mitochondrial damage. Experiments were conducted to investigate the safety and efficacy of intracoronary delivery of mitochondria in clinically relevant porcine models.

[0156] method: Adult pigs were anesthetized, and autologous mitochondria were isolated. The animals were sedated with terazole (2.2-4.4 mg / kg) / xylazine (1-2 mg / kg) and intubated. General anesthesia was maintained with a 0.5-2% isoflurane-oxygen mixture. A median sternotomy was performed, and the heart was suspended in a pericardial cradle. Next, angiographic access to the left coronary artery (LCA) was established by fluoroscopy-guided 5F JR angiography catheter (Merit Medical Sys, UT) through the right carotid artery (5F sheath) to the left coronary artery orifice. Mitochondrial uptake and in vivo distribution were investigated. 18 F-rhodamine-6G labeled mitochondria were injected into the left coronary artery by angiography and then evaluated by positron emission tomography (PET) (n=3). The safety profile of intra-coronary mitochondrial injection was evaluated under normal conditions, during coronary vasoconstriction, and during tachycardia (n=18). To evaluate the therapeutic effect of intra-coronary mitochondrial transplantation, the left anterior descending artery was snagged for 30 minutes. At the start of reperfusion, animals received either mitochondria (n=8) or vehicle solution (n=8), followed by 2 hours of reperfusion.

[0157] result: Intra-coronary delivery of mitochondria resulted in rapid uptake and specific in vivo distribution of mitochondria throughout the heart. Coronary artery patency and myocardial function were maintained under all test conditions. Intra-coronary injection of mitochondria resulted in a concentration-dependent increase in coronary blood flow (CBF). Mitochondrial-induced congestion required mitochondrial viability, ATP production, and, in part, activation of inward-directing rectifying potassium channels (KIRs). Intra-coronary delivery of mitochondria resulted in a significant enhancement of post-ischemic myocardial function, improved CBF, and reduced infarct size compared to controls. Intracoronary mitochondrial transplantation is a safe and effective method for improving myocardial perfusion, myocardial function, and cardiac tissue survival.

[0158] Other embodiments Although the present invention has been described in conjunction with its detailed description, it should be understood that the foregoing description is intended to describe, and not limit, the scope of the invention as defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. A method for treating or preventing a target heart failure, comprising administering a therapeutically effective amount of a composition comprising isolated mitochondria or a combination of mitochondrial agents to the target.

2. The method according to claim 1, wherein the composition is administered to a subject by intramyocardial injection.

3. The method according to claim 1, wherein the subject has or is at risk of developing heart failure - right ventricular hypertrophy (RVH), left ventricular hypertrophy (LVH), right ventricular failure (RVF), or left ventricular failure (LVF).

4. The method according to claim 1, wherein the subject has a lung disease.

5. The method according to claim 4, wherein a lung disease affects right ventricular function.

6. The method according to claim 1, wherein the composition is administered to a target by injecting the composition into a target blood vessel.

7. The method according to claim 1, wherein the mitochondria are in-house.

8. The method according to claim 1, wherein the mitochondria are homogeneous.

9. The method according to claim 1, wherein the mitochondria are of a different species.

10. A method for maintaining right ventricular (RV) contractility, maintaining RV capillary density, preventing RV dilation, or delaying the onset of RVF in a subject, comprising administering a therapeutically effective amount of a composition containing isolated mitochondria or a combination of mitochondrial agents to the subject.

11. The method according to claim 10, wherein the composition is administered to a subject by intramyocardial injection.

12. The method according to claim 10, wherein the subject has or is at risk of developing right ventricular hypertrophy (RVH) or right ventricular failure (RVF).

13. The method according to claim 10, wherein the subject has a lung disease.

14. The method according to claim 10, wherein a lung disease affects right ventricular function.

15. The method according to claim 10, wherein the composition is administered to a target by injecting the composition into a target blood vessel.

