The incidence of post-cardiac arrest myocardial dysfunction (PAMD) is high, and there is currently no effective treatment available. This study aims to investigate the protective effects of exogenous mitochondrial transplantation. Exogenous mitochondrial transplantation can enhance myocardial function and improve the survival rate. Mechanistic studies suggest that mitochondrial transplantation can limit impairment in mitochondrial morphology, augment the activity of mitochondrial complexes II and IV, and raise ATP levels. As well, mitochondrial therapy ameliorated oxidative stress imbalance, reduced myocardial injury, and thus improved PAMD after cardiopulmonary resuscitation (CPR).
eLife assessment
This study presents valuable findings on how mitochondrial transplantation affects post-cardiac arrest myocardial dysfunction (PAMD). The authors demonstrate that mitochondrial transplantation enhances cardiac function, increases survival rates after the return of spontaneous circulation (ROSC). While the findings are promising, the organization of the paper, along with the analysis and interpretation of the results, are inadequate and need revision.
Cardiac arrest (CA) is a life-threatening event that is followed by post-cardiac arrest myocardial dysfunction (PAMD) after the return of spontaneous circulation (ROSC). PAMD exhibits degrees of systolic and diastolic dysfunction. The heart requires up to 30 kg of ATP daily to maintain systolic function (Hall, et al., 2014). Due to the high energy requirements of the myocardium, mitochondria make up approximately one-third of the volume of cardiomyocytes (Cao, et al., 2023). The occurrence of PAMD is associated with mitochondrial damage with increased mitochondrial volume, accumulation of calcium ions, a diminution in complex activity, and a decrease in phosphate synthesis (Huang, et al., 2015; McCully, et al., 2023; Su, et al., 2021; Tsai, et al., 2021). Myocardium, as a high energy-consuming tissue, is very susceptible to energy depletion due to mitochondrial dysfunction. After mitochondrial injury, cytoplasmic and mitochondrial calcium overload prevents full relaxation of cardiomyocytes and can release cytochrome C to activate mitochondrial apoptotic pathways. This process contributes to the development of PAMD (Gazmuri and Radhakrishnan, 2012; Su, et al., 2021). Therefore, maintaining cardiomyocyte mitochondrial function after cardiopulmonary resuscitation (CPR) could be useful for cell survival and tissue homeostasis.
Transplantation of mitochondria isolated from non-ischemic or allogeneic tissues promoted myocardial recovery after regional ischemia (McCully, et al., 2009). Although the safety and efficacy of this approach have been verified (Ali, et al., 2021; Jia, et al., 2022), effectiveness of mitochondrial transplantation in treating myocardial injury following cardiac arrest remains to be demonstrated. Herein, we performed mitochondrial transplantation in rats following CPR to observe its effects on cardiac function and prognosis and explored its potential mechanism of action.
Results
CA-CPR model
No significant differences in weight or temperature were found between any groups before modeling. There was no significant difference in CA induction or CPR duration among the NS, Vehicle, and Mito groups. Compared to the Sham group, the other three groups tended to receive less anesthetic (Table 1).
Baseline characteristics of rats and resuscitation characteristics .
Mitochondrial transplantation improves cardiac function and does not alter hemodynamics in rats
Echocardiography revealed no differences in cardiac function among the groups prior to CA-CPR. Compared to the baseline, the left ventricular ejection fraction of rats subjected to CA-CPR significantly decreased after ROSC at 1 hour, and gradually improved over time (P<0.05; Fig. 2A, 2B). Of note, the level of cardiac-function impairment in the Mito group was significantly reduced four hours after ROSC compared to the NS and Vehicle groups (P<0.05).
Experimental design of the in vivo study.
Rats in all groups except the sham group underwent CA for 5 minutes and then received the corresponding intervention immediately after ROSC. ROSC was defined as the spontaneous restoration of sinus rhythm with a systolic blood pressure of >60 mmHg that was maintained for 10 minutes. Four hours after ROSC, the 4-hour group was used to collect myocardial tissue and blood samples for detection, whereas the 72-hour group was used for survival detection. BL, baseline; CA, cardiac arrest; ROSC, return of spontaneous circulation.
