Treating heart failure

Abstract

The present disclosure relates to compositions comprising isolated mitochondria or combined mitochondrial agents, and methods of using such compositions to treat disorders. The described methods are based, at least in part, on the discovery that isolated mitochondria themselves, and isolated mitochondria bound to therapeutic, diagnostic, and / or imaging agents, can be delivered to the tissues of a patient by injecting them into the patient's blood vessels.

Description

[Technical Field]

[0001] Priority claims This application claims the benefit of U.S. Provisional Application No. 62 / 806,473, filed February 15, 2019, the entire contents of which are incorporated herein by reference.

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

[0003] Mitochondria are double-membrane-bound organelles found in the cytoplasm of nucleated eukaryotic cells. They are found in nearly all cells in the human body except red blood cells. They are the primary site of cellular energy metabolism, producing adenosine triphosphate (ATP) for various cellular functions. Normally, over 90% of a cell's demand for ATP is supplied by the cell's own mitochondria.

[0004] Mitochondria consist of two concentric membranes with specialized functions: the inner mitochondrial membrane contains the ATP synthase protein, and the outer mitochondrial membrane, which contains numerous 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 have originated from an ancient symbiosis when a nucleated cell engulfed an aerobic prokaryote. In this 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] Due to the key function of mitochondria in cellular metabolism, they have the potential to be used in the treatment of various disorders. There is also a need to harness mitochondria for drug delivery and several other therapeutic and diagnostic purposes. Summary of the Invention

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

[0008] Provided herein are, inter alia, methods for treating or preventing heart failure in a subject, 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 suffers from a pulmonary disease. In some embodiments, the pulmonary disease affects right ventricular function. In some embodiments, the composition is administered to the subject by injecting the composition into a blood vessel of the subject. In some embodiments, the mitochondria are autologous. In some embodiments, the mitochondria are allogeneic. In some embodiments, the mitochondria are xenogeneic.

[0009] Provided herein are, inter alia, 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 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 suffers from a pulmonary disease. In some embodiments, the pulmonary disease affects right ventricular function. In some embodiments, the composition is administered to the subject by injecting the composition into a blood vessel of the subject. In some embodiments, the mitochondria are autologous. In some embodiments, the mitochondria are allogeneic. In some embodiments, the mitochondria are xenogeneic.

[0010] Provided herein are methods for, among other things, 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 methods 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 suffers from a pulmonary disease. In some embodiments, the pulmonary disease affects left ventricular function. In some embodiments, the composition is administered to the subject by injecting the composition into a blood vessel of the subject. In some embodiments, the mitochondria are autologous. In some embodiments, the mitochondria are allogeneic. In some embodiments, the mitochondria are xenogeneic.

[0011] Provided herein, among other things, is a method of preserving ventricular contractility in a subject, the method comprising: Identifying those in need, and administering to a subject a therapeutically effective amount of a composition comprising isolated mitochondria or a combination of mitochondrial agents. In some embodiments, the subject is identified by measuring end-systolic pressure volume (ESPV).

[0012] Provided herein, among other things, is a method of maintaining ventricular capillary density in a subject, the method comprising: Identifying those in need, and administering to a subject a therapeutically effective amount of a composition comprising isolated mitochondria or a combination of mitochondrial agents. Includes.

[0013] Provided herein, among other things, is a method of reducing the risk of ventricular dilation in a subject, the method comprising: Identifying those in need, and administering to a subject a therapeutically effective amount of a composition comprising isolated mitochondria or a combination of mitochondrial agents. Includes.

[0014] In some embodiments, the subject is identified as having diabetes, obesity, high blood pressure, alcohol abuse, cocaine use and abuse, bacterial infection, viral infection, fungal infection, parasitic infection, exposure to toxins (e.g., lead, mercury, or cobalt), cardiac arrhythmia, or late pregnancy complications.

[0015] Provided herein, among other things, is a method of delaying the onset of heart failure in a subject, the method comprising: Identifying those in need, and administering to a subject a therapeutically effective amount of a composition comprising isolated mitochondria or a combination of mitochondrial agents. In some embodiments, the subject is identified as having right ventricular hypertrophy or left ventricular hypertrophy.

[0016] Provided herein are, among other things, methods for treating heart failure, delaying the onset of heart failure, or reducing the risk of developing heart failure in a subject, the methods 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 method comprises identifying the subject as at risk for developing heart failure. In some embodiments, the subject suffers from a lung disease. In some embodiments, the composition is administered to the subject by injecting the composition into a blood vessel to the heart.

[0017] Provided herein are, among other things, methods for treating cardiac hypertrophy, delaying the onset of cardiac hypertrophy, or reducing the risk of developing cardiac hypertrophy in a subject, the methods 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 method comprises identifying the subject as at risk for developing cardiac hypertrophy. In some embodiments, the subject suffers from a lung disease. In some embodiments, the composition is administered to the subject by injecting the composition into a blood vessel to the heart.

[0018] In certain embodiments, the blood vessel is a blood vessel or part of the vascular system that carries blood to a target site, target organ, or target region, e.g., a subject's coronary arteries, a subject's hepatic portal vein, a subject's great pancreatic artery, or a subject's prostatic artery.

[0019] In certain embodiments, the mitochondria can have different sources, for example, the mitochondria can be autologous, allogeneic, or xenogeneic. In certain embodiments, autologous mitochondria can have exogenous mtDNA. In some embodiments, the mitochondria are derived from a first-degree relative of the subject.

[0020] In some embodiments, the described methods include collecting the isolated mitochondria from the cells prior to administration. The isolated mitochondria or combined mitochondrial agents can be administered to the subject immediately after the isolated mitochondria are collected from the cells.

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

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

[0023] As used herein, the term "isolated mitochondria" means functional, intact mitochondria free from extraneous eukaryotic cellular material.

[0024] A "combined mitochondrial agent" is an isolated mitochondria artificially combined with a pharmaceutical, diagnostic, imaging, or other agent. The agent may be combined with the mitochondria in any manner, such as by being bound to the mitochondria (e.g., chemically or electrostatically bound), attached to the mitochondria, embedded in the mitochondrial membrane, substantially enclosed within the mitochondria, or completely encapsulated by the mitochondria, so long as the mitochondria and agent are in physical contact with each other. The combined mitochondrial agent is designed so that the mitochondria function as a "carrier" capable of transporting the agent into a patient's tissues after injection.

[0025] The terms "subject" and "patient" are used throughout this specification to describe a human or non-human animal to which treatment according to the methods of the present disclosure is provided. Veterinary applications are expressly contemplated by this disclosure. The terms include, but are not limited to, birds, reptiles, amphibians, and mammals, such as humans, other primates, pigs, rodents such as mice and rats, rabbits, guinea pigs, hamsters, cows, horses, cats, dogs, sheep, and goats. Preferred subjects are humans, farm animals, and domestic pets such as cats and dogs.

[0026] The term "treating" is used herein to refer to delaying the onset of, inhibiting, reducing the effects of, or increasing the longevity of a patient afflicted with a condition, such as a disease described herein.

[0027] An "ischemia-related disease" is a disease involving ischemia. As used herein, ischemia is a reduction in blood flow to an organ and / or tissue. The reduction in blood flow can be caused by any suitable mechanism, including, inter alia, partial or complete occlusion (blockage), narrowing (constriction), and / or leakage / rupture of one or more blood vessels supplying blood to the organ and / or tissue.

[0028] "Immediately after the mitochondria are harvested from the cells" means immediately after the mitochondria are harvested from the cells, before any substantial loss of mitochondrial viability can occur.

[0029] As used herein, the term "transplantation" is used throughout the specification as a general term to describe the process of transplanting an organ, tissue, cell mass, individual cell, or organelle into a recipient. The term "cell transplantation" is used throughout the specification as a general term to describe the process of transferring at least one cell, such as a pancreatic islet cell or stem cell, into a recipient. For example, such a transplant can be performed by removing beta cells (or intact pancreatic islets) from a donor's pancreas and placing them into a recipient patient whose pancreas cannot produce sufficient insulin. These terms include all categories of transplantation known in the art, except for blood transfusion. Transplants are classified by the site and genetic relationship between the donor and recipient. This term includes, for example, autotransplantation (the removal and transfer of cells or tissue from one location in a patient to the same or another location in the same subject), allotransplantation (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 commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. Furthermore, the materials, methods, and examples are illustrative only and are not intended to be limiting.

