The Protective Effect Of Exogenous Mitochondria Slowly Released Through ECM Hydrogels After Myocardial Ischemia-reperfusion Injury

BackgroundCardiovascular disease remains one of the leading causes of death worldwide.Ischemic injury caused by acute obstruction of myocardial tissue has a poor prognosis.Although reperfusion therapy is currently the most effective treatment for rapidly restoring coronary blood flow to the myocardium,additional myocardial damage may occur during reperfusion therapy.Transplanting exogenous mitochondria to the ischemic area to replace damaged mitochondria is a new therapy to slow myocardial ischemia-reperfusion(IR)injury and improve cardiac function.In the reported clinical trials and animal experiments,myocardial IR injury was alleviated by a single injection of exogenous mitochondria.However,studies have shown that there is persistent mitochondrial dysfunction during reperfusion,and long-term protection of mitochondrial function is particularly important.Compared with a single administration of mitochondria,it is attractive to investigate multiple slow releases of mitochondria for the maintenance of long-term efficacy in the treatment of myocardial IR injury.Hydrogels have been used to encapsulate various cells and biomolecules for the treatment of myocardial ischemic diseases,achieving targeted release and concentration of biologics in effective areas,and are ideal biological carriers.The extracellular matrix(ECM)hydrogel itself constitutes a natural extracellular environment after being injected into the tissue,which has good biocompatibility and can retain the structure and function of biomolecules.In addition,studies have confirmed that mitochondria are encapsulated by hydrogels and preserve their functional activities,so the slow release of exogenous mitochondria through ECM hydrogel has a theoretical basis and potential long-term efficacy in the treatment of myocardial IR injury.Objectives1.Functionally active exogenous mitochondria were isolated.After repeated administration of mitochondrial therapy,the effect on the function and metabolism of cardiomyocytes after IR injury was studied.2.Preparation of injectable ECM hydrogel.To investigate the feasibility of infusion of mitochondria through hydrogel for the treatment of myocardial ischemia injury.3.To study the effects of slow-release of exogenous mitochondria from ECM hydrogel on myocardial injury,cardiac repair and inflammation regulation after myocardial IR injury.Methods1.Culture of cardiomyocytes and adipose stem cells: Ventricular tissues were obtained from Sprague-Dawley(SD)rats,cut into 1x1 mm tissue blocks,and digested with0.1% trypsin solution to obtain neonatal rat cardiomyocytes(NRCMs).After filtration and removal of fibroblasts,DMEM medium containing double-antibody and 20% Fetal bovine serum(FBS)was added and cultured in a 37°C incubator containing 5% CO2.In addition,adipose stem cells were obtained by crawling out of the adipose tissue block of SD rats by the tissue adherence method.2.Isolation and identification of exogenous mitochondria:According to the operating protocol of a commercially available cell mitochondrial isolation kit,exogenous mitochondria were isolated from rat adipose stem cells,and the homogenate was filtered using a standard sieve to improve the purity of mitochondria.Mitochondrial integrity was detected by Mito Tracker Red CMXROS(mitochondrial red fluorescent probe)staining,and mitochondrial functionality was detected by flow cytometry analysis following mitochondrial membrane potential staining(JC-1 staining).3.Construction of cardiomyocyte OGD/R model and mitochondrial treatment: NRCMs were added to serum-free,sugar-free DMEM medium and cultured in a hypoxic incubator for 12 hours,and then replaced with high-glucose DMEM medium containing 20% FBS in normal Incubate for 12 hours.Finally,an oxygen and glucose deprivation/reperfusion(OGD/R)model was constructed after reoxygenation treatment.Under the condition of giving the same amount of exogenous mitochondria,the mitochondria and NRCMs were co-cultured with 1 or 3 times respectively during reoxygenation.4.Detection of energy metabolism of NRCMs: In vitro,mitochondria were co-cultured with NRCMs.Then,the uptake of mitochondria by NRCMs was detected by fluorescent phalloidin staining.After the treatment of mitochondrial transplantation,the ATP value generated by NRCMs in each group was detected by a commercially available Adenosine triphosphate(ATP)detection kit.After loading NRCMs with commercially available reactive oxygen species(ROS)fluorescent probes,the levels of ROS were calculated by analyzing the fluorescence signal intensity.5.Preparation and characteristic detection of ECM hydrogel: After vacuum freeze-thaw and decellularization,a bovine pericardial matrix was obtained,and then ECM hydrogel was prepared by digesting bovine pericardial matrix with pepsin solution.In vitro,the biocompatibility of ECM hydrogels was tested by seeding various cells on the ECM hydrogel surface;in vivo,the biocompatibility of ECM hydrogel was tested by HE staining and immunohistochemical staining after subcutaneous embedding;The biodegradability of ECM hydrogel was tested by measuring the size after subcutaneous embedding.6.Feasibility test of the release of exogenous mitochondria after hydrogel injection into the heart: In vitro,mitochondria were co-cultured with NRCMs.Then,the uptake of mitochondria by NRCMs was detected by fluorescent phalloidin staining.In vivo,after prestained mitochondria were injected alone or together with ECM hydrogel into ischemic regions of rat ventricles,three-color immunofluorescence staining of tissue