Observational results at 4 and 72 hours after cardiopulmonary resuscitation in rats.
(A) Echocardiograms of rats in each group from baseline to 4 hours following ROSC (n = 7 animals per group). (B) EF of rats in each group from baseline to 4 hours following ROSC (n = 7 animals per group). (C) HR and MAP changes during post-ROSC in 4 hours (n = 7 animals per group). (D) Survival rate during the first 72 hours following ROSC (n = 10 animals per group). Myocardial function between groups was compared by time-based measurements in each group using repeated-measures ANOVA. The survival rate between groups was compared using the Kaplan–Meier survival analysis test. * P < 0.05 vs. sham group and # P < 0.05 vs. Mito group. BL, baseline; EF, ejection fraction; HR, heart rate; MAP, mean arterial pressure; bpm, beats per minute; mmHg, millimeters of mercury; AMV, after mechanical ventilation; ROSC, return of spontaneous circulation.
Compared to the Sham group, the mean arterial pressure (MAP) and heart rate (HR) of the other groups decreased at 1, 2, 3, and 4 hours after ROSC, reaching their nadir at 1 hour. MAP and HR then increased gradually but remained lower than in the Sham group at four hours. MAP and HR did not differ at any observational timepoints (P>0.05, Fig. 2C).
Mitochondrial transplantation improves the 72-h survival rate after cardiopulmonary resuscitation in rats
In animals not treated with mitochondria, Kaplan-Meier analysis found a rapid drop in the survival rate of rats within 24 hours after ROSC. In contrast, the survival rate of the Mito group was significantly higher than that of the NS and Vehicle groups (P<0.05, Fig.2D).
Viability and purity of isolated mitochondria
Under conditions of elevated mitochondrial membrane potential, JC-1 was noted to aggregate and produced red fluorescence. Nearly all of the mitochondria isolated from healthy rats a high membrane potential, suggesting more viability (Fig. 3A). Mitochondria purity was confirmed by immunoblotting for COX-IV (a mitochondrial-specific protein) in both skeletal samples and isolated mitochondrial proteins. GAPDH was only detected in skeletal muscle proteins (Fig. 3B).
Assessment of the viability and purity of isolated mitochondria.
(A) JC-1 staining of mitochondria after isolation from muscle. The staining of isolated mitochondria by JC-1 is visible as either red for J-aggregates or green for J-monomers. The intensity of the red color indicates that the isolated mitochondria had a high membrane potential, confirming their quality for transplantation. Scale bar = 100 µm. (B) SDS/PAGE analysis of fractions obtained during the purification of muscle mitochondria. GAPDH is only expressed in muscle, confirming its purity for transplantation.
Effects of mitochondrial transplantation on cardiomyocyte mitochondria
Transplanted Mitochondria are internalized by cardiomyocytes
Animals received mitochondria pre-labeled with MitoTracker Red CMXRos. Four hours after injection, analysis of tissue sections found labelled mitochondria in epicardium, muscle, and endocardium (Fig. 4A).
Administration of mitochondria ameliorates ischemia/reperfusion– mediated mitochondrial alterations in cardiomyocytes 4 hours after ROSC.
(A) Localization and uptake of transplanted mitochondria in the heart; myocardial tissue was stained for anti-α-actinin 2 (green) and nuclei (blue); the pre-stained isolated mitochondria were labeled red (n = 3 animals per group). Scale bar = 100 µm. (B and C) Changes in myocardial mitochondrial complex II and IV enzyme activities in hearts (n = 7 animals per group). (D) The ATP content in myocardial tissue was measured via colorimetry (n = 7 animals per group). The detection of fluorescence intensity of Tom20: (E) image and (F) quantitative data. Scale bar = 100 µm (n = 3 animals per group). (G) Representative photographs of mitochondrial morphology by TEM examination; arrows indicate calcium accumulation (n = 3 animals per group). Scale bars = 500 nm. Analyses were performed using ANOVA with Tukey’s post hoc test. The data are expressed as the mean ± standard deviation (SD). * P < 0.05 vs. sham group and # P < 0.05 vs. Mito group. ROSC, return of spontaneous circulation.