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

[0032] [Figure 1] FIG. 1 is a schematic diagram showing the method for mitochondrial isolation. [Figure 2] FIG. 2 is a schematic diagram illustrating disease outcomes associated with ventricular overload. [Figure 3] FIG. 3 is a schematic diagram outlining a method for mitochondrial transplantation in a subject. [Figure 4] FIG. 4 is a schematic diagram showing an animal model study utilizing pulmonary artery banding (PAB). [Figure 5] Figure 5 is a schematic diagram showing the measurement and analysis timeline for the experiment. [Figure 6] Figure 6 is a line graph showing the functional area of ​​change (FAC) as a percentage at baseline, 1 month after PAB, and at euthanasia for the control (C, also referred to as the "sham" group), PAB-V (vehicle), and PAB-M (mitochondrial) groups. [Figure 7] Figure 7 is a line graph showing tricuspid annular plane contractile excursion (TAPSE) in mm for the control (C, also referred to as the "sham" group), PAB-V (vehicle), and PAB-M (mitochondrial) groups at baseline, 1 month after PAB, and at euthanasia. [Figure 8] Figure 8 is a line graph showing right ventricular (RV) wall thickness in cm at baseline, 1 month after PAB, and at euthanasia for the control (C, also referred to as the "sham" group), PAB-V (vehicle), and PAB-M (mitochondrial) groups. [Figure 9] Figure 9 is a box plot showing dP / dt max (mmHg / sec) at the time of euthanasia for the control (C, also referred to as the "sham" group), PAB-V (vehicle), and PAB-M (mitochondrial) groups. [Figure 10] Figure 10 is a line graph showing dP / dt max in mmHg / sec at baseline and at euthanasia for the control (C, also referred to as the "sham" group), PAB-V (vehicle), and PAB-M (mitochondrial) groups. [Figure 11A] Figure 11A is an immunofluorescence image showing TUNEL staining to highlight apoptotic cells (white arrows) in the TUNEL-positive control group. Cardiomyocytes are stained with desmin stain, and nuclei are stained with DAPI stain. [Figure 11B] Figure 11B is an immunofluorescence image showing TUNEL staining to highlight apoptotic cells (white arrows) in the control / sham group. Cardiomyocytes are stained with desmin stain, and nuclei are stained with DAPI stain. [Figure 11C] Figure 11C is an immunofluorescence image showing TUNEL staining to highlight apoptotic cells (white arrows) in the PAB-V group. Cardiomyocytes are stained with desmin staining, and nuclei are stained with DAPI staining. [Figure 11D] Figure 11D is an immunofluorescence image showing TUNEL staining to highlight apoptotic cells (white arrows) in the PAB-M group. Cardiomyocytes are stained with desmin staining, and nuclei are stained with DAPI staining. [Figure 11E] FIG. 11E is a bar graph showing the ratio % of desmin per field / number of nuclei per field (P<0.01**). [Figure 12A]Figure 12A is an immunofluorescence image showing CD31 staining to highlight capillary density (white arrow) in the control / sham group. Cardiomyocytes are stained with desmin stain, and nuclei are stained with DAPI stain. [Figure 12B] Figure 12B is an immunofluorescence image showing CD31 staining to highlight capillary density (white arrow) in the PAB-V group. Cardiomyocytes are stained with desmin staining, and nuclei are stained with DAPI staining. [Figure 12C] Figure 12C is an immunofluorescence image showing CD31 staining to highlight capillary density (white arrow) in the PAB-M group. Cardiomyocytes are stained with desmin staining, and nuclei are stained with DAPI staining. [Figure 13A] FIG. 13A is an electron microscopy study showing the number and shape of mitochondria in the control / sham group. [Figure 13B] FIG. 13B is an electron microscopy study showing the number and shape of mitochondria in the PAB-V group. [Figure 13C] FIG. 13C is an electron microscopy study showing the number and shape of mitochondria in the PAB-C group. [Figure 14] FIG. 14 is a schematic diagram showing disease outcomes and outcomes associated with mitochondrial transplantation therapy. [Figure 15] FIG. 15 is an immunofluorescence image showing photographs of control, RV hypertrophy (RVH), and RVH with mitochondrial transplantation. [Figure 16] Figure 16 is a box plot showing ATP levels in control cardiomyocytes, untreated hypertrophied cardiomyocytes (without H Mito), and mitochondria-treated hypertrophied cardiomyocytes (with H Heart Mito, H Gastrocnemius Mito, and H Soleus Mito). *p=0.05 vs. control, #p=0.001 vs. untreated hypertrophied cardiomyocytes (without H Mito). [Figure 17A] Figure 17A shows immunofluorescence images showing TUNEL staining to highlight apoptotic cells (white arrows) in the control / sham, PAB-V, and PAB-M groups. Cardiomyocytes are stained with desmin stain, and nuclei are stained with DAPI stain. [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] FIG. 17C is a microscopic examination showing representative histological sections to detect fibrosis in the control / sham, PAB-V, and PAB-M groups. [Figure 17D] FIG. 17D is a box plot showing the percent fibrosis per field at study endpoint (*p=0.01 vs. control and #p=0.05 PAB-V vs. PAB-M). [Figure 18A] FIG. 18A is a box plot showing RV wall thickness (cm) at baseline in the control (C, also called "sham"), PAB-V, and PAB-M groups. [Figure 18B] FIG. 18B is a box plot showing RV wall thickness (cm) at 1 month after PAB for the control (C, also called "sham"), PAB-V, and PAB-M groups. [Figure 18C] FIG. 18C is a box plot showing RV wall thickness (cm) at the time of euthanasia for the control (C, also called "sham"), PAB-V, and PAB-M groups. [Figure 19A] FIG. 19A is a box plot showing the Functional Area Change (FAC) at baseline as a percentage for the control (C, also called "sham"), PAB-V, and PAB-M groups. [Figure 19B] FIG. 19B is a box plot showing the functional area of ​​change (FAC) as a percentage at 1 month after PAB for the control (C, also called "sham"), PAB-V, and PAB-M groups. [Figure 19C] FIG. 19C is a box plot showing the functional area change (FAC) as a percentage at euthanasia for the control (C, also called "sham"), PAB-V, and PAB-M groups. [Figure 20A] FIG. 20A is a box plot showing tricuspid annular plane contractile excursion (TAPSE) in mm at baseline for the control (C, also called "sham"), PAB-V, and PAB-M groups. [Figure 20B] FIG. 20B is a box plot showing tricuspid annular plane contractile excursion (TAPSE) in mm at 1 month after PAB for the control (C, also called "sham"), PAB-V, and PAB-M groups. [Figure 20C] FIG. 20C is a box plot showing tricuspid annular plane contractile excursion (TAPSE) in mm at euthanasia for the control (C, also called "sham"), PAB-V, and PAB-M groups. [Figure 21A] FIG. 21A is a box plot showing dP / dt max (mmHg / sec) at baseline for the control (C, also called "sham"), PAB-V, and PAB-M groups. [Figure 21B] FIG. 21B is a box plot showing dP / dt max (mmHg / sec) at euthanasia for the control (C, also called "sham"), PAB-V, and PAB-M groups. [Figure 22] FIG. 22 is a schematic diagram showing animal model studies utilizing pulmonary artery banding (PAB) and a summary of some clinical findings. DETAILED DESCRIPTION OF THE INVENTION

[0033] Detailed Description Right ventricular hypertrophy (RVH) and failure (RVF) are major causes of cardiac morbidity and mortality, affecting long-term outcomes in patients with abnormally overloaded right ventricles (RVs) due to pulmonary hypertension, outflow tract obstruction, or when the RV functions as the systemic ventricle. As an initial compensatory step, the RV adapts to these hemodynamic changes by increasing wall thickness, providing greater contractility to overcome increased afterload. Ultimately, these mechanisms are insufficient, and hypertrophy progresses to diastolic and systolic dysfunction. Clinical observations have shown that these compensatory changes maintain contractile function more effectively and 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 RV adaptation to long-term increased pressure load is related to the inability of mitochondria and calcium handling mechanisms to keep up with the demands of myocardial tissue hypertrophy. Wasteful calcium cycling, accompanied by adenosine triphosphate (ATP) consumption and dysfunction of the electron transport chain (ETC), further limits ATP synthesis and leads to bioenergetic failure (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 ultimately 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 important roles in RV dysfunction, as they gradually decrease from hypertrophy to failure. In response to these findings, this disclosure used a combination of microarray and proteomic analyses of matched samples to demonstrate that mitochondrial function, particularly in terms of mitochondrial quantity / mass, is equally important as myocardial tissue development in the adaptation of thin-walled RVs to pathological loads. Furthermore, activation of proapoptotic pathways, particularly those related to mitochondria, and downregulation of calcium signaling pathways are associated with progression to failure. The initial upregulation of oxidative phosphorylation and associated calcium handling for mitochondrial stabilization corresponds to meeting the energy demands of cardiac muscle growth, allowing thin-walled RVs to adapt to pressure overload. However, prolonged exposure to increased pressure loads renders mitochondria unable to adapt, leading to rapid deterioration accompanied by a decline in contractile function. Progression to heart failure is associated with a decline in energy storage capacity, and compensatory mechanisms can no longer support the imbalance between the reduced energy supply and the increased demands of 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 pulmonary function rather than directly interfering with the critical myocardial energy deficit resulting from mitochondrial dysfunction. Previous studies have demonstrated a successful and safe technique for therapeutic transplantation of autologous, breathing-competent mitochondria, which replaces and / or replenishes the pool of native, damaged mitochondria with viable mitochondria isolated from healthy tissue. However, therapeutic success has primarily been demonstrated in established models of acute ischemia-reperfusion injury (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). Whether adaptive maintenance of mitochondrial function due to progressive pressure-overload hypertrophy, as well as long-term mitochondrial dysfunction, can be achieved through mitochondrial transplantation is largely unknown. Furthermore, the combination of metabolic adaptive changes and mitochondrial dysfunction may play a key role in determining the source of mitochondria for transplantation. As reported, mitochondria adapt to the roles demanded by the tissue they are transplanted from (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, compared with slow-twitch skeletal muscle, are accustomed to high glucose metabolism, allowing them to rapidly adapt to increasing energy demands.It is well established that hypertrophied and failing myocardium switches 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 defective energy production and mitochondrial dynamics in the failing heart. Because cardiac mitochondria are already impaired in the failing heart, mitochondria from other cellular sources must be harvested for transplantation.

[0036] The present disclosure is based, in part, on the surprising discovery that mitochondria can be used to prevent, treat, and / or alleviate one or more symptoms of heart failure, even before heart failure occurs. Accordingly, in one aspect, the present disclosure provides methods for minimizing heart failure, reducing the risk of heart failure, ameliorating at least one symptom of heart failure, and preventing or treating cellular, tissue, and / or organ damage associated with heart failure in a subject at risk for heart failure.

[0037] In some embodiments, the methods described herein for treating or preventing heart failure in a subject include 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 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), or right ventricular failure (RVF), or left ventricular failure (LVF).

[0038] The present disclosure is also based, at least in part, on the discovery that isolated mitochondria, and isolated mitochondria bound to therapeutic, diagnostic, and / or imaging agents, can be delivered to a patient's tissues by injecting them into the patient's blood vessels. Using relatively simple medical procedures, those skilled in the art can distribute mitochondria locally and / or generally to a patient's tissues and / or cells for a variety of purposes. Furthermore, mitochondria can be used, for example, as a carrier agent for delivering therapeutic, diagnostic, and / or imaging agents to a patient's tissues. It is further noted that, compared to some conventional therapeutic regimens, including nanoparticles, mitochondria are non-toxic and do not induce substantial adverse immune or autoimmune responses.

[0039] Without intending to be bound by any theory, it is believed that injected mitochondria extravasate through the capillary wall by first attaching to the endothelium. After they are injected or infused into arteries, mitochondria may cross the vascular endothelium and be taken up by tissue cells through an endosomal actin-dependent internalization process.

[0040] Combined mitochondrial agents Combined mitochondrial agents include mitochondria physically associated with agents such as therapeutic agents, diagnostic agents, and / or imaging agents.

