Application of mitochondrial transplantation in regulating immune-metabolic networks for the treatment of myocardial dysfunction after cardiac arrest

Abstract

This invention belongs to the field of medical technology, specifically relating to the application of mitochondrial transplantation to regulate the immune-metabolic network in the treatment of myocardial dysfunction after cardiac arrest. The mitochondrial transplantation is performed within 0.5-2 hours after the restoration of spontaneous cardiac circulation. This technical solution, through multi-omics integrated analysis, proposes that mitochondrial transplantation exerts a cardioprotective effect by regulating the immune-metabolic-mitochondrial triaxial synergistic network, providing a new research direction for the precision treatment of myocardial dysfunction after cardiac arrest.

Description

Technical Field

[0001] This invention belongs to the field of medical technology, specifically relating to the application of mitochondrial transplantation to regulate the immune-metabolic network in the treatment of myocardial dysfunction after cardiac arrest. Background Technology

[0002] Cardiac arrest followed by myocardial dysfunction is a core factor contributing to poor patient outcomes. Despite advancements in cardiopulmonary resuscitation (CPR) techniques in recent years, the survival rate of cardiac arrest patients remains unsatisfactory, with myocardial dysfunction being a key limiting factor. Studies have shown that cardiomyocytes undergo severe mitochondrial dysfunction after cardiac arrest, including energy metabolism disorders, oxidative stress damage, and abnormal mitochondrial dynamics. These changes directly lead to a decline in myocardial contractile function.

[0003] Mitochondrial transplantation, as an emerging therapeutic strategy, has demonstrated cardioprotective effects in various ischemia-reperfusion injury models. However, current research mainly focuses on direct effects such as energy replenishment and mitigation of oxidative damage. A deeper understanding of how mitochondrial transplantation systematically modulates the complex pathological network following cardiac arrest, particularly its intervention in the two interrelated core injury mechanisms of immune inflammatory response and cellular metabolic disorders, remains lacking. Clarifying the specific targets and pathways involved in the cardioprotective effects of mitochondrial transplantation after cardiac arrest is crucial for optimizing treatment strategies, identifying targets for combination therapies, and developing prognostic biomarkers. Summary of the Invention

[0004] The purpose of this invention is to provide an application of mitochondrial transplantation to regulate the immune-metabolic network in the treatment of myocardial dysfunction after cardiac arrest, so as to solve the problems in the background art mentioned above.

[0005] To achieve the above objectives, this application employs the following technical solution:

[0006] Application of mitochondrial transplantation in regulating the immune-metabolic network for the treatment of myocardial dysfunction after cardiac arrest.

[0007] Furthermore, the optimal time window for mitochondrial transplantation was determined based on the dynamic changes in immune and metabolic pathways.

[0008] Furthermore, mitochondrial transplantation is performed within 0.5–2 hours after the heart's spontaneous circulation is restored.

[0009] Furthermore, after transplanted mitochondria are taken up by immune cells, their phenotype can be regulated to transform from the pro-inflammatory M1 type to the anti-inflammatory and repairing M2 type.

[0010] Furthermore, this also includes combining mitochondrial transplantation with immunomodulators or metabolic modulators.

[0011] Furthermore, this also includes the combined use of mitochondrial transplantation and hypothermia.

[0012] Furthermore, the route of administration for the mitochondrial transplantation is any of the following:

[0013] A. Transpulmonary transport pathway via the central vein to the pulmonary circulation and then to the left atrium;

[0014] B. An extracorporeal tubing accumulation-slow-release pathway based on VA-ECMO, specifically, encapsulating mitochondria in biocompatible microcapsules and placing them in series with the blood storage sac or bioreactor in the ECMO tubing.

[0015] C. The airway to myocardial permeation pathway via endotracheal intubation.

[0016] Furthermore, it also includes mitochondrial pretreatment, specifically the following steps: extracting mitochondria from an allogeneic source and pretreating donor cells in vitro to ensure that the extracted mitochondria contain a set level of specific miRNA or heat shock protein.