Mitochondrial transplantation does not alter the mass of myocardial mitochondria
To investigate the potential increase in mitochondrial mass in myocardial cells through mitochondrial transplantation, the expression of TOM20 (a commonly used marker for mitochondrial mass) was assessed in each group using tissue immunofluorescence staining. After four hours of ROSC, there was no significant difference in the TOM20 level (as reflected by fluorescence intensity) among the groups, suggesting that mitochondrial transplantation did not increase the mass of mitochondria in the hearts. (Figures 4E, 4F).
In the NS and Vehicle groups, the myocardial myofibrils exhibited a disordered arrangement on EM, with variation in sarcomeres length and loss of Z-lines. Translucent and swollen mitochondria were visible, exhibiting a larger gap between membranes, distorted cristae, and accumulated calcium. In contrast, the mitochondria sarcomeres of the Mito group were more distinct, and the myocardial myofibrils were arranged in a more orderly fashion. The M-lines and Z-lines were clearly visible, and the number of mitochondrial cristae had increased, with their structure appearing to be complete (Fig. 4G).
Mitochondrial transplantation improves mitochondrial metabolism of cardiac tissue after CA-CPR
The activity of mitochondrial complexes II and IV and ATP content in cardiac tissue was characterized. Compared to the Sham group, complex II and IV activity and ATP content were significantly attenuated in the other groups (P<0.05). In contrast, the activities of complexes II and IV and ATP content were significantly elevated in the Mito group compared to the NS and Vehicle groups, suggesting that transfer of exogenous mitochondria enhanced function and energy metabolism or resident mitochondria (P<0.05, Fig. 4B, 4C, 4D).
Effects of mitochondrial transplantation on cardiomyocytes
Mitochondrial transplantation alleviates myocardial oxidative stress injury after CA-CPR
MDA levels in the myocardial tissue of rats four hours after ROSC was decreased in animals given exogenous mitochondria CPR (P<0.05) (Fig. 5A). Additionally, the activity of SOD was significantly increased in the Mito group relative to the NS and Vehicle groups (P<0.05).
Mitochondrial transplantation reduces myocardial damage 4 hours after ROSC.
(A) Changes in superoxide dismutase activities and malondialdehyde levels in cardiac tissue (n = 7 animals per group). (B) Myocardial apoptosis levels were examined using TUNEL (n = 3 animals per group). Scale bar = 100 µm. (C) The percentage of myocardial apoptosis was examined using flow cytometry (n = 3 animals per group). (D) Quantitative analysis of myocardial TUNEL apoptosis index and flow apoptosis rate. (E) H&E staining was used to detect myocardial injury (n = 3 animals per group). Scale bar = 100 µm. (F) The changes in CK-MB and cTn-I levels in the serum of rats were examined using ELISA (n = 7 animals per group). Analyses were performed using ANOVA with Tukey’s post hoc test. The data were expressed as mean ± standard deviation (SD). * P < 0.05 vs. sham group and # P < 0.05 vs. Mito group. ROSC, return of spontaneous circulation; CK-MB, creatine kinase-MB fraction; cTn-I, cardiac troponin-I.
Apoptotic indices and apoptotic rate as assessed by flow cytometry increased four hours after ROSC. Compared with the NS and Vehicle groups, the apoptotic index and apoptotic rate by in the Mito group were lower (P<0.05). This links exogenous mitochondrial transplantation to reduced cardiomyocyte apoptosis after CPR (Fig. 5B, 5C, 5D).
Mitochondrial transplantation reduces the markers of myocardial injury
ELISA was used to detect the expression of myocardial injury markers, CK-MB and cTn-I, at four hours of ROSC. Compared to the Sham group, the levels of CK-MB and cTn-I in the serum of the other groups showed a significant rise (P<0.05). However, both CK-MB and cTn-I levels in the serum of the Mito group were lower than those in the NS and Vehicle groups, suggesting that myocardial injury was reduced (P<0.05, Fig. 5F).