[0041] A therapeutic agent can be any agent with therapeutic or prophylactic use. Exemplary therapeutic agents include, for example, agents for treating ischemia-related disorders, cytotoxic agents for treating cancer, and the like, among many others. In some cases, mitochondria can deliver therapeutic agents to specific cells, such as tumor cells. A therapeutic agent can be, for example, an intracellular inhibitor, inactivator, toxin, arresting agent, and / or cytostatic / cytotoxic agent that, upon entry into the cell, inhibits, destroys, arrests, denatures, and / or modifies the cell so that it can no longer function normally and / or survive. A therapeutic agent can be, for example, an agent for restoring proper function of a cell, such as a DNA vector for gene therapy. A therapeutic agent can be, for example, an inorganic or organic compound; a small molecule (less than 500 daltons) or large molecule; a proteinaceous molecule such as a peptide, polypeptide, protein, post-translationally modified protein, or antibody; or a nucleic acid molecule such as double-stranded DNA, single-stranded DNA, double-stranded RNA, single-stranded RNA, or triple-helical nucleic acid molecule. In some embodiments, therapeutic agents can be derived from libraries of natural products or synthetic molecules derived from any known organism (e.g., animals, plants, bacteria, fungi, protists, or viruses). In some embodiments, therapeutic agents can be monomeric or polymeric compounds. Some exemplary therapeutic agents include cytotoxic drugs, 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 diagnostic agents to cells, in some embodiments, the diagnostic agents can be designed to measure intracellular conditions, such as intracellular pH and oxidative stress.

[0043] Imaging agents are agents utilized for use in imaging techniques. Techniques or modalities include, but are not limited to, X-ray, computed tomography (CT), magnetic resonance imaging (MRI), scintigraphy, fluorescence, ultrasound, and the like. Imaging agents can be fluorescent and / or radioactive. In some embodiments, imaging agents can also be diagnostic agents. Exemplary imaging agents include MitoTracker fluorophores (Thermo Fisher Scientific Inc.), CellLight® RFP, BacMam 2.0 (Thermo Fisher Scientific Inc.), pH-sensitive pHrodo fluorescent dyes (Thermo Fisher Scientific Inc.), 18 F‐rhodamine 6G, 18 These include, but are not limited to, F-labeled rhodamine B, magnetic iron oxide nanoparticles, and gold- and platinum-based nanoparticles.

[0044] As described above, a combined mitochondrial agent includes a mitochondria and a drug that are in direct and / or indirect physical contact with each other. For example, the drug may bind to the mitochondria, attach to the mitochondria, be embedded in the mitochondrial membrane, or be fully or partially enclosed in the mitochondria. In some cases, the pharmaceutical agent can be covalently bound to the mitochondria. In some cases, the drug is bound to a component of the mitochondrial membrane directly via a covalent bond (e.g., a carboxamide bond and a disulfide bond) or indirectly via a linker (e.g., a peptide linker) or another covalently bound drug. In other examples, the drug can be non-covalently bound to the mitochondria, for example, via 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, e.g., two different types of therapeutic agents, three different types of imaging agents, one therapeutic agent and one imaging agent, a therapeutic agent and a diagnostic agent, etc. Those skilled in the art will understand that any variations are possible.

[0046] One particularly useful linker for linking mitochondria and drugs provides sustained release of the drug upon injection.This can be achieved, for example, by using a hydrazone functional group.For example, the hydrazone is formed to covalently bind the drug to a component on the mitochondrial membrane.When this combined mitochondrial drug is taken up into cells, a change in pH causes the hydrazone to hydrolyze, releasing the drug bound to the cell.

[0047] In some embodiments, therapeutic, diagnostic, and / or imaging agents can be attached to the outer mitochondrial membrane using functionalized surface chemistries. In some cases, heterobifunctional chemistry can attach therapeutic, diagnostic, and / or imaging agents to the mitochondrial surface, and upon internalization, these agents can be released through interactions with intracellular esterases (e.g., via interactions with acetoxymethyl esters) or via UV or near-infrared light activation strategies. UV light activation and near-infrared light activation strategies are described, 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; 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 ("Ultraviolet Light-Mediated Drug Delivery: Principles, Applications, and Challenges"), each of which is incorporated by reference in its entirety.

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

[0049] The pharmaceutical compositions described herein can include mitochondria and / or combined mitochondrial agents and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, 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 Omnipaque, or a mixture thereof. In some embodiments, the pharmaceutically acceptable carrier is sterile mitochondrial buffer (300 mM sucrose; 10 mM K-HEPES (potassium buffered (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, pH 7.2); 1 mM K-EGTA, (potassium buffered ethylene glycol tetraacetic acid, pH 8.0)). In some embodiments, the pharmaceutically acceptable carrier is respiration buffer (250 mM sucrose, 2 mM KHPO, 10 mM MgCl, 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, which include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), sublingual, transdermal (e.g., topical), transmucosal, and rectal administration.

[0051] Pharmaceutical compositions can be formulated for various clinical uses, such as imaging, wound healing, injury treatment, organ preservation, improving mitochondrial function of organs or tissues, and skin care. In some cases, the pharmaceutically acceptable carrier is a contrast agent for imaging purposes. In some embodiments, the pharmaceutical composition may include a disinfectant, an antibacterial agent (e.g., antibiotic), an antifungal agent, a bactericide, an analgesic, an anesthetic, a steroid, a nutritional supplement, an essential oil, or the like. An anesthetic is an agent 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, polymyxins, rifamycins, spectinomycin, tetracyclines, vancomycin, teicoplanin, streptogramins, antifolates, sulfonamides, trimethoprim, pyrimethamine, nitrofurans, methenamine mandelate, methenamine hippurate, nitroimidazoles, quinolones, fluoroquinolones, isoniazid, ethambutol, pyrazinamide, para-aminosalicylic acid, cycloserine, capreomycin, ethionamide, prothionamide, thiacetazone, and viomycin. Antiseptics are antimicrobial substances that can be applied to living tissue / skin to reduce the likelihood of infection, sepsis, or putrefaction. Exemplary antiseptics 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, alclometasone dipropionate, fluocinolone acetonide, dexamethasone, methylprednisolone, desonide, pivorate, clocortolone pivorate, triamcinolone acetonide, prednicarbate, fluticasone propionate, flurandrenolide, mometasone furoate, desoximetasone, 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, vitamin B family, vitamin C, vitamin D family, vitamin E, and vitamin K. Ethereal oils include, but are not limited to, mint, sage, fir, lavender, basil, lemon, juniper, rosemary, eucalyptus, marigold, chamomile, orange, and the like. Many of these agents are described, for example, in WO2008152626, which is incorporated by reference in its entirety. Compositions containing mitochondria and / or combined mitochondrial agents can be formulated in any form, for example, liquid, semi-solid, or solid. Exemplary compositions include, inter alia, liquids, creams, salves, ointments, oils, emulsions, and liposomal formulations.

[0052] Methods of Making Compositions Comprising Mitochondria and / or Combined Mitochondrial Agents Isolation of mitochondria Mitochondria for use in the methods described herein can be isolated or provided from any source, such as 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 the like, and any mixture thereof. Exemplary tissues include, but are not limited to, liver tissue, skeletal muscle, heart, brain, and adipose tissue, among others. Mitochondria can be isolated from cells of autologous, allogeneic, and / or xenogeneic origin. In some cases, mitochondria are isolated from genetically modified cells, such as cells with 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. Exemplary methods for isolating mitochondria are described, for example, in 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); Frezza, C., Cipolat, S., & Scorrano, L, Organelle isolation: functional mitochondria from mouse liver, muscle and cultured filtroblasts. Nature protocols, 2(2), 287-295. (2007) (Organelle Isolation: Functional Mitochondria from Mouse Liver, Muscle, and Cultured Fibroblasts); and PCT applications entitled "Products and Methods for Isolating Mitochondria" (PCT / US2015 / 035584; WO2015192020), each of which is incorporated by reference.

[0054] Methods for creating combined mitochondrial agents One of skill in the art will appreciate that an agent can be bound to mitochondria in any number of ways, for example, by being attached to the mitochondria, by being partially or completely embedded in the mitochondrial membrane, by being enclosed in the mitochondria, or by being encapsulated within the mitochondria.

[0055] Without intending to be bound by theory or a particular approach, it is believed that the outer membrane of mitochondria is adhesive and therefore particularly suitable for combination with various drugs. In some embodiments, pharmaceutical agents can be attached to the outer membrane of mitochondria simply by incubation. For example, an effective amount of pharmaceutical agent can be thoroughly mixed with isolated mitochondria in a buffer solution, such as a respiratory buffer, at a temperature favorable to the isolated mitochondria, for example, 0°C to 26°C, 0°C to 4°C, or about 0°C, 4°C, or 26°C. This procedure helps to attach an effective amount of pharmaceutical agent (such as nanoparticles, DNA vectors, RNA vectors, etc.) to mitochondria.

[0056] In some embodiments, organic cations (e.g., rhodamine and tetramethylrosamine) are readily sequestered by functioning mitochondria due to the electrical potential on the mitochondrial membrane. Healthy mitochondrial membranes maintain a potential difference between the inside and outside of the organelle, referred to as the membrane potential. This membrane potential is a direct result of mitochondrial functional processes and can be lost if 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 aqueous matrix space. Similarly, in some other embodiments, anions can attach to the outer membrane of mitochondria due to their negative charge. To bind these pharmaceutical agents to mitochondria, an effective amount of the pharmaceutical agent should be thoroughly mixed with isolated mitochondria in a buffer, e.g., a respiratory buffer, at a temperature favorable to the isolated mitochondria, e.g., about 0°C or 4°C.

[0057] Therapeutic, diagnostic, and / or imaging agents can be attached to phospholipids, peptides, or proteins on the mitochondrial membrane via chemical bonds. For example, molecules including fluorophores (e.g., pHrodo Red (Thermo Fisher Scientific, Inc.)) and metal particles (e.g., 30 nm magnetic iron oxide nanoparticles (Sigma)) can be covalently attached to exposed amine groups on proteins and peptides exposed on the outer membrane of intact mitochondria using succinimidyl ester conjugates. These reactive reagents react with unprotonated aliphatic amine groups, such as the amine termini of proteins or the ε-amino groups of lysine residues, to form stable carboxamide bonds. In another example, when pharmaceutical agents, such as MitoTracker® OrangeCM™ Ros (Invitrogen, Carlsbad, CA, now Thermo-Fisher Scientific, Cambridge, MA), are 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 available for attaching therapeutic, diagnostic, and / or imaging agents to the surface of mitochondria (e.g., carboxylic acids, amine functionalization, etc.).