[0017] The beneficial effects of this invention are:

[0018] 1. This technical solution has a clear therapeutic effect. The protective effect of mitochondrial transplantation on myocardial dysfunction after cardiac arrest has been systematically verified at the cellular and animal levels. This includes improving cardiac function, reducing myocardial damage, reducing cell apoptosis and oxidative stress, providing a solid basis for clinical application.

[0019] 2. Innovative mechanism hypothesis: For the first time, through multi-omics integrated analysis, a new hypothesis was proposed that mitochondrial transplantation exerts cardioprotective effects by regulating the "immune-metabolic-mitochondrial" triaxial synergistic network. This breaks through the traditional understanding of a single mechanism and provides a new systematic theoretical framework that integrates immune and metabolic regulation for research in this field.

[0020] 3. Complete treatment plan: This technical plan provides a complete process from mitochondrial preparation and quality control to dosing regimen, with a clear clinical translation pathway.

[0021] 4. Broad application prospects: Based on mechanism discovery, this technical solution proposes combined treatment strategies and efficacy prediction biomarkers, providing a new research and development direction for the precision treatment of myocardial dysfunction after cardiac arrest. Attached Figure Description

[0022] Figure 1 This is a schematic diagram of rapid mitochondrial extraction in 30 minutes.

[0023] Figure 2 This image shows the membrane potential of extracted mitochondria detected using a JC-1 microscopy device under a confocal microscope, indicating that the mitochondria are active.

[0024] Figure 3This is a schematic diagram of the activity of extracted mitochondria detected by flow cytometry using a Mito Tracker.

[0025] Figure 4 This diagram illustrates how mitochondrial transplantation improves cell viability at the cellular level.

[0026] Figure 5 This diagram illustrates how mitochondrial transplantation reduces reactive oxygen species levels at the cellular level.

[0027] Figure 6 This diagram illustrates how mitochondrial transplantation increases ATP levels at the cellular level.

[0028] Figure 7 This diagram illustrates how mitochondrial transplantation enhances mitochondrial membrane potential at the cellular level.

[0029] Figure 8 A schematic diagram for constructing an animal model of cardiac arrest.

[0030] Figure 9 A flowchart of mitochondrial transplantation in animals undergoing asphyxiation-induced cardiac arrest.

[0031] Figure 10 A flowchart for mitochondrial transplantation in animals with electrically stimulated cardiac arrest.

[0032] Figure 11 A schematic diagram illustrating how mitochondrial transplantation improves cardiac function after resuscitation.

[0033] Figure 12 A schematic diagram illustrating how mitochondrial transplantation improves the levels of myocardial injury markers after resuscitation.

[0034] Figure 13 HE staining suggests that mitochondrial transplantation alleviated cardiomyocyte swelling and myofibril dissolution to varying degrees, and reduced inflammatory cell infiltration.

[0035] Figure 14 This image shows the damage-causing structural changes in cardiomyocytes, such as swelling and cristae breakage, caused by mitochondrial transplantation, as revealed by transmission electron microscopy.

[0036] Figure 15 This is a cluster analysis diagram of the transcriptome and proteome of myocardial tissue.

[0037] Figure 16 This is a graph showing the GO enrichment analysis of the myocardial tissue transcriptome.

[0038] Figure 17 This is a graph showing the GO enrichment analysis of the myocardial tissue proteome.

[0039] Figure 18 This is a diagram showing the KEGG enrichment analysis of the myocardial tissue transcriptome.

[0040] Figure 19 This is a KEGG enrichment analysis diagram of the myocardial tissue proteome. Detailed Implementation

[0041] The technical solution of the present invention will be described in detail below with reference to the accompanying drawings. The following embodiments are merely exemplary and can only be used to explain and illustrate the technical solution of the present invention, and should not be construed as limiting the technical solution of the present invention.