Four hours of ROSC, Tissue samples from sham group exhibited a well-organized structure of myocardial fibers, with no apparent inflammatory cell infiltration into the stroma (Fig.5E). In the NS and Vehicle groups, the myocardial fibers were disordered, with some myocardial cells exhibiting thickening, rupture, and vacuolization. Also, there was noticeable inflammatory cell invasion of the stroma. The myocardial arrangement in the Mito group was more neatly arranged than that in the NS and Vehicle groups although thickening of myocardial cells was noted.
Discussion
PRMD is a common complication following resuscitated. It is characterized by systolic and diastolic insufficiency, arrhythmias, and recurrent cardiac arrest. Circulatory dysfunction occurs in approximately two-thirds of cardiac arrest subjects is associated with a poor outcome (Han and Lee, 2022). Unlike myocardial depression caused by regional ischemia characterized by localized ventricular wall-motion abnormalities, myocardial depression caused by PAMD after CA manifest as global left ventricular systolic dysfunction accompanied by reduced ejection fraction, left ventricular diastolic dysfunction, and right ventricular dysfunction (Lazzarin, et al., 2022; Xu, et al., 2008). Of these, global left ventricular systolic dysfunction is the most significant manifestation.
In the present study, we test the hypothesis that administration of exogenous mitochondrial is cardioprotective effects after cardiopulmonary resuscitation. The rationale was to enhance the energy-dependent contractility of the myocardium. Mitochondria from non-ischemic gastrocnemius muscle of health donor animals were isolated and a manner that maximized their healing potential. Of import, uptake of the delivered mitochondria occurred in the heart and myocytes, perhaps via micropinocytosis (Fig. 6) (Kami and Gojo, 2020). AS well, mitochondrial internalization occurred through actin-dependent endocytosis (Pacak, et al., 2015). Internalized mitochondria promote degradation of resident via lysosomes to maintain an energy mitochondrial balance (Jia, et al., 2022; Liu, et al., 2022). Internalized mitochondria facilitated respiratory restoration in recipient cells with depleted mtDNA, and improved cellular function by increasing ATP levels and oxygen-consumption rates (Pacak, et al., 2015).
Potential mechanisms by which mitochondrial transplantation preserves cardiac function following ROSC.
Exogenous mitochondria enter the cell through actin-dependent endocytosis or macropinocytosis. A small number of mitochondria bind to lysosomes and are degraded. Most exogenous mitochondria can escape from the donor cells and play a role in recipient cells, altering the dynamic equilibrium of the receptor cells and exerting their protective function. ROSC, return of spontaneous circulation. By Figdraw.
Contrary to expectations, increased numbers of mitochondria were not found in the hearts of treated animals. Possibly CPR caused ischemia/reperfusion injury to organs throughout the body, with non-specific distribution and uptake of the delivered mitochondria. The results of our study are consistent with other date that the energy metabolism of myocardial mitochondria improved after mitochondrial transplantation along with increased complex activity and ATP content (Jia, et al., 2022; Pacak, et al., 2015). Not surprisingly, mitochondrial transplantation is being pursued as a therapeutic strategy in part to effects coronary patency, autoimmunity, and arrhythmia attacks (Masuzawa, et al., 2013; Shin, et al., 2019). The clinical application of mitochondrial transplantation can enhance the prognosis of pediatric patients requiring extracorporeal membrane oxygenation following cardiac surgery, the improvement of cardiac function in patients helps them successfully wean off ECMO support (Emani and McCully, 2018). It is worth noting that the method of mitochondrial isolation may better meet the needs of clinical settings where an interventional time below 60 min is desired (Ali, et al., 2021). After the isolation, mitochondria can be immediately used for transfer and internalization (Ali, et al., 2021). Still concerns regarding mitochondrial hemocompatibility and stability in serum have been raised. In this line, serum calcium did not adversely impact isolated mitochondria (Maleki, et al., 2023).