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

[0060] In many cases, therapeutic, diagnostic, and imaging agents can be simply mixed with isolated mitochondria and incubated in a buffer (e.g., respiration buffer) for a sufficient time (e.g., several minutes, 5 minutes, 10 minutes, or 1 hour) under preferred conditions (e.g., 0°C to 26°C, 0°C to 4°C, or about 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 autologous mitochondria protects the heart from ischemia-reperfusion injury). Each of the foregoing is incorporated by reference in its entirety.

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

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

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

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

[0066] Heart failure, also known as chronic heart failure, refers to a disorder in which the heart cannot 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 in the legs. Limited exercise capacity is also common in patients with heart failure. When used in this context, "treat" means improving at least one symptom of the disorder associated with the disease. Treatment often improves blood supply and improves one or more symptoms (e.g., shortness of breath, excessive fatigue, swelling in the legs).

[0067] Generally, the methods involve administering a composition described herein (e.g., a composition comprising isolated mitochondria or a composition comprising a combined mitochondrial agent) to a subject in need of, or determined to be in need of, such treatment.

[0068] In some aspects, the methods described herein are also 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] The present disclosure provides methods for minimizing heart failure, reducing the risk of heart failure, ameliorating at least one symptom of heart failure, and preventing or treating cell damage, tissue damage, and / or organ damage associated with heart failure in a subject at risk for heart failure.

[0070] As used herein, the term "at risk of heart failure" refers to an elevated risk of heart failure compared to the risk of heart failure of the average person in a 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 risk of heart failure of 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 risk of heart failure of 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 can be due to a variety of factors, such as genetic factors (e.g., genetic mutations), environmental factors (e.g., occupational risks, pollution), various diseases, medical procedures (e.g., surgery, organ / tissue transplant), behaviors (e.g., smoking, inactivity), etc. Once a subject is identified as being at risk for heart failure, a therapeutically effective amount of a composition described herein can be administered to the subject to reduce the risk of heart failure. Risk can also arise from potential medical procedures. As used herein, the term "medical procedure" refers to a course of action aimed at achieving a result in the delivery of health care. Medical procedures can include, for example, diagnostic procedures, therapeutic procedures, and surgical procedures. Some medical procedures include, for example, extracorporeal membrane oxygenation (ECMO), chemotherapy, radiation therapy, endotracheal intubation, gene therapy, anesthesia, resection, amputation, cardiopulmonary resuscitation (CPR), cryosurgery, endoscopic surgery, hemilaminectomy, image-guided surgery, knee cartilage replacement therapy, laminectomy, laparoscopic surgery, lithotomy, lithotripsy, lobotomy, neovaginoplasty, radiosurgery, stereotactic surgery, vaginoplasty, transplantation (e.g., tissue or organ transplant), xenotransplantation, etc. A healthcare provider 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 high blood pressure, myocardial infarction, abnormal heart valves, cardiomyopathy, a family history of heart disease, and diabetes. In these cases, a therapeutically effective amount of a composition described herein can be administered to the subject prior to these procedures to minimize risk.

[0072] In some embodiments, the methods described herein can be used to treat or prevent heart failure in a subject, hi 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 enlargement of the myocardium surrounding the right ventricle. RVH usually occurs due to chronic lung disease or a structural cardiac defect. One of the most common causes of RVH is pulmonary hypertension (PH). Pulmonary hypertension is characterized by elevated blood pressure in the blood vessels supplying the lungs. Pulmonary hypertension can lead to elevated pulmonary artery pressure. The right ventricle attempts to compensate for this increased pressure by changing its shape and size. Hypertrophy of individual myocytes 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 develops in response to a structural cardiac defect. 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 midwall. Other risk factors for RVH include smoking, sleep apnea, and strenuous activity.

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

[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 a disease involving the heart. Disease processes that can cause LVH include any condition that increases the afterload the heart must contract, as well as several primary diseases of the heart muscle. Causes of increased afterload that can lead to LVH include aortic stenosis, aortic regurgitation, and hypertension. The primary disease of the heart muscle 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] Thus, in one aspect, the present disclosure provides a method for reducing the risk of developing left ventricular hypertrophy. The method includes identifying a subject as being at risk of developing left ventricular hypertrophy and administering a composition described herein to the subject. In some embodiments, the method includes identifying the 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, occurs when the heart cannot pump enough to maintain blood flow to meet the body's needs. Signs and symptoms of heart failure typically include shortness of breath, excessive fatigue, and leg swelling. Limited exercise capacity is also a common feature. Common causes of heart failure include previous myocardial infarction (heart attack), high blood pressure, atrial fibrillation, valvular heart disease, excessive alcohol use, infection, 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 fails, blood backs up (congestion) into the lungs, causing respiratory symptoms and fatigue due to an insufficient supply of oxygenated blood. Right-sided heart failure is often caused by pulmonary heart disease, which is usually caused by difficulties with pulmonary circulation such as pulmonary hypertension or pulmonary valve stenosis.

[0078] Thus, in one aspect, the present disclosure provides a method for reducing the risk of developing heart failure. The method includes identifying a subject as being at risk of developing heart failure and administering a 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 methods described herein can 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, an electrocardiogram (ECG) is often used to show signs of elevated voltage from the heart in patients with LVH.

[0080] The present disclosure also provides methods for treating ischemic heart 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 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, not during reperfusion, and maintaining mitochondrial respiratory function promotes contractile recovery and reduces myocardial infarction size.

[0081] The method described herein can be used to treat ischemic heart. For example, an effective amount of isolated mitochondria can be injected into the blood vessel of a subject, for example, into the coronary vasculature of a subject. For example, about 1 × 10 7The mitochondria can be administered to the coronary vasculature of a subject. After transplantation, the injected mitochondria are internalized by cardiomyocytes, increasing oxygen consumption, upregulating chemokines that enhance cardiac function after infarction, and upregulating the expression of protein pathways important for maintaining myocardial energy. In another example, an effective amount of mitochondria can be injected directly into the area at risk (region of focal ischemia). The injection can be repeated several times at different sites in the heart.

[0082] Reperfusion injury is tissue damage caused by the blood supply when blood returns to the tissue after a period of ischemia or oxygen deprivation. The lack of oxygen and nutrients during the ischemic period leads to inflammation and oxidative damage when blood flow is restored. The inflammatory response further leads to tissue reperfusion injury. Therefore, in some cases, treatment also includes administering an immunosuppressant to the patient. The immunosuppressant can be administered, for example, separately or as a co-treatment with the mitochondrial agent. Alternatively, or in addition, the immunosuppressant can be bound to mitochondria to form a combined mitochondrial agent that can be used for treatment. A particularly useful immunosuppressant is a bisphosphonate.

[0083] Ischemia / reperfusion injury in several other organs is often associated with mitochondrial damage and 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, cyanosis, and gangrene. The described methods can also be used to treat ischemic injury in these organs / tissues. For these treatments, isolated mitochondria and / or combined mitochondrial agents can be injected directly into the organ tissue or into blood vessels carrying blood to the target organ / tissue or injury site in a subject.

[0084] cardiac surgery Isolated mitochondria and / or combined mitochondrial agents can be delivered to the heart to reduce shock and allow the heart to recover from a surgical procedure (e.g., cardioplegia) without increasing the heart's heart rate or oxygen demands. In some embodiments, the methods involve direct injection of isolated mitochondria and / or combined mitochondrial agents into the heart. In some methods, the isolated mitochondria and / or combined mitochondrial agents are injected into a coronary artery.

[0085] Imaging In many cases, mitochondria can be attached to imaging agents by co-incubating them with the imaging agent. Such imaging agents include MitoTracker and pHrodo fluorophores from ThermoFisher Scientific Inc. 18 F-rhodamine 6G, and iron oxide nanoparticles.

[0086] The combined mitochondrial agent containing the imaging agent can be injected into tissue, for example, cardiac tissue, or perfused through blood vessels.Tissue containing labeled mitochondria can be examined using imaging techniques such as positron emission tomography (PET), micro-computed tomography (μCT), magnetic resonance imaging (MRI), bright-field microscopy, 3D super-resolution microscopy, etc.Those skilled in the art will understand that other imaging techniques or modalities can be used.These include, but are not limited to, X-ray, scintigraphy, fluorescence, and ultrasound.

[0087] Administration The isolated mitochondria and combined mitochondrial agents can be administered to patients by intravenous, intraarterial, intraperitoneal, intramuscular injection, and / or via intraosseous infusion. In some embodiments, the isolated mitochondria and combined mitochondrial agents can be delivered by direct injection or vascular infusion.

[0088] When mitochondria are injected into tissue, they are taken up by cells around the injection site. Thus, in some embodiments, the injection site is the target site. In some other embodiments, mitochondria are injected into a blood vessel that carries blood to the target site, such as an organ, tissue, or injury site. Without intending to be bound by any theory, evidence suggests that mitochondria delivered by direct injection are internalized by cells via actin-dependent endocytosis. However, the uptake of mitochondria via vascular delivery appears to be more complex. The rapid and widespread uptake of mitochondria when delivered by vascular injection suggests that a mechanism is involved that allows mitochondria to rapidly cross the vascular wall. Several studies support the notion that cells can routinely escape from the circulation. It has been shown that certain cardiac and mesenchymal stem cells appear to actively exit the vasculature in a process distinct from extravasation (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 (Vascular perosis as an alternative mechanism of cell extravasation). Stem cell migration through the vascular wall requires extensive endothelial remodeling.Mitochondria may use similar remodeling mechanisms to cross blood vessel walls. Another possible mechanism of mitochondrial uptake may be leakage. Some cells routinely escape from the circulation. For example, leukocyte extravasation (i.e., leakage) between venous endothelial cells is a well-understood process involving cell adhesion proteins. Furthermore, injected mitochondria may extravasate through the capillary wall through the interendothelial space. After mitochondria cross the vascular endothelium, they are taken up by tissue cells through an actin-dependent process of endosomal internalization.