[0042] like Figures 1 to 19 As shown, this application provides the application of mitochondrial transplantation to regulate the immune-metabolic network in the treatment of myocardial dysfunction after cardiac arrest.

[0043] In this application, the optimal time window for mitochondrial transplantation is determined based on the dynamic changes in immune and metabolic pathways.

[0044] In this application, mitochondrial transplantation was performed within 0.5–2 hours after the heart’s spontaneous circulation was restored.

[0045] In this application, after the transplanted mitochondria are taken up by immune cells, their phenotype can be regulated to transform from the pro-inflammatory M1 type to the anti-inflammatory and repair M2 type.

[0046] This application also includes the combined use of mitochondrial transplantation with immunomodulators or metabolic modulators.

[0047] This application also includes the combined use of mitochondrial transplantation and hypothermia.

[0048] This application provides a complete chain of evidence to systematically verify the therapeutic effect of mitochondrial transplantation on myocardial dysfunction after cardiac arrest, and reveals its potential mechanism of action network through multi-component bioanalysis, and proposes a treatment strategy based on immune-metabolic axis regulation.

[0049] 1. Cellular level validation:

[0050] In a cardiomyocyte (H9C2 cell line and primary cardiomyocyte) ischemia-reperfusion model, mitochondrial transplantation was shown to significantly improve cell survival, reduce reactive oxygen species (ROS) levels, increase ATP content, and improve mitochondrial membrane potential (JC-1 staining).

[0051] 2. Animal-level verification:

[0052] Two rat models of cardiac arrest were established: one of asphyxia and the other of electrical stimulation-induced ventricular fibrillation. In both models, intravenous transplantation of active mitochondria demonstrated that: it improved post-resuscitation function; compared with the control group, mitochondrial transplantation improved LVEF and FS values ​​at 3 and 6 hours post-resuscitation; it reduced myocardial tissue pathological damage: HE staining showed reduced cardiomyocyte swelling, myofibril dissolution, and decreased inflammatory cell infiltration; transmission electron microscopy showed that mitochondrial transplantation reduced damaging structural changes in cardiomyocytes, such as mitochondrial swelling and cristae breakage; it reduced serum myocardial injury markers: levels of myocardial injury markers such as cTnI and CK-MB were significantly reduced (p<0.05); it reduced the trend of cardiomyocyte apoptosis: TUNEL staining showed a decreasing trend in the proportion of apoptotic cells; and it improved myocardial oxidative stress: MDA levels decreased, and SOD activity increased.

[0053] 3. Multi-omics analysis:

[0054] Transcriptomic and proteomic analyses of myocardial tissues from mitochondrial transplantation groups and control groups were performed to screen for significantly altered genes and proteins. Pathway enrichment analysis was conducted to identify misidentification processes, and network analysis was used to construct key regulatory networks. Multi-omics integration analysis revealed the regulatory role of mitochondrial transplantation on myocardial immune inflammation after cardiac arrest.

[0055] Potential suppression of adaptive immunity: Proteomics data showed that the expression of key molecules in the MHC class II antigen processing and presentation pathway (such as RT1-Db / Bb) in the myocardium of the mitochondrial transplant group was downregulated (enriched in GO:0002504 / 0042613, KEGG: rno04612).

[0056] Extensive Impact on Innate Immunity and Inflammatory Signaling: Transcriptomic analysis further confirmed the initiation of a strong innate immune and inflammatory response in myocardial tissue after cardiac arrest. Pathway enrichment analysis revealed significant activation of core pro-inflammatory signaling pathways, including the tumor necrosis factor (TNF) signaling pathway, the interleukin-17 (IL-17) signaling pathway, the cytokine-cytokine receptor interaction pathway, and the NF-κB signaling pathway. Significant enrichment was also observed in typical pathological immune-related pathways such as rheumatoid arthritis and graft-versus-host disease. Furthermore, proteins related to the Toll-like receptor 7 / 9 (TLR7 / 9) signaling pathway (such as Unc93b1 and Pik3ap1) were also identified.

