Baseline characteristics of rats and resuscitation characteristics (x̄ ± s)

Experimental design of the in-vivo study, Rats in all groups except the Sham group underwent cardiac arrest for 5 minutes and then received the corresponding intervention 10 minutes after ROSC. Four hours after ROSC, the 4-hour group was used to collect myocardial tissue and blood samples for detection, while 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). (B) EF of rats in each group from baseline to 4 hours following ROSC (n=7). (C) HR and MAP changes during post -ROSC in 4 hours (n=7). (D) Survival rate during the first 72 hours following ROSC (n=10). Myocardial function between groups was compared by time-based measurements in each group using repeated-measures ANOVA. The survival rate between groups was compared by the Kaplan-Meier survival analysis test. * P<0.05 vs. the Sham group and # P<0.05 vs. the 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 either as 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.

Localization and uptake of transplanted mitochondria in endocardium, myocardial tissue was stained for anti-α-actinin 2 (ACTN2; green) and nuclei (blue); the pre-stained isolated mitochondria were labeled red (n=3). Scale bar = 80 µm.

Administration of mitochondria ameliorates ischemia reperfusion-mediated mitochondrial alterations in cardiomyocytes four hours after ROSC. (A and B) A and B represent the detection of fluorescence intensity of TOM20. A represents the image, while B represents the quantitative data. Scale bar = 100 µm (n = 3). (C and D) C shows representative photographs of mitochondrial morphology obtained through TEM examination, with arrows indicate calcium accumulation. D illustrates the degree of mitochondrial damage (n=3). Scale bars = 500 nm. (E and F) Changes in myocardial mitochondrial complex II and IV enzyme activities in hearts (n=7). (G) The ATP content in myocardial tissue was measured by colorimetry (n=7). (H and I) mPTP opening was detected by Calcein staining. I represent the quantitative analysis of the mean fluorescent intensity acquired in H (n = 3). Analyses were performed using ANOVA with Tukey’s post hoc test. mPTP opening detection between groups was compared by time-based measurements in each group using repeated-measures ANOVA. The data were expressed as the mean ± standard deviation (SD). * P<0.05 vs. the Sham group and # P<0.05 vs. the Mito group.

Mitochondrial transplantation reduces myocardial damage 4 hours after ROSC. (A) Detection of mitochondrial ROS in various groups (n=3). (B and C) Changes in malondialdehyde and superoxide dismutase activity level in cardiac tissue (n=7). (D) Myocardial apoptosis level was examined using TUNEL (n=3). Scale bar = 100 µm. (E) The percentage of myocardial apoptosis was examined using flow cytometry (n=3). (F) Quantitative analysis of myocardial TUNEL apoptosis index and flow apoptosis rate (n=3). (G and H) Immunoblotting and quantitative analysis of the expression level of cleaved caspase-3 in the myocardium 4 hours after ROSC (n=3). (I and J) The changes in CK-MB and cTn-I level in the serum of rats were examined using ELISA (n=7). (K and L) Representative histological sections of the myocardium stained with hematoxylin and eosin. Myocardium from each experimental group were subjected to histological evaluation (n=3), Scale bar = 100 µm. 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. CK-MB, creatine kinase-MB fraction; cTn-I: cardiac troponin-I.

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 CPR.