[0089] The mitochondria or combined mitochondrial agents can be administered to a subject as a single one-time treatment, or as multiple treatments, e.g., a course of treatment continuing intermittently or continuously for about 1, 2, 5, 8, 10, 20, 30, 50, or 60 days, a year, indefinitely, or until a physician determines that administration of the 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, e.g., cardiac tissue. The injection can optionally be repeated several times at different sites in 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, and each injection site can be filled with, for example, about 1.2 x 10 6 can receive mitochondria.

[0091] Those skilled in the art will understand that the amount of mitochondria and / or combined mitochondrial agents, e.g., a composition containing mitochondria and / or combined mitochondrial agents, to be administered to a patient will vary depending, for example, on the type of disorder being treated, the route of administration, the duration of treatment, the size of the area being treated, and / or the location of the treatment site on the patent, among other factors. Those skilled in the art will be able to determine the dosage to be administered depending on these and other variables. For example, a total of about 1 x 10 7can be administered to a subject's blood vessels, for example, to treat focal ischemia of the myocardium. As another example, for larger organs or affected areas, more mitochondria, for example, 1×10 10 From 1×10 14 mitochondria can be injected into the bloodstream. Conversely, for small, localized lesions, 1x10 3 From 1x10 6 can be injected into a patient. Thus, an effective amount of mitochondria or a combined mitochondrial agent (or a composition comprising same) is the total amount of mitochondria or combined mitochondrial agents sufficient to produce the desired therapeutic effect. An effective amount can be, for example, at least or about 1 x 10 2 mitochondria, or a combination of mitochondrial agents, e.g., about 1 x 10 3 to approximately 1 × 10 14 , about 1×10 4 to approximately 1 × 10 13 , about 1×10 5 From about 1x10 12 , approximately 1x10 6 From about 1x10 11 , approximately 1x10 7 From about 1x10 10 , approximately 1x10 3 From about 1x10 7 , approximately 1x10 4 From about 1x10 6 , approximately 1x10 7 From about 1x10 14 , or about 1x10 8 From about 1x10 13 , approximately 1x10 9 From about 1x10 12 , approximately 1x10 5 From about 1x10 8 , or at least or about 1x10 3 , 1x10 4 , 1x10 5 , 1×10 6 , 1x10 7 , 1x10 8 , 1x10 9 , 1x10 10 , 1x10 11 , 1x10 12, 1x10 13 , or at least or about 1x10 14 , or for example, 1 x 10 14 As used herein, the term "total amount" in the context of administration to a patient can refer to the total amount of mitochondria or combined mitochondrial agents in a single administration (e.g., one injection, one dose administered by infusion) or in multiple administrations (e.g., multiple injections), depending on the dosing regimen being followed.

[0092] The isolated mitochondria and / or combined mitochondrial agents can be administered to a subject by various routes, e.g., direct injection, vascular delivery, every 12-24 hours. In some embodiments, the isolated mitochondria or combined mitochondrial agents can be administered to a subject by various routes, e.g., direct injection, vascular infusion, every 5-10 minutes (e.g., every 5 minutes, every 10 minutes).

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

[0094] The isolated mitochondria and / or combined mitochondrial agents can be administered to a subject at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, or 30 days, or at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, or at least or 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 can 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, 24 months, or within about 1, 2, 3, 4, or 5 years after the subject is identified as being at risk for 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 can 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, 24 months, or within about 1, 2, 3, 4, or 5 years after the subject is identified as having a cardiovascular disorder or some other disorder that may lead to heart failure (e.g., obesity, right ventricular hypertrophy, etc.).

[0096] In some embodiments, isolated mitochondria and / or combined mitochondrial agents may be administered to a subject at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, or 30 days, or at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, or at least or about 1, 2, 3, 4, or 5 years after 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 can be injected directly into tissues or organs through needles of gauges 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, and 34. In some other cases, isolated mitochondria or combined mitochondrial agents can be delivered to the target site by catheter.

[0098] In some cases, the mitochondria are freshly isolated and viable. The mitochondria or combined mitochondrial agents can be administered to a subject within about 5 minutes, about 10 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 60 minutes, about 70 minutes, about 80 minutes, about 90 minutes, about 100 minutes, about 110 minutes, or about 120 minutes after the mitochondria are isolated. In some cases, the mitochondria or combined mitochondrial agents are administered to a subject within about 5 minutes, about 10 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 60 minutes, about 70 minutes, about 80 minutes, about 90 minutes, about 100 minutes, about 110 minutes, or about 120 minutes after the mitochondrial isolation process is initiated. The mitochondria and / or combined mitochondrial agents can optionally be stored for a short period of time (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) before use.

[0099] The mitochondria for treatment can be isolated from cells or tissues of autologous source, allogeneic source and xenogeneic source.In some cases, mitochondria are collected from the cultured cells or tissues of a subject, and these mitochondria are administered to the same subject.In other cases, mitochondria are collected from the cultured cells or tissues of a second subject, and these mitochondria are administered to the first subject.In some cases, mitochondria are collected from the cultured cells or tissues of different species (mouse, pig, yeast, etc.). [Example]

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

[0101] Example 1: Exemplary methods for isolating mitochondria from tissue samples or cultured cells preparation The following solutions can be prepared to isolate intact, viable, and respiration-capable mitochondria. To successfully isolate mitochondria using the methods of the present invention, solutions and tissue samples are kept on ice to maintain mitochondrial viability. Even when kept on ice, isolated mitochondria will exhibit 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] 1 M K‐HEPES stock solution (adjust pH to 7.2 with KOH). 0.5MK‐EGTA stock solution (adjust pH to 8.0 with KOH). 1M KH2PO4 stock solution. 1M MgCl2 stock solution.

[0103] Homogenization buffer (pH 7.2): 300 mM sucrose, 10 mM K-HEPES, and 1 mM K-EGTA. The buffer can be stored at 4°C. Respiration 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. 10X PBS stock solution: Dissolve 80 g NaCl, 2 g KCl, 14.4 g NaHPO, and 2.4 g KHPO in 1 L double-distilled HO (pH 7.4). 1XPBS is prepared by pipetting 100 mL of 10XPBS into 1 L of double distilled HO. Subtilisin A stock is prepared by weighing 4 mg of subtilisin A into a 1.5 mL microfuge tube. The stock can be stored at -20°C until use. Prepare BSA stock by weighing 20 mg of BSA into a 1.5 mL microfuge tube. Stocks can be stored at -20°C until use.

[0104] Isolating mitochondria from tissue A diagram outlining the steps in isolating mitochondria using tissue dissociation and differential filtration is shown in Figure 1. Transfer two 6 mm biopsy sample punches to 5 mL of homogenization buffer in a Dissociation C tube and homogenize the sample using the 1-minute homogenization program on the tissue dissociator (A). Add 250 µL of subtilisin A stock solution to the homogenate in the Dissociation C tube and incubate on ice for 10 minutes (B). Centrifuge the homogenate at 750 x G for 4 minutes (an optional step). Filter the homogenate through a pre-wetted 40 µm mesh filter in a 50 mL conical centrifuge tube on ice, then add 250 µL of BSA stock solution to the filtrate (C). Re-filter the filtrate through a new pre-wetted 40 µm mesh filter in a 50 mL conical centrifuge on ice (D). The filtrate is refiltered through a new, pre-wetted 10 μm mesh filter in a 50 mL conical centrifuge tube on ice (E). The filtrate is refiltered through a new, pre-wetted 6 μm mesh filter in a 50 mL conical centrifuge tube on ice (F). The resulting filtrate can be used immediately or concentrated by centrifugation. For concentration, the filtrate is transferred to a 1.5 mL microfuge tube and centrifuged at 9000 x g for 10 min at 4 °C (F). The supernatant is removed, and the pellet containing mitochondria is resuspended and placed in 1 mL of respiration buffer (G).

[0105] Immediately prior to isolation, dissolve subtilisin A in 1 mL of homogenization buffer. Immediately prior to isolation, dissolve BSA in 1 mL of homogenization buffer. Collect two fresh tissue samples using a 6 mm biopsy sample punch and store them in 1X PBS in a 50 mL conical centrifuge tube on ice. 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 attaching the Dissociation C Tube to the tissue dissociator and selecting the pre-set mitochondrial isolation cycle (60 seconds of homogenization).

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

[0107] Add 250 μL of freshly prepared BSA stock solution 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-wet the filter with homogenization buffer, and filter the homogenate into the 50 mL conical centrifuge tube on ice. Place a 10 μm filter in a 50 mL conical centrifuge tube on ice, pre-wet the filter with homogenization buffer, and filter the homogenate into the 50 mL conical centrifuge tube on ice. Transfer the filtrate into two pre-chilled 1.5 mL microfuge tubes and centrifuge at 9000 x g for 10 minutes at 4 °C. Remove the supernatant and resuspend the pellet in 1 mL of ice-cold respiration buffer. Mitochondria isolated from tissues can be 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, e.g., human fibroblasts, are used rather than a biopsy sample.

[0109] Mitochondrial number The number of viable mitochondria was measured by labeling an aliquot (10 μl) of isolated mitochondria with MitoTracker Orange CMTMRos or MitoTracker Red CMXMRos (5 μmol / l; Invitrogen, Carlsbad, CA, now Thermo-Fisher Scientific, Cambridge, MA). An aliquot of labeled mitochondria was spotted onto a slide and counted using a spinning-disk confocal microscope equipped with a 63x C-apochromat objective (1.2 W Korr / 0.17 NA, Zeiss). Mitochondria were counterstained with the mitochondrial-specific dye MitoFluor Green or MitoTracker Deep Red FM (Invitrogen, Carlsbad, CA, now Thermo-Fisher Scientific, Cambridge, MA). Appropriate wavelengths were selected to measure autofluorescence and background fluorescence using unstained cells or tissues. Briefly, 1 μl of labeled mitochondria was placed on a microscope slide and covered. Mitochondrial number is measured using MetaMorph Imaging Analysis software at low magnification (×10) covering the entire specimen area.