[0057] Reprogramming of the overall immune-inflammatory background: The multi-omics data consistently showed that the model group's myocardium was significantly enriched in pathways representing a state of intense immune inflammation. Mitochondrial transplantation altered this aberrant enrichment pattern, suggesting its modulatory role in the pathological immune-inflammatory background.

[0058] Multi-omics analysis revealed an important phenomenon: metabolic reprogramming induced by mitochondrial transplantation has the most significant signal at the proteomic level, revealing a profound adjustment from cofactor metabolism to energy supply mode.

[0059] Activation of proteome-driven metabolic pathways:

[0060] KEGG pathway analysis revealed significant enrichment of the retinol metabolism and one-carbon pool metabolism pathways at the proteomic level (e.g., rno00830 and rno00670). These two pathways are crucial for maintaining cellular redox homeostasis and supporting DNA synthesis and methylation modification. Notably, these two pathways did not appear in the list of most significantly enriched pathways in the transcriptome, suggesting a key role of posttranscriptional regulation in mediating mitochondrial transplantation metabolic effects.

[0061] Adaptive regulation of energy metabolism patterns:

[0062] Further GO functional enrichment analysis revealed that the positive regulation of glycolysis was enhanced after mitochondrial transplantation (GO:0045821). This suggests that while supplementing mitochondrial function, cells may also upregulate glycolysis, a rapid energy-producing pathway, resulting in a more flexible and robust energy supply network.

[0063] Potential crossover between metabolism and immunity:

[0064] The affected metabolic pathways (such as retinol metabolism) are closely related to immune regulation. This remodeling of metabolic state may provide the necessary metabolic substrates and signaling molecules for improving the aforementioned immune-inflammatory environment, forming a potential basis for "immuno-metabolic synergistic regulation".

[0065] Based on the above findings, this application proposes that mitochondrial transplantation, by simultaneously regulating immune inflammatory responses and cellular metabolic states, synergistically acts on mitochondrial function, forming a "immune-metabolic-mitochondrial" triaxial synergistic regulatory network, reprogramming the pathological state after cardiac arrest into a repair state. This model provides an integrated framework for understanding the systemic protective effect of mitochondrial transplantation and offers a direct theoretical basis for its combined application with immune / metabolic modulators.

[0066] The mitochondria used in this application were derived from skeletal tissues such as the gastrocnemius muscle. The process was performed using differential filtration, entirely on ice, and completed within 30 minutes. The mitochondrial membrane potential (JC-1 staining) was >85%, and the concentration was 3.0 × 10⁻⁶. 7 per ml.

[0067] After cardiac arrest resuscitation, patients undergo a unique pathophysiological phase—"post-cardiac arrest syndrome." This technical protocol proposes that mitochondrial transplantation is most effective when performed within a very specific time window after the restoration of spontaneous circulation (e.g., within 30 minutes or 2 hours after ROSC).

[0068] Following cardiac arrest, a systemic cytokine storm is a key factor leading to myocardial dysfunction. Mitochondrial transplantation not only provides energy to cardiomyocytes, but more importantly, after the transplanted mitochondria are taken up by immune cells (such as macrophages), they can regulate the transformation of their phenotype from the pro-inflammatory M1 type to the anti-inflammatory and repairing M2 type, thereby suppressing the systemic inflammatory response and reducing myocardial damage.

[0069] In this application, the route of administration is one of the following three:

[0070] A. The "transpulmonary transport" pathway via central venous catheter (CVC) to the left venous system. This utilizes the most convenient and readily available access route already present after cardiac arrest resuscitation. The isolated mitochondria are rapidly injected via the CVC.

[0071] Scientific basis: 1. Mitochondria are small in size and have the potential to cross capillaries. 2. After cardiac arrest, the permeability of pulmonary capillaries increases significantly, creating conditions for mitochondria to "cross" the pulmonary circulation and enter the left ventricle.