sections was used to examine the feasibility of mitochondrial release after infusion into heart by ECM hydrogel.7.Myocardial ischemia-reperfusion animal model and experimental intervention: The myocardial IR model was constructed by blocking the blood flow of the proximal left anterior descending artery in SD rats for 30 minutes and then recanalizing the blood supply.During reperfusion,mitochondria,ECM hydrogel,or an equivalent mixture of both were injected separately into the ischemic area of myocardial ischemia to complete the experimental intervention.8.Detection of the protective effect of myocardial IR injury: after 4 weeks of ischemia recovery,the detection of myocardial repair after myocardial IR injury was performed by triphenyltetrazole chloride(TTC)on tissue sections.Staining,Terminal-deoxynucleotidyl transferase-mediated nick end labeling(TUNEL)staining,Masson’s trichrome staining,and immunohistochemical staining.In addition,the levels of cardiac troponin I(Cardiac troponin I,c Tn I)and creatine kinase isoenzyme-MB(Creatine Kinase Isoenzyme-MB,CK-MB)were detected after 1 and 3days of ischemia recovery.After 4 weeks of blood recovery,serum was collected to detect the levels of inflammatory cytokines such as interleukin-6(Interleukin-6,IL-6),interleukin-10(Interleukin-10,Interleukin-10),and tumor necrosis factor-α(Tumor necrosis factor-α,TNF-α).Results1.The effect of multiple administration of exogenous mitochondria on the functional metabolism of cardiomyocytes after hypoxia-ischemia injuryMitochondria isolated from adipose stem cells were labeled with Mito-Traxker Red CMXROS to confirm that mitochondria with intact morphology and similar size were extracted.The isolated exogenous mitochondria were further confirmed to be functional by using the mitochondrial membrane potential detection kit JC-1 staining.Isolation and culture of NRCMs were confirmed by fluorescent staining using phalloidin and the observed spontaneous beating.Mitochondria were taken up into the cytoplasm after co-culture with NRCMs.After a hypoxic-ischemic injury,a single administration of mitochondria and multiple administrations of mitochondria significantly increased the production of ATP and reduced the accumulation of ROS after ischemia-hypoxic injury in cardiomyocytes.Under the condition of the same amount of mitochondria,multiple administration of mitochondria resulted in more ATP and reduced ROS accumulation after hypoxia-ischemia injury in cardiomyocytes than a single administration of mitochondria.2.To explore the feasibility of exogenous mitochondrial release after hydrogel injection into the heartIn vitro,a variety of cells maintained good viability and biocompatibility after seeding on ECM hydrogels.In vivo,ECM hydrogel is biodegradable after subcutaneous embedding,and immunohistochemical staining confirmed that a large number of vascular endothelial and smooth muscle cells have grown in ECM hydrogel,which is a promising biological carrier.In vivo,compared with the injection of mitochondria alone,mixing and injection with ECM hydrogel into the ischemic area of the left ventricle resulted in the retention of more mitochondria,and mitochondria were gradually released to surrounding tissues as the ECM hydrogel degraded in vivo.It is feasible to achieve the targeted release of mitochondria and the concentration of mitochondria in the effective area.3.The protective effect of exogenous mitochondria slowly released through ECM hydrogel after myocardial IR injuryFIRst,exogenous mitochondria injected alone or slowly released through ECM hydrogel significantly decreased both c Tn I and CK-MB in serum after IR injury 1 and 3days compared with the vehicle group,and mitochondrial transplantation effectively alleviated myocardial injury after IR.Second,compared with the vehicle and mitochondria groups,exogenous mitochondria after slow release from the hydrogel significantly reduced myocardial ischemia area and infarct size in hearts 28 days after myocardial IR injury,and similarly reduced fibrosis formation in ischemic areas,increased the amount of neovascularization,and decreased cardiomyocyte apoptosis in the ischemic area.These promote cardiac repair after cardiac IR injury.Finally,compared with the vehicle and mitochondrial groups,exogenous mitochondrial slow release from hydrogels significantly decreased the levels of pro-inflammatory cytokines IL-6 and TNF-α in serum 28 days after myocardial IR injury,Conversely,the level of the anti-inflammatory cytokine IL-10 was significantly increased.It is shown that the slow release of mitochondria through the ECM hydrogel alleviates inflammation.ConclusionIn general,the treatment of exogenous mitochondrial transplantation resulted in the production of more ATP and reduced accumulation of ROS in cardiomyocytes after hypoxia-ischemiac injury.Under the condition of giving the same amount of mitochondria,the energy metabolism of cardiomyocytes after hypoxia-ischemia injury was improved by multiple administration of mitochondria compared with a single administration of mitochondria.In addition,after co-injection of mitochondria and ECM hydrogel into the myocardial ischemia area,it is feasible to achieve targeted release of mitochondria and concentration of mitochondria in the effective area.Most importantly,in a rat model of myocardial ischemia-reperfusion,the slow release of mitochondria through hydrogel alleviated myocardial injury,promoted cardiac repair in ischemic areas,and alleviated inflammation compared with mitochondrial transplantation alone.Overall,the slow release of mitochondria through the hydrogel exerted a long-term therapeutic effect on myocardial IR injury.