[0110] Example 2: Exemplary methods for preparing combined mitochondrial agents Mitochondria and 18 F-Rhodamine 6G combined with potential 18F-Rhodamine 6G (40–100 μCi in a volume of 20 μl) was diluted to a volume of 1.0 mL with mitochondria isolation solution A (homogenization buffer: 300 mM sucrose, 10 mM K-HEPES, and 1 mM K-EGTA, pH 7.2) at 4 °C, and then the isolated mitochondria (0.5 ml, 1 × 10) were collected in mitochondria isolation solution A. 7 -1x10 8 The mixture is thoroughly mixed with 18 F-rhodamine 6G is electrophoretically distributed across the inner mitochondrial membrane into the mitochondrial matrix depending on the potential and is therefore sequestered by functioning mitochondria. 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, resuspending the pellet each time in mitochondrial isolation solution A. After the final wash, resuspend the pellet in respiration buffer.

[0111] Combining mitochondria with iron oxide nanoparticles via the outer mitochondrial membrane Iron oxide nanoparticles containing succinimidyl esters (10 mg) were suspended in respiration buffer at 4 °C and isolated mitochondria (1 x 10 7 ~1x10 8 Mix thoroughly with 1.0 ml of 1% ammonium hydroxide solution containing 1% iron oxide. Iron oxide binds to mitochondrial amine groups on the outer mitochondrial membrane via a succinimidyl ester-amine 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, resuspending the pellet each time in mitochondrial isolation solution A. After the final wash, resuspend the pellet in respiration buffer.

[0112] Combining mitochondria with two medicines 18 F-rhodamine 6G (40–100 μCi in a volume of 20 μl) was combined with iron oxide nanoparticles containing succinimidyl esters (10 mg) and diluted to a volume of 1.0 mL with mitochondria isolation solution A at 4 °C, followed by dissolving isolated mitochondria in mitochondria isolation solution (0.5 mL. 1 × 107 -1x10 8 Mix thoroughly with mitochondrial isolation solution A (containing mitochondrial DNA). Incubate the mixture on ice for 10-30 minutes. Wash the mixture three times by centrifuging at 9,000 rpm (10,000 g) for 10 minutes, resuspending the pellet each time in mitochondrial isolation solution A. After the last wash, resuspend the pellet in respiration buffer.

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

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

[0115] method: The RVH / RVF model was created in immature piglets (n = 6 / group) by 50% pulmonary artery banding (gradient = 15-20 mmHg). Sham-operated 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 vehicle (PAB-V) by injection into the RV free wall. At the time of euthanasia, tissues were analyzed histologically 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 the time of euthanasia.

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

[0117] Conclusion: Mitochondrial transplantation maintained RV hypertrophic adaptation and preserved contractile function. Directly addressing myocardial dysfunction by targeting mitochondrial dysfunction can be used to treat patients with pulmonary diseases that affect right heart function.

[0118] Example 4: Transplantation of autologous mitochondria 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 the progression to RVF is cardiomyocyte apoptosis due to mitochondrial dysfunction. Because transplantation of respiratory-competent mitochondria is available, the purpose of this study was to determine whether localized intramyocardial injection of autologous mitochondria can treat heart failure.

[0119] The beneficial effects of transplanted mitochondria from different sources were measured in cultured hypertrophied cardiomyocytes. A pulmonary artery ligation model of RVH / RVF in premature piglets with sham-operated controls (n = 6 / group) was used for treatment with autologous mitochondria isolated from the piglets' own calf muscles (PAB-M) and vehicle (PAB-V) injected into the RV free wall. Animals were followed for 8 weeks by echocardiography (free wall thickness, contractile function), and Dp / Dtmax were measured at the study endpoint, where histological analysis of cardiomyocyte hypertrophy, fibrosis, and apoptosis was performed. Neither internalization nor ATP levels were significantly different depending on the source of mitochondria 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, whereas PAB-V was already severely dilated (0.5 ± 0.04 cm vs. 0.28 ± 0.08 cm; P = 0.01). Contractile function at baseline was not different but was significantly reduced in PAB-V hearts compared with a significant improvement in PAB-M. This was also reflected in Dp / Dtmax at the study endpoint. There was minimal apoptotic cardiomyocyte loss and fibrosis in C, but significant loss and fibrosis, with the greatest number of hypertrophied PAB-V 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). Addressing myocardial dysfunction directly via mitochondrial transfer preserves RV hypertrophic adaptation and preserves contractile function.

[0120] Methods and Results: Animal models Immature male Yorkshire piglets (N = 18) weighing 5–10 kg underwent either pulmonary artery banding (PAB) or sham surgery. Piglets were sedated with Telazol (4.5 mg / kg im), xylazine (2 mg / kg im), and atropine (0.04 mg / kg im). After tracheal intubation, ventilation with isoflurane (1–3%) and air was initiated. EKG, blood oxygen saturation (maintained at >97%), body temperature, and end-tidal carbon dioxide were monitored. Femoral arterial and venous lines were placed. Piglets were positioned on their right side, draped, and a left thoracotomy was performed at the fourth intercostal space. Lidocaine (1%, iv) was administered before thoracotomy to prevent ventricular fibrillation. The pulmonary artery (PA) was dissected from the ascending aorta and ligated while monitoring RV and distal PA pressures via needle puncture. The PA was ligated to 50% of its original diameter. A 4F angiography catheter (Merit Medical Systems, South Jordan, UT) was inserted into the PA and connected to a PowerLab data acquisition system (DAQ, ADInstruments, Series 16 / 35) to obtain data for baseline pressure calculations. Baseline echocardiograms were obtained epicardially before and after placement of the PA band. The thoracotomy was closed in three layers, and pleural air was evacuated via a chest tube. Bupivacaine (0.25%; <0.03 mg / kg) was infused as local analgesia, and postoperative systemic analgesia was provided via Benamin / Glunixin meglumine (1-2 mg / kg im) and a fentanyl patch (1-4 μg / kg transdermal) for the first 72 hours. Piglets were allowed to recover in a 37°C incubator and then immediately returned to their pens. Sham surgery (C, N = 6) involved opening and closing the chest with local manipulation at the site of the PA. Following intraoperative echocardiography, the progression of RV hypertrophy was measured biweekly. During these procedures, 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 RV free wall, following the same anesthesia protocol as above. Under sterile conditions, muscle biopsies were taken from the gastrocnemius muscles of piglets, and mitochondria were isolated as described above. Under direct vision 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 of the RV free wall using a 30G needle. Echocardiography was performed before injection and immediately after the mitochondria / vehicle injection. The incision was closed in layers, and the animals were allowed to recover.

[0122] All animals survived for an additional 4 weeks (8 weeks after the initial PAB) and were monitored by echocardiography every other week. At the study endpoint, piglets were anesthetized in the same manner as above, except that anesthesia was maintained via face mask rather than intubation. A median sternotomy was performed, and PA (proximal and distal to the PA band) and aortic pressures were invasively measured using a 4F angiography catheter (Merit Medical Systems, South Jordan, UT). Additionally, PA and RV pressure-volume curves were calculated from data obtained using a 7F Scisense pressure-volume (PV) loop catheter (Transonic, Ithaca, NY) 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, NY). After all measurements were taken, the animals were euthanized with Fetal Plus®, and the hearts were excised and flushed with phosphate-buffered saline (PBS) on ice. RV free wall biopsies were obtained, snap-frozen, and stored in liquid nitrogen until further use. Individual RV free wall tissues were embedded in optimal cutting temperature (OCT) compound, snap-frozen, and stored at -80°C until sectioning. Fresh RV free wall tissue was used for wet / dry weight calculations.

[0123] Isolated cardiomyocyte culture model Using a cell culture model of neonatal rat cardiomyocytes, we measured mitochondrial internalization in hypertrophied cardiomyocytes. Furthermore, the functional benefits of different sources of mitochondria were tested in this model. Pharmacologically induced hypertrophied samples were compared with nonhypertrophied control samples. All isolated cell experiments were performed in duplicate.

[0124] Briefly, 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). After 2 days of culture, 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, were collected by punch biopsy from the gastrocnemius and soleus muscles obtained from dam rats and compared with RV myocardial mitochondria. This method yields >99.5% viable mitochondria from a 100 mg tissue sample. Muscle tissue was homogenized with a commercially available tissue dissociator in homogenization buffer (300 mM sucrose, 10 mM K-HEPES, and 1 mM K-EGTA, pH 7.2 at 4°C), followed by the addition of 250 μl of a buffer solution containing subtilisin A to 1 ml of homogenization buffer. The homogenate was mixed by inversion, incubated on ice for 10 min, and then differentially filtered. The filtrate was transferred to two pre-chilled microfuge tubes and centrifuged at 9,000 x g for 10 min at 4°C. The supernatant was removed, and the combined pellets were resuspended in 1 ml of ice-cold respiration 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, IN).

[0126] Measurement of internalization of transplanted mitochondria Mitochondria were prelabeled with pHrodo red particle label (ThermoFisher, Waltham, MA) for 10 minutes at 4°C and washed four times with respiration buffer. PHrodo fluorescence provides a positive indicator of internalization because it only follows uptake by viable mitochondria. Labeled mitochondria were resuspended in fresh respiration buffer, and the final wash supernatant was saved. This wash solution was used to determine nonspecific labeling by incubating control cells with this supernatant (data not shown). Labeled mitochondria (1x100 / well) were co-cultured with cardiomyocytes (~50,000 / well). After 24 hours, the medium was removed, the 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 then incubated with primary antibodies diluted 1:1000 in 10% fetal bovine serum (FBS) in PBS for 1 hour. Cardiomyocyte desmin was used as the primary antibody, along with a species-appropriate Alexa Fluor 488-conjugated secondary antibody (ThermoFisher, Waltham, MA). Nuclei were simultaneously stained using 4',6-diamidino-2-phenylindole (DAPI) (ThermoFisher). Detection of internalization was based on red-fluorescent mitochondria into green-destroyed cardiomyocytes. Internalization was assessed using a Zeiss fluorescence microscope.

[0127] Measurement of mitochondrial function ATP content was measured using the ATPlite Luminescent ATP Detection Assay System (Perkin Elmer, Waltham, MA). A separate set of cells was used for these experiments because fluorescent dyes that label mitochondria may interfere with mitochondrial function.