[0072] Mitochondria return to the right heart via veins, enter the pulmonary circulation, and then, with increased capillary permeability, pass through the lungs into the pulmonary veins, returning to the left heart and distributing to the myocardium via coronary blood flow.

[0073] B. Integrating mitochondria into the VA-ECMO circulation circuit. For example, mitochondria can be encapsulated in biocompatible microcapsules and placed in the "blood reservoir" of the ECMO circuit, or a special "bioreactor" can be designed and connected in series in the circuit. With the ECMO running, mitochondria are continuously and controllably released into the bloodstream and directly reinfused through the arterial end, achieving a "drip irrigation" treatment for failing hearts.

[0074] C. Using the endotracheal intubation required by 100% of cardiac arrest patients, mitochondria are formulated into a nebulized inhaler and administered via ventilator tubing. The left atrium is close to the pulmonary veins and lung tissue. After the nebulized mitochondria are taken up by the alveolar epithelium, they may enter the myocardium through direct diffusion from the alveolar interstitium to the myocardial interstitium.

[0075] This application also relates to a synergistic approach combining mitochondrial transplantation with hypothermia therapy, the only proven effective neuroprotective measure after cardiac arrest. This application combines mitochondrial transplantation with hypothermia, where hypothermia may stabilize mitochondria and increase their survival rate; mitochondria may also compensate for the inhibition of myocardial energy metabolism caused by hypothermia.

[0076] In this application, mitochondria are extracted from allogeneic sources (such as mesenchymal stem cells from healthy donors) and "pretreated" in vitro, for example, by pretreating donor cells with mild stress stimuli (hypoxia, drugs) to make the extracted mitochondria contain higher levels of specific miRNAs or heat shock proteins, thus having greater "repair potential," and then used for treatment with these "enhanced" mitochondria.

[0077] The above description is merely an embodiment of this application and is not intended to limit this application in any way. Although this application discloses preferred embodiments as described above, it is not intended to limit this application. Any changes or modifications made by those skilled in the art without departing from the scope of the technical solution of this application using the disclosed technical content are equivalent to equivalent implementation cases and fall within the scope of the technical solution.

Claims

1. Application of mitochondrial transplantation in regulating the immune-metabolic network for the treatment of myocardial dysfunction after cardiac arrest.

2. The application according to claim 1, characterized in that, The optimal time window for mitochondrial transplantation was determined based on the dynamic changes in immune and metabolic pathways.

3. The application according to claim 2, characterized in that, Mitochondrial transplantation is performed within 0.5–2 hours after the heart’s spontaneous circulation is restored.

4. The application according to claim 1, characterized in that, After transplanted mitochondria are taken up by immune cells, their phenotype can be regulated to transform from the pro-inflammatory M1 type to the anti-inflammatory and repairing M2 type.

5. The application according to claim 1, characterized in that, This also includes combining mitochondrial transplantation with immunomodulators or metabolic modulators.

6. The application according to claim 1, characterized in that, It also includes the combined use of mitochondrial transplantation and hypothermia.

7. The application according to claim 1, characterized in that, The administration route for the mitochondrial transplant is any of the following: A. Transpulmonary transport pathway via the central vein to the pulmonary circulation and then to the left atrium; B. An extracorporeal tubing accumulation-slow-release pathway based on VA-ECMO, specifically, encapsulating mitochondria in biocompatible microcapsules and placing them in series with the blood storage sac or bioreactor in the ECMO tubing. C. The airway to myocardial permeation pathway via endotracheal intubation.

8. The application according to claim 7, characterized in that, It also includes mitochondrial pretreatment, the specific steps of which are: extracting mitochondria from allogeneic sources and pretreating donor cells in vitro to ensure that the extracted mitochondria contain a set level of specific miRNA or heat shock protein.

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