ROS is generated with ischemia and then targets the normal physiologic functions of cells by damaging the structures of proteins, lipids, and even DNA (Zhu, et al., 2024). As well, oxidative stress promotes cardiac ischemia-reperfusion injury and was implicated in pro-inflammatory responses and apoptosis, both of which worsen myocardial cell injury (Patel and Karch, 2020). In a regional ischemia-reperfusion model, mitochondrial transplantation reduced oxidative stress in cells following reperfusion (Zhang, et al., 2019). Similaerly, in the present study, mitochondrial transplantation decreased the levels of myocardial oxidative markers and increased the expression of antioxidant enzymes after four hours of ROSC.
Ischemia/reperfusion injury of myocardial tissue releases CK-MB and cTn-I, commonly used indicators of early myocardial injury in clinical settings (Zhang, et al., 2021). Although the concentrations of CK-MB and cTn-I rose significantly four hours after resuscitation in the CA-CPR model, mitochondrial transplantation significantly reduced this effect. During ischemia, the loss of mitochondrial membrane potential leads to mitochondrial swelling and the release of cytochrome C followed by caspase-mediated cell death during reperfusion (Lu, et al., 2023; Milliken, et al., 2022). Apoptosis is the process by which cardiomyocytes die (Kunapuli, et al., 2006). Increase apoptosis was found in rats after CA-CPR along with increased caspase-3 expression after resuscitation (Wu, et al., 2021). Similar results were reported in pigs that underwent CA-CPR (Wang, et al., 2023). We found that the level of apoptosis in cardiomyocytes increased significantly after CPR whereas administration of healthy mitochondria significantly reduced cardiomyocyte apoptosis. Exogenous mitochondrial transplantation significantly reduced the disordering of myocardial fiber arrangement and inflammatory cell infiltration.
In conclusion, exogenous mitochondrial transplantation improved cardiac function after CPR. The specific mechanism involved may be related to the improvement in mitochondrial function, thus reducing the oxidative-stress response and apoptosis of myocardial cells. These dates suggest possible advantage in mitochondrial transplantation following cardiopulmonary resuscitation.
Limitations
Hemodynamic observation was carried out only for four hours after cardiopulmonary resuscitation, Thus, the impact of mitochondrial transplantation on long-term cardiac function following cardiopulmonary resuscitation could not be assessed. In this study, the animal model consisted of healthy rats. However, in clinical practice, CA is primarily caused by cardiac issues. Healthy rats tend to exhibit higher recovery ability after CA, which may lead to an overestimation of the therapeutic effects of mitochondrial transplantation. In our next phase, we plan to utilize various cardiac arrest models in conducting comprehensive studies to validate the effectiveness of mitochondrial transplantation in different pathological models.
Methods
Animals
The experimental protocol (Fig.1) was approved by the Animal Experiment Committee of the General Hospital of Central Theater Command (No.2023017) and conformed to the Guide for the Care and Use of Experimental Animals published by the National Institutes of Health, USA (NIH Publication No. 5377-3, 1996). Adult male SD (7 to 8 weeks old) rats weighing 250 to 350g were purchased from Hunan Silaidajing Experimental Animal Co., Ltd. (No. 430727231102711675, Changsha, China). Male rats were selected to avoid estrous cycle interference. Animals were housed in standard cages with a 12-hour light-dark cycle at a room temperature 22°C.
Animals were randomly assigned to four groups using the random number table method: a sham group, NS group, Vehicle group, and Mito group (n=17 per group). Each group was further divided into two survival-analysis subgroups: a four-hour group (n=7) and a 72-hour group (n=10). Rats in the NS, Vehicle and Mito groups underwent cardiac arrest induced by asphyxia and CPR, while rats in the sham group underwent tracheal intubation and arteriovenous puncture only, without CA and CPR.