[0128] Echocardiography Echocardiographic measurements were obtained at baseline, before treatment (4 weeks after ligation), and at the study endpoint (8 weeks after ligation). All studies were performed using a Philips iE33 device (Philips Healthcare, Amsterdam, The Netherlands) equipped with an S8-3 transducer, and all cycles were recorded with simultaneous ECG recordings. RV function by fractional area change (FAC) and tricuspid annular plane systolic excursion (TAPSE) was assessed from four-chamber views. RV free wall thickness was measured at end-diastole on M-mode recordings obtained from the parasternal long axis (PLAX) and parasternal short axis (PSA) views.

[0129] Invasive pressure-volume (PV) measurement PA and RV pressure-volume curves were calculated from data obtained using a 7F Scisense pressure-volume (PV) loop catheter (Transonic Systems Inc., Ithaca, NY). This catheter was inserted via the right adnexa and secured with 4-0 prolene sutures (Ethicon Inc., Somerville, NJ). The PV loop catheter was connected and automatically calibrated using an ADVantage pressure-volume system (ADV500, Transonic, Ithaca, NY). Measurements were obtained at baseline and at the time of euthanasia. Data were acquired using a Powerlab data acquisition system (DAQ, ADInstruments, Series 16 / 35) and analyzed with the provided LabChart 7 Acquisition software (AD Instruments, Sydney, Australia). RV peak developed 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] Histological analysis of RV tissues RV tissues were embedded in optimal cutting temperature (OCT) compound, flash-frozen, and stored at -80°C until further use. Sections were taken, and the frozen slides were stored at -80°C until used for staining. All slides were visualized using a Zeiss Observer.Z1 fluorescence microscope equipped with a Nikon 20x objective (NA = 20x / 0.45). Ten randomly selected fields from each slide were photographed with a Leica digital color camera and analyzed using ImageJ (version 2.0.0‐rc‐43, obtained from the National Institutes of Health, Bethesda, MD).

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

[0132] Measurement of myocardial apoptosis Without being bound by theory, cardiomyocyte apoptosis has been shown to be primarily the result of mitochondrial dysfunction. Therefore, cardiomyocyte apoptosis was measured by TUNEL staining using the ApopTagPlus Fluorescein In Situ Apoptosis Detection Kit (MilliporeSigma, Burlington, MA). Cardiomyocytes were counterstained with desmin (1:50, Santa Cruz Biotechnology Inc., Desert Sun, TX) and nuclei with DAPI (1:1000, Dako, Carpinteria, CA). TUNEL-positive nuclei were counted manually. The total number of nuclei per field was measured using ImageJ. Data were expressed as the ratio of apoptotic nuclei per 1000 cardiomyocyte nuclei.

[0133] Measurement of myocardial fibrosis Another set of tissue sections was stained with Masson's trichrome, resulting in fibrotic (collagen-rich) areas appearing blue and myocardium appearing red. The blue and red areas (fibrosis and myocardium) were measured, and the ratio served as an estimate of fibrosis. Slides were visualized with a Nikon 10x objective. Ten randomly selected fields were acquired per tissue sample and analyzed with 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 normal distribution of the data, ANOVA and Bonferroni post hoc analysis for multiple group comparisons were performed to obtain statistical significance (SPSS 23, IBM Corporation, Armonk, NY). Probability values ​​of 0.05 or less were considered statistically significant.

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

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

[0137] Animal models All 18 piglets survived to the study endpoint. The mean gradient (mmHg, mean ± SEM) measured across the PA band at the study endpoint was not significantly different between the PAB-V (12.1 ± 1.6) and PAB-M (9.7 ± 1.9) groups (P = 0.8). Body weight (kg) did not differ among 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 study endpoint (C: 17.3 ± 3.1, PAB-V: 16.6 ± 1.2, PAB-M: 17.8 ± 0.9; P = 0.4). However, total heart weight (grams) at the study endpoint was significantly higher in PA-banded animals compared with sham-operated controls (C: 100.6 ± 4.7 vs. PAB-V: 132.4 ± 13 and PAB-M: 141.6 ± 12.8; P < 0.05). RV wet / dry weight ratios (wet / dry) were not significantly different 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 assessed histologically by calculating the ratio of myocardial area to the number of nuclei per field. Both PAB groups showed a significant increase in muscle mass compared with sham-operated 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 PAB-V hearts exhibiting the greatest number of apoptotic 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 also exhibited swollen mitochondria and decreased cristae (Figures 13A-13C). These findings were also supported by a significant increase in myocardial fibrosis in vehicle-treated hypertrophied hearts compared with mitochondria-treated hypertrophied hearts (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 assessment of myocardial hypertrophy, right ventricular wall thickness in centimeters was measured by end-diastolic M-mode recording. Baseline wall thickness was not significantly different between groups before intervention (C: 0.25 ± 0.01, PAB-V: 0.24 ± 0.01, PAB-M: 0.25 ± 0.01; P = 0.48), but 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 study endpoint, mitochondria-treated hearts maintained their wall thickness, while vehicle-treated hearts returned to baseline, exhibiting dilation (Pab-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 systolic excursion (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 with sham controls 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 treatment with mitochondrial therapy (PAB-V vs. PAB-M; P = 0.9). Four weeks after mitochondrial transplantation, treated hypertrophied hearts were significantly more functionally superior to vehicle-treated hearts (PAB-V: 6.7 ± 0.2 vs. PAB-M: 12.2 ± 0.4; P < 0.001), with no difference between mitochondria-treated hearts and sham-operated controls (PAB-V: 13 ± 0.5 vs. PAB-M: 12.2 ± 0.4; P = 0.42) (Figures 6 and 19A-19C).

[0141] The functional area change (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 was significantly different for the hypertrophy group 4 weeks after ligation compared with sham-operated controls (C: 43 ± 1.6, PAB-V: 23.7 ± 1.5, PAB-M: 25 ± 2.5; P < 0.001). Both hypertrophy groups were not different from each other before mitochondrial treatment, but at the study endpoint, systolic function of vehicle-treated hearts was significantly reduced compared with mitochondrial-treated and sham-operated controls (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 with groups C and PAB-M at the study endpoint, but the difference did not reach significance (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 compared with sham-operated controls at the study endpoint, but did not reach significance (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 began 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 in mitochondria-treated hypertrophied hearts compared with vehicle-treated hearts at the study endpoint (PAB-V: 506.6 ± 88.1 vs. PAB-M: 894.9 ± 119.23; P < 0.05). Meanwhile, 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 RV hypertrophic adaptation and preserved contractile function. Directly addressing myocardial dysfunction by targeting mitochondrial dysfunction can be used to treat patients with pulmonary diseases that affect right heart function.

[0145] The goal of this study was to delay the onset of heart failure by targeting defects affecting mitochondrial energetics. Without being bound by theory, cardiac mitochondrial dysfunction is crucial in heart failure, in part due to the increased energy demands of the hypertrophied RV. Our intervention aimed to improve mitochondrial function to maximize energy production through the transplantation of respiratory-competent mitochondria. We established that autologous exogenous mitochondria obtained from skeletal muscle sources internalized into hypertrophied cardiomyocytes and equivalently increased mitochondrial function as mitochondria obtained from RV myocardium. We also established that transplantation of autologous mitochondria in a large animal model of pulmonary artery banding protected cardiomyocytes from apoptotic cell loss. Furthermore, maintaining hypertrophic growth led to the preservation of contractile function compared to untreated hypertrophied hearts that showed signs of dilation and systolic dysfunction. Thus, in some embodiments, methods are described herein for preventing or reducing apoptotic cell loss in cardiomyocytes and preserving or improving cardiac contractile function, comprising administering to a patient a composition comprising mitochondria.

[0146] Without being bound by theory, the right ventricle is a major determinant of prognosis in pulmonary heart failure. RV adaptation and maladaptation are crucial in the disease process. Initially, RV contractility increases due to changes in muscle properties and compensatory hypertrophy until a certain uncoupling point occurs where afterload exceeds contractility. This indicates maladaptation, a hallmark of ventricular dilatation. In right heart failure, treatment is primarily limited to targeting pulmonary function rather than directly interfering with the critical energy deficit of the RV myocardium, which is a consequence of 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; 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; 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 bound by theory, mitochondrial dysfunction, resulting in a reduced ability to generate ATP, is known to affect cardiac function because approximately 90% of cellular 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, bioenergetics, calcium transport, reactive oxygen species (ROS) generation, and activation of cell death pathways. This results in a vicious feedforward cycle that leads to cardiomyocyte death by apoptosis (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 injury. Studies of humans with end-stage heart failure have shown that markers of energy metabolism are decreased in the failing heart, giving rise to the idea that the failing heart is an engine that runs 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, rather than starving failing myocardium of energy, mitochondria play a more complex role in regulating metabolism and cell death. A piglet model of right heart failure demonstrates impaired oxidative phosphorylation and significant structural damage, highlighting the importance of mitochondrial function and structural quality in pressure overload-induced right heart failure (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). Our data demonstrate that cardiomyocytes were better preserved from apoptotic cell loss after treatment with mitochondria, whereas vehicle-treated hypertrophied hearts exhibited more apoptotic cell death.

[0148] Without being bound by theory, the metabolic demands of the hypertrophied RV are greatly increased, requiring the thin RV to increase muscle mass to compensate for the increased pressure load, thereby immediately increasing the necessary 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; 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). In our animal model, pressure overload is not reversed, and mitochondrial adaptation to the hypertrophied RV muscle is required to maintain function. Based on our results, this compensatory adaptation to accommodate increased pressure load does not occur over the long term, as indicated by the reduction in wall thickness in vehicle-treated hypertrophied hearts. In contrast, mitochondrial transplantation maintains RV hypertrophic adaptive growth. Furthermore, our data demonstrated that the source of mitochondria for transplantation is not a determining factor in their benefit. It made no difference which mitochondrial source was used. Skeletal muscle mitochondria responded similarly to mitochondria isolated from cardiac muscle. Therefore, cardiac muscle mitochondria are not required for the treatment of RVH / RVF-mediated mitochondrial dysfunction.Exogenous autologous skeletal muscle mitochondria maintain contractile function of the dysfunctional RV.

