Baseline characteristics of rats and resuscitation characteristics .

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.

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.

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 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.

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.