Experimental protocol
CA-CPR model
Establishment of the CA-CPR model was as published previous (Huang, et al., 2020). Rats were anesthetized with pentobarbital sodium (45 mg/kg) i.p. A 16-G intravenous catheter was used for endotracheal intubation. The left femoral artery and the right femoral vein were subsequently connected using a 24-G venous indwelling needle for dynamic blood pressure detection and venous-access establishment. Blood pressure, heart rate, and rectal temperature were monitored using the ALC-MPA monitoring system (Alcott Biotech, Shanghai, China). After 10 minutes of mechanical ventilation, asphyxia-mediated cardiac arrest was induced by blocking the tracheal tube. CA was defined as a decrease in systolic blood pressure to 25 mmHg. Five minutes after CA, external chest compression (200 bpm) and mechanical ventilation (tidal volume, 0.60 ml/100 g, respiratory rate, 80 bpm) were initiated, and epinephrine (40 mcg/kg) was administered intravenously. Return of spontaneous circulation (ROSC) was defined as the spontaneous restoration of sinus rhythm with a systolic blood pressure of >60 mmHg that was maintained for 10 minutes. Subsequently, corresponding treatments were administered. Animals that did not regain autonomic circulation within five minutes were excluded from the study. After ROSC, hemodynamics and echocardiograms were recorded for 4 hours. Basal body temperature was maintained at 37°C throughout the intervention. Pentobarbital sodium (10 mg/kg) was given if animals urinated or showed increased limb-muscle tension. Twenty-eight animals of four-hour group completed the full protocol and were humanely euthanized with excess pentobarbital sodium 4 hours after ROSC. Myocardial tissue and blood samples were collected for detection. The remaining 40 animals of 72-hour group were monitored for 72-hour to track survival.
Intervention measures
Ten minutes after ROSC, 0.5 ml of saline, 0.5 ml of respiration buffer, and 0.5 ml of 1×109/mL mitochondrial suspension were injected through the femoral vein in the NS, Vehicle, and Mito groups, The Sham group received 0.5 ml of saline at 30 minutes postoperatively.
Echocardiography
To assess myocardial function, an ultra-high resolution, small-animal ultrasound imaging system (SigmaVET, Esaote, Genoa, Italy) was used to capture M-mode echocardiographic images at the parasternal long axis and/or parasternal short axis near the papillary muscle at 1-, 2-, 3-, and 4-hours post-ROSC.
Survival analysis
In the 72-hour survival study cohort, the catheters were extracted four hours post-ROSC, and the surgical incisions were closed. Animals were placed back in their cages and administered subcutaneous injections of 0.5ml of 1% lidocaine for analgesia. Animals were monitored every 2 hours during the initial 24 hours following ROSC, and then at 48 and 72 hours. Animals that exhibited signs such as wheezing or a respiratory rate below five breaths per minute were deemed moribund and humanely euthanized (Su, et al., 2021). At the end of the time rats were euthanized.
Isolation and identification of mitochondria
Mitochondrial isolation and labeling
Mitochondria were isolated as published. 31 In brief, 180 mg of gastrocnemius muscle obtained from healthy rats was placed into a centrifuge tube containing 5 ml of pre-chilled homogenizing buffer (300 mM sucrose, 10 mM K-HEPES, and 1 mM K-EGTA), homogenized, and incubated on ice with Bacillus subtilis A protease (P5380, Sigma Aldrich, Darmstadt, Germany) for 10 minutes. The mixture was filtered through a 40-μm cell filter, bovine serum albumin (ST023-50 g, Beyotime, Shanghai, China) was added and incubated for five minutes. The homogenate was then filtered through a 40-um screen and then through a 10-um screen, and centrifuged at 9000 g for 10 minutes at 4℃. The precipitate was reconstituted in 1 ml of pre-chilled respiration buffer (250 mM sucrose, 2 mM potassium dihydrogen phosphate, 10 mM magnesium chloride, 20 mM K-HEPES, and 0.5 mM K-EGTA). Mitochondrial counts were as previously published and were quantified using a fluorescence microscope (BX53, OLYMPUS, Tokyo, Japan) (McCully, et al., 2009). The isolated mitochondria were incubated with MitoTracker Red CMXRos (C1049B-50ug, Beyotime, Shanghai, China) and washed with respiratory buffer prior to administration.
Detection of mitochondrial membrane potential
A mitochondrial membrane potential detection kit (JC-1) (C2003S; Beyotime, Shanghai, China) was used to characterize function. Purified mitochondria were incubated with JC-1 staining solution at 4℃ for a duration of 60 minutes followed by imaging using a fluorescence microscope.