[0149] Mitochondrial transplantation as a therapeutic intervention targets all aspects of mitochondrial function and structure. Mitochondrial metabolic manipulation alone is not sufficient to treat a failing right ventricle, as mitochondrial structural integrity must also be addressed. Furthermore, mitochondrial transplantation targets mitochondrial dynamics, which are impaired in the hypertrophied / failed right heart. A potential mechanism under investigation is the disruption of mitochondrial biogenesis and the generation of new mitochondria as an early event in the pathophysiology of heart failure. During the early stages of compensatory hypertrophy, mitochondrial biogenesis signaling is maintained. In contrast, as decompensated heart failure becomes evident, mitochondrial biogenesis signaling declines.

[0150] In conclusion, this study advances our understanding of the benefits of mitochondrial transplantation. In particular, the results demonstrate that exogenous autologous skeletal muscle mitochondria preserve contractile function in failing hearts.

[0151] Example 5: Autologous mitochondrial transplantation by intracoronary injection for myocardial protection Experiments were conducted to investigate preischemic intracoronary autologous mitochondrial transfer (MT) as a therapeutic strategy for preventive myocardial protection in a porcine model.

[0152] method: The left coronary artery of Yorkshire pigs was cannulated (n = 26). Mitochondria (1 × 10 9 ) or buffer (vehicle [Veh]) was administered as a single bolus (MT S ) or continuous (10 injections in 60 minutes; MT SS A single injection was delivered as a bolus antegrade into the left main coronary artery (1 × 10 in 6 mL). 9 ). Sequential injections (1 × 10 in 6 mL of respiration buffer for each injection) 9Ten injections of 10 ribosomal RNA (1000 mg / kg / day) were administered every 5 minutes. Fifteen minutes after injection, the heart underwent transient regional ischemia (RI) by snare of the left anterior descending coronary artery. Thirty minutes after RI, the snare was released, and the heart was reperfused for 120 minutes.

[0153] result: Coronary blood flow (CBF) and myocardial function transiently increased during the pre-RI period. Thirty minutes after RI, MT S and MT SS Hearts had a significant increase in CBF that persisted throughout reperfusion (Veh vs MT). S and MT SS ;P=0.04). MT S and M.T. SS significantly improved ejection fraction (Veh vs. MT) S , P<0.001; Veh vs. MTSS, P=0.04) and developed pressure (Veh vs. MT S , P<0.001; Veh vs. MT SS , P = 0.03). S , P = 0.03; Veh vs. MT SS , P<0.001), shortening fraction (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 Regional function, assessed by MRI, was also significantly improved (P = 0.003). There was no difference in the area at risk between the treatment groups, but infarct size was significantly reduced in both MT groups (Veh vs. MT). S and MT SS , P<0.001).

[0154] Conclusion: Pre-ischemic MT by single or continuous intracoronary injection provides preventative myocardial protection, significantly reduces infarct size, and enhances global and regional cardiac function.

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

[0156] method: Adult pigs were anesthetized to isolate autologous mitochondria. Animals were sedated with Telazol (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. Angiographic access to the left coronary artery (LCA) was then established by floating a 5F JR angiographic catheter (Merit Medical Systems, UT) through the right carotid artery (5F sheath) into the left coronary ostium under fluoroscopic guidance. Mitochondrial uptake and biodistribution were evaluated. 18 F-rhodamine-6G-labeled mitochondria were injected angiographically into the left coronary artery and then evaluated by positron emission tomography (PET) (n = 3). The safety profile of intracoronary mitochondrial injection was evaluated under normal conditions, during coronary vasoconstriction, and during tachycardia (n = 18). To evaluate the therapeutic effect of intracoronary mitochondrial transplantation, the left anterior descending artery was snare for 30 minutes. At the onset of reperfusion, animals received either mitochondria (n = 8) or vehicle solution (n = 8), followed by 2 hours of reperfusion.

[0157] result: Intracoronary delivery of mitochondria resulted in rapid uptake and specific biodistribution of mitochondria throughout the heart. Coronary artery patency and myocardial function were maintained under all test conditions. Intracoronary injection of mitochondria resulted in a concentration-dependent increase in coronary blood flow (CBF). Mitochondria-induced hyperemia required mitochondrial viability, ATP generation, and, in part, activation of the vascular inwardly rectifying potassium channel (KIR). Intracoronary mitochondrial delivery resulted in a significant enhancement of postischemic myocardial function, improved CBF, and reduced infarct size compared with controls. Intracoronary mitochondrial transplantation is a safe and effective method for improving myocardial perfusion, myocardial function, and cardiac tissue survival.

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

Claims

1. 1. A composition comprising isolated mitochondria or a combined mitochondrial agent for use in treating or preventing heart failure in a subject, wherein the combined mitochondrial agent comprises mitochondria that act as carriers to transport the agent to a tissue of the subject, the composition being administered in a therapeutically effective amount to a subject in need thereof, the subject having a pulmonary disease.

2. 10. The composition of claim 1, wherein the subject has or is at risk of developing heart failure - right ventricular hypertrophy (RVH) or right ventricular failure (RVF).

3. 10. The composition of claim 1, wherein the pulmonary disease affects right ventricular function.

4. The composition of claim 1, wherein the pulmonary disease is pulmonary hypertension.

5. 1. A composition comprising isolated mitochondria or a combined mitochondrial agent for use in maintaining right ventricular (RV) contractility, maintaining RV capillary density, preventing RV dilation, or delaying the onset of RVF in a subject, wherein the combined mitochondrial agent comprises mitochondria that act as carriers to transport the agent to a tissue of the subject, the composition being administered in a therapeutically effective amount to a subject in need thereof, the subject having a lung disease.

6. 6. The composition of claim 5, wherein the subject has or is at risk of developing right ventricular hypertrophy (RVH) or right ventricular failure (RVF).

7. 6. The composition of claim 5, wherein the pulmonary disease affects right ventricular function.

8. The composition of claim 5, wherein the pulmonary disease is pulmonary hypertension.

9. A composition comprising isolated mitochondria or a combined mitochondrial agent for use in maintaining right ventricular (RV) contractility in a subject, wherein the combined mitochondrial agent comprises mitochondria that act as carriers to transport the agent to a tissue of the subject, the composition being administered to a subject in a therapeutically effective amount, the subject being identified as being in need thereof.

10. 10. The composition of claim 9, wherein the subject is identified by measuring pulmonary artery pressure, RV fractional area change (FAC), tricuspid annular plane contractile excursion (TAPSE), RV end-systolic pressure volume (ESPV), RV peak developed pressure, RV end-diastolic pressure, or RV dP / dt (change in pressure over time).

11. The composition of claim 9, wherein the subject is identified as having pulmonary hypertension.

12. A composition comprising isolated mitochondria or a combined mitochondrial agent for use in maintaining right ventricular (RV) capillary density in a subject, wherein the combined mitochondrial agent comprises mitochondria that act as carriers to transport the agent to the tissue of the subject, and wherein the composition is administered to a subject in need thereof in a therapeutically effective amount.

13. 13. The composition of claim 12, wherein the RV capillary density is measured by magnetic resonance imaging (MRI) or angiographic imaging of the microvascular circulation.

14. 13. The composition of claim 12, wherein the subject is identified by measuring pulmonary artery pressure, RV fractional area change (FAC), tricuspid annular plane contractile excursion (TAPSE), RV end-systolic pressure volume (ESPV), RV peak developed pressure, RV end-diastolic pressure, or RV dP / dt (change in pressure over time).

15. The composition of claim 12, wherein the subject is identified as having pulmonary hypertension.

16. 1. A composition comprising isolated mitochondria or a combined mitochondrial agent for use in reducing the risk of right ventricular (RV) dilation in a subject, wherein the combined mitochondrial agent comprises mitochondria that act as carriers to transport the agent to a tissue of the subject, and wherein the composition is administered to a subject in need thereof in a therapeutically effective amount.

17. 17. The composition of claim 16, 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 a toxin, cardiac arrhythmia, or late pregnancy complications.

18. 17. The composition of claim 16, wherein the subject is identified as having pulmonary hypertension.

19. 17. The composition of claim 16, wherein the RV dilation is measured by RV maximum volume (Vmax) and end-diastolic volume (Ved).

20. A composition comprising isolated mitochondria or a combined mitochondrial agent for use in delaying the onset of right heart failure in a subject, wherein the combined mitochondrial agent comprises mitochondria that act as carriers to transport the agent to the tissues of the subject, the composition being administered in a therapeutically effective amount to a subject in need thereof, wherein the subject is identified as having right ventricular hypertrophy.

21. 21. The composition of claim 20, wherein the subject is identified as having pulmonary hypertension.

22. 1. A composition comprising isolated mitochondria or a combined mitochondrial agent for use in treating right heart failure, delaying the onset of right heart failure, or reducing the risk of developing right heart failure in a subject, wherein the combined mitochondrial agent comprises mitochondria that act as carriers to transport the agent to a tissue of the subject, the composition being administered in a therapeutically effective amount to a subject in need thereof, the subject having a lung disease.

23. 23. The composition of claim 22, wherein the subject is at risk of developing right heart failure.

24. 23. The composition of claim 22, wherein the pulmonary disease is pulmonary hypertension.

25. 1. A composition comprising isolated mitochondria or a combined mitochondrial agent for use in treating right cardiac hypertrophy, delaying the onset of right cardiac hypertrophy, or reducing the risk of developing right cardiac hypertrophy in a subject, wherein the combined mitochondrial agent comprises mitochondria that act as carriers to transport the agent to a tissue of the subject, the composition being administered in a therapeutically effective amount to a subject in need thereof, the subject having a lung disease.

26. 26. The composition of claim 25, wherein the pulmonary disease is pulmonary hypertension.

27. 26. The composition of claim 25, wherein the subject is at risk of developing cardiac hypertrophy.

28. 27. The composition of any one of claims 1 to 26, comprising isolated mitochondria.

29. 27. The composition of any one of claims 1 to 26, comprising a combined mitochondrial agent.

30. 27. The composition of any one of claims 1 to 26, wherein the mitochondria are autologous, allogeneic, or xenogeneic.

31. The composition of any one of claims 1 to 26, wherein the composition is administered to a subject by intramyocardial injection.

32. The composition of any one of claims 1 to 26, wherein the composition is injected into a blood vessel of a subject in need thereof.

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