Mitochondrial transplantation prolongs cold ischemia time in murine heart transplantation

Abstract

Background: Cold ischemia time (CIT) causes ischemia‒reperfusion injury to the mitochondria and detrimentally effects myocardial function and tissue viability. Mitochondrial transplantation replaces damaged mitochondria and enhances myocardial function and tissue viability. Herein we investigated the efficacy of mitochondrial transplantation in enhancing graft function and viability after prolonged CIT.

Methods: Heterotopic heart transplantation was performed in C57BL/6J mice. Upon heart harvesting from C57BL/6J donors, 0.5 ml of either mitochondria (1 × 10⁸ in respiration buffer; mitochondria group) or respiration buffer (vehicle group) was delivered antegrade to the coronary arteries via injection to the coronary ostium. The hearts were excised and preserved for 29 ± 0.3 hours in cold saline (4°C). The hearts were then heterotopically transplanted. A second injection of either mitochondria (1 × 10⁸) or respiration buffer (vehicle) was delivered antegrade to the coronary arteries 5 minutes after transplantation. Grafts were analyzed for 24 hours. Beating score, graft function, and tissue injury were measured.

Results: Beating score, calculated ejection fraction, and shortening fraction were significantly enhanced (p < 0.05), whereas necrosis and neutrophil infiltration were significantly decreased (p < 0.05) in the mitochondria group as compared with the vehicle group at 24 hours of reperfusion. Transmission electron microscopy showed the presence of contraction bands in vehicle but not in mitochondria grafts.

Conclusions: Mitochondrial transplantation prolongs CIT to 29 hours in the murine heart transplantation model, significantly enhances graft function, and decreases graft tissue injury. Mitochondrial transplantation may provide a means to reduce graft failure and improve transplantation outcomes after prolonged CIT.

Keywords: cold ischemia time; heart transplantation; ischemia–reperfusion; mitochondria; mitochondrial transplantation.

Fig. 1.

Experimental protocol. (A) C57BL/6J male mice (n = 17, 7–9 weeks) were used for all experimental groups. Donor hearts received 1 × 10⁸ mitochondria in 0.5 mL respiration buffer (Mitochondria, n = 8) or 0.5 mL respiration buffer (Vehicle, n = 9) delivered antegrade to the coronary arteries via injection to the coronary ostium using a tuberculin syringe with a 40 G needle, 10 minutes before organ harvest. Heart grafts were then preserved in normal saline solution containing 1% Penicillin and 1% Streptomycin at 4 °C for 29 ± 0.3 hours prior to heterotopic heart transplantation. Five minutes after transplantation, heart grafts received a second injection of 1 × 10⁸ mitochondria in 0.5 mL respiration buffer (Mitochondria) or 0.5 mL respiration buffer (Vehicle) delivered antegrade to the coronary arteries via injection to the coronary ostium using a tuberculin syringe with a 40 G needle. Mice were allowed to recover for 24 hours, and tissue was then collected for further analysis. Serial beating score assessment and echocardiography were obtained for analysis. (B) C57BL/6J male mice (n = 8, 7–9 weeks) were used for sham control (Sham, n = 4). Donor mice heart grafts were immediately transplanted with no CIT and no mitochondrial or respiration buffer injection. Recovery and analysis were identical as mentioned above.

Fig. 2.

Uptake and distribution of transplanted mitochondria. ¹⁸F-Rhodamine 6G labeled mitochondria (1 × 10⁹ in 0.5 mL respiration) were delivered antegrade to the coronary arteries via injection to the coronary ostium using a tuberculin syringe with a 30 G needle. Ten minutes after the injection animals were euthanized in CO₂ chamber and examined using positron emission tomography (PET) and microcomputed tomography (μCT). ¹⁸F-Rhodamine 6G labeled mitochondria were distributed throughout the heart and were not detectable in any other region of the body. Images are shown for (A) transverse, (B) sagittal, (C) coronal and (D) 3D reconstructed views.

Fig. 3.

Beating score in heart grafts at 10 minutes, 3 and 24 hours of reperfusion in Vehicle, Mitochondria and Sham groups. Beating score was determined as; 0, no contraction; 1, contraction barely visible or palpable; 2, obvious decrease in contraction strength, but still contracting in a coordinated manner; rhythm disturbance; 3, strong, coordinated beat but noticeable decrease in strength or rate; 4, strong contraction of both ventricles, regular rate. All analysis was performed by a blinded observer. Results show significant difference in BS between Mitochondria and Vehicle heart grafts at 24 hours of reperfusion. All values are mean ± SEM. *P < 0.05 vs. Sham. **P < 0.05 vs. Vehicle. n = 8 for Mitochondria, 9 for Vehicle, 4 for Sham.

Fig. 4.

Echocardiographic analysis in heart grafts. (A) Calculated ejection fraction (EF) and (B) shortening fraction (FS) were measured at 3 and 24 hours of reperfusion in Vehicle, Mitochondria and Sham groups. Results show significant difference in EF and FS between Mitochondria and Vehicle heart grafts at 3 and 24 hours of reperfusion. All values are mean ± SEM. *P < 0.05 vs. Sham. **P < 0.05 vs. Vehicle. n = 8 for Mitochondria, 9 for Vehicle, 4 for Sham.

Fig. 5.

Myocardial tissue injury and neutrophil infiltration at 24 hours of reperfusion. (A) Representative H&E micrographs of heart grafts tissue sections. Tissue sections from Mitochondria heart grafts show significantly less severe necrosis and inflammatory cells infiltration as compared to Vehicle heart grafts. Sham heart grafts show only minimal myocardial injury and inflammatory cells infiltration. Scale bars, 100 μm. (B) Representative micrographs of tissue sections from heart grafts stained for myeloperoxidase content to quantify neutrophil infiltration. Tissue sections from Mitochondria heart grafts show decreased neutrophil infiltration as compared to Vehicle heart grafts. Tissue sections from Sham heart grafts show no neutrophil infiltration. Scale bars, 100 μm. (C) Representative transmission electron micrographs. Transmission electron microscopy analysis shows similar profile in Mitochondria and Sham heart grafts, with no observed contraction bands, while Vehicle heat grafts show contraction bands indicating myocardial injury. Scale bars, 1 μm. (D) Area of necrosis and inflammatory cells infiltration in the entire transversal tissue sections of Vehicle, Mitochondria and Sham heart grafts. Results show significantly less necrosis and inflammatory cells infiltration in Mitochondria heart grafts as compared to Vehicle. (E) Neutrophil count determined by positive myeloperoxidase staining counted in 5 random (20x) visual fields per tissue section. Results show significantly lower neutrophil infiltration in Mitochondria heart grafts as compared to Vehicle. All values are mean ± SEM. *P < 0.05 vs. Sham. **P < 0.05 vs. Vehicle. n = 8 for Mitochondria, 9 for Vehicle, 4 for Sham.

Fig. 6.

Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay of the heart grafts tissue sections at 24 hours of reperfusion to determine severity of apoptosis in Vehicle, Mitochondria and Sham heart grafts. Twenty-seven visual fields per section at 20x were analyzed. Results show no significant difference in TUNEL positive nuclei between Mitochondria and Vehicle heart grafts. All values are mean ± SEM. *P < 0.05 vs. Sham. **P < 0.05 vs. Vehicle. n = 8 for Mitochondria, 9 for Vehicle, 4 for Sham.