Western blotting
Total protein was extracted from skeletal muscle and purified mitochondria. Protein concentration was determined by the BCA method. Protein was boiled in a water bath for 10 minutes. Electrophoresis was performed using a 12% SDS-PAGE gel (P0012A, Beyotime, Shanghai, China), followed by transfer to a PVDF membrane (FFP70, Beyotime, Shanghai, China). Non-specific binding was blocked with 5% skim milk powder at room temperature for two hours. Membranes were exposed to primary antibodies overnight at 4℃: COX -Ⅳ (1:1000; AC610, Beyotime, Shanghai, China) and GAPDH (1:3000; LF206, Epizyme, Shanghai, China) on a shaker at 4℃ for 12 hours. After washing, membranes were incubated with horseradish peroxidase-labeled secondary antibody for two hours and developed with the ECL chemiluminescence method.
Mitochondrial distribution and uptake
Mitochondrial co-localization was determined as published (Masuzawa, et al., 2013). Tissue sections were incubated with 0.25% Triton-X-100 for 10 minutes at room temperature, blocked with normal bovine serum albumin, and incubated with primary antibody generated against alpha-actinin 2 (1:500; GTX103219; GeneTex, Irvine, USA) at 4℃ overnight. After washing with PBST, sections were incubated with an Alexa Fluor 488-labeled secondary antibody (1:1000, HZ0176; Huzhen, Shanghai, China) for one hour. DAPI (P0131-5 ml; Beyotime, Shanghai, China) was applied to stain nuclei. Images were acquired using the Olympus fluorescence microscope.
Analysis of cardiac mitochondria
Complex activity
Mitochondria were extracted from 0.1 g of myocardial tissue using differential centrifugation. The mitochondrial suspension was disrupted by ultrasonic waves in an ice bath releasing the mitochondrial respiratory chain complex. The working solution from mitochondrial complex activity test kit (AKOP008M, AKOP006M; Boxbio, Beijing, China) was mixed with the samples and the absorbance at 605 nm and 550 nm measured values at various timepoints. The variance between the two timepoints was calculated and recorded as ΔA1 and ΔA2. Protein concentration (C) of each sample was measured. Mitochondrial complex II activity was calculated as 476.19 × ΔA1/C, and mitochondrial complex IV activity was calculated as 1099.48 × ΔA2/C.
ATP determination
ATP levels were determined using an ATP-content test kit (A095-1-1; Nanjing Jiancheng, Nanjing, China).
Tom20 labeling
Tissue sections were labeled with Tom20 antibody (1:100, AF5206; Affinity, Changzhou, China), incubated with a horseradish peroxidase-coupled secondary antibody (1:400, 5220-0336; SeraCare, Massachusetts, USA), subjected to tyramide signal amplification (TSA), stained with DAPI and images acquired using an Olympus fluorescence microscope.
Transmission electron microscope
Fixed hearts were rinsed with phosphate-buffered saline (PBS) for 45 minutes and exposed to 1% osmium tetroxide for two hours. After dehydration in graded ethanol concentrations, tissue samples were immersed in epoxy resin, embedded, and cut into ultrathin sections (60-80 nm). The sections were stained with uranium and lead, and examined and imaged using a transmission electron microscope (HT7800; Hitachi, Tokyo, Japan).
Quantification of MDA and SOD detection
The SOD activity and MDA levels of myocardial samples were determined with a malondialdehyde (MDA) test kit (A003-1; Nanjing Jiancheng, Nanjing, China) and superoxide dismutase (SOD) test kit (A001-3; Nanjing Jiancheng, Nanjing, China).
Detection of myocardial injury
TUNEL assay
Paraffin-embedded tissue sections were processed with an in-situ cell-death detection kit (11684795910; Roche, Basel, Switzerland) for terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling. The ratio of TUNEL-positive cells to total cells was determined.