16. The method according to claim 10, wherein the mitochondria are in-house.

17. The method according to claim 10, wherein the mitochondria are homogeneous.

18. The method according to claim 10, wherein the mitochondria are of a different species.

19. A method for maintaining left ventricular (LV) contractility, maintaining LV capillary density, preventing LV dilation, or delaying the onset of left ventricular failure (LVF) in a subject, comprising administering a therapeutically effective amount of a composition containing isolated mitochondria or a combination of mitochondrial agents to the subject.

20. The method according to claim 19, wherein the composition is administered to a subject by intramyocardial injection.

21. The method according to claim 19, wherein the subject has left ventricular hypertrophy (LVH) or left ventricular failure (LVF), or is at risk of developing it.

22. The method according to claim 19, wherein the subject has a lung disease.

23. The method according to claim 19, wherein a lung disease affects left ventricular function.

24. The method according to claim 19, wherein the composition is administered to a target by injecting the composition into a blood vessel of the target.

25. The method according to claim 19, wherein the mitochondria are in-house.

26. The method according to claim 19, wherein the mitochondria are homogeneous.

27. The method according to claim 19, wherein the mitochondria are of a different species.

28. A method for maintaining the ventricular contractility of the target, Identifying those who need it, and Administer a therapeutically effective dose of a composition containing isolated mitochondria or combined mitochondrial agents to the target. A method that includes this.

29. The method according to claim 28, wherein the subject is identified by measuring end-systolic pressure volume (ESPV), LV peak pressure, ejection fraction, systolic shortening, LV end-diastolic pressure, or dP / dt (change in pressure over time).

30. A method for maintaining ventricular capillary density in a subject, Identifying those who need it, and Administer a therapeutically effective dose of a composition containing isolated mitochondria or combined mitochondrial agents to the target. A method that includes this.

31. The method according to claim 30, wherein the ventricular capillary density is measured by magnetic resonance imaging (MRI) or microvascular angiography.

32. A method for reducing the risk of ventricular dilation in a subject, Identifying those who need it, and Administer a therapeutically effective dose of a composition containing isolated mitochondria or combined mitochondrial agents to the target. A method that includes this.

33. The method according to claim 32, wherein the subject is identified as having diabetes, obesity, hypertension, alcohol abuse, cocaine use and abuse, bacterial infection, viral infection, fungal infection, parasitic infection, exposure to toxins (e.g., lead, mercury, or cobalt), arrhythmia, or late pregnancy complications.

34. A method for delaying the onset of heart failure in the subject, Identifying those who need it, and Administer a therapeutically effective dose of a composition containing isolated mitochondria or combined mitochondrial agents to the target. A method that includes this.

35. The method according to claim 34, wherein the subject is identified as having right ventricular hypertrophy or left ventricular hypertrophy.

36. A method for treating heart failure, delaying the onset of heart failure, and reducing the risk of developing heart failure in a subject, comprising administering a therapeutically effective amount of a composition containing isolated mitochondria or a combination of mitochondrial agents to the subject.

37. The method according to claim 36, wherein the composition is administered to a subject by intramyocardial injection.

38. The method according to claim 36, wherein the method includes identifying a subject as one who is at risk of developing heart failure.

39. The method according to claim 36, wherein the subject has a lung disease.

40. The method according to claim 36, wherein the composition is administered to the subject by injecting the composition into the blood vessels leading to the heart.

41. A method for treating cardiac hypertrophy, delaying the onset of cardiac hypertrophy, and reducing the risk of developing cardiac hypertrophy in a subject, comprising administering a therapeutically effective amount of a composition containing isolated mitochondria or a combination of mitochondrial agents to the subject.

42. The method according to claim 41, wherein the composition is administered to a subject by intramyocardial injection.

43. The method according to claim 41, wherein the method includes identifying a subject as one who is at risk of developing cardiac hypertrophy.

44. The method according to claim 41, wherein the subject has a lung disease.

45. The method according to claim 41, wherein the composition is administered to the subject by injecting the composition into the blood vessels leading to the heart.

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