Flow cytometry
To prepare a single-cell suspension, myocardium tissue samples were digested with collagenase II at 37°C and filtered. Cells were washed with PBS, suspended in the binding buffer and stained with Annexin-V (Annexin-V PI cell double-staining apoptosis-detection kit; A211-02; Vazyme, Nanjing, China). Apoptosis was assessed using flow cytometry.
Markers of ischemic injury
Blood from rats was obtained the femoral artery and the serum separated. The levels of myocardial-injury markers were measured using the Creatine Kinase MB isoenzyme (CK-MB) kit (MM-0625R1; Meimian, Yancheng, China) and the Cardiac Troponin I (cTn-I) kit (MM-0426R2; Meimian, Yancheng, China).
Histology
Hearts was fixed in 4% paraformaldehyde for 24 hours, dehydrated, rendered transparent, embedded in paraffin and cut into thin sections. After dewaxing and rehydration, the sections were stained with hematoxylin and eosin, and images acquired using a light microscope.
Statistics and data analysis
IBM SPSS Statistics software (V26.0, IBM Corp, New York, USA) was used for data analysis. All data are expressed as mean ± standard deviation (Mean ± SD). Sample size for the four-hour group was calculated as published (Dell, et al., 2002). The ejection fraction of the first three surviving rats among the groups was measured. The population standard deviation of the variable is 11.34. With a magnitude of difference set at 15, an error rate of 0.1, and a power of 80%, the smallest acceptable sample size was determined to be five animals. Considering an animal loss of approximately 20% after four hours of CPR, seven animals were selected for each group. The sample size for the 72-hour group was calculated using StatBox (StatBox-Open 0.1.0; Cloud Powered Clinical Trial, Hebei, China). Set the survival rate at 20% for survival rate 1 and 80% for survival rate 2 (with power = 0.8 and α = 0.05). Accordingly, 10 was the smallest acceptable animal sample size. The comparison of time-based measurements was evaluated using repeated-measures tow-way ANOVA, with groups as the between-subjects factor and time as the within-subjects factor. One-way ANOVA and Tukey’s multiple-comparison test were employed to compare multiple groups. Survival was characterized using the Kaplan-Meier survival analysis test. Statistical significance was defined at P<0.05.
Acknowledgements
We thank LetPub (www.letpub.com) for linguistic assistance and pre-submission expert review.
Additional information
Funding
This work was supported by the National Natural Science Foundation of China (82372210, 81901932) and Natural Science Foundation of Hubei Province (2021CFB492).
Author contributions
X.Z. and G.S.G. supervised the study and designed the experiments. Z.W. and J.Z performed the experiments. Z.W and J.Z. collected, analyzed and interpreted the data. M.D.X. and J.Y.Y. supervised the experiments. X.Y.M. and M.Z.S. accessed and verified the data. Z.W. and J.Z. wrote the manuscript. X.Z. and G.S.G. revised the manuscript. All authors read and approved the final version of the manuscript.
Ethics
The animal experiments were approved by the Animal Experiment Committee of the General Hospital of Central Theater Command (No.2023017).
Data availability
The data underlying this article will be shared on reasonable request to the corresponding author.
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Article and author information
Zhen Wang
The First School of Clinical Medicine, Southern Medical University, Guangzhou, China
Jie Zhu
The First School of Clinical Medicine, Southern Medical University, Guangzhou, China
Mengda Xu
Department of Anesthesiology, General hospital of central theater command of PLA, Wuhan, China
Xuyuan Ma
Base of Central Theater Command of People’s Liberation Army, Hubei University of Medicine, Wuhan, China
Maozheng Shen
Base of Central Theater Command of People’s Liberation Army, Hubei University of Medicine, Wuhan, China
Jingyu Yan
Department of Anesthesiology, General hospital of central theater command of PLA, Wuhan, China
Guosheng Gan
The First School of Clinical Medicine, Southern Medical University, Guangzhou, China, Department of Anesthesiology, General hospital of central theater command of PLA, Wuhan, China
For correspondence:
526193186@qq.com
Xiang Zhou
The First School of Clinical Medicine, Southern Medical University, Guangzhou, China, Department of Anesthesiology, General hospital of central theater command of PLA, Wuhan, China
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