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. 2018 Feb 20;8(1):3330.
doi: 10.1038/s41598-018-21539-y.

Delivery of exogenous mitochondria via centrifugation enhances cellular metabolic function

Mi Jin Kim  1 , Jung Wook Hwang  1 , Chang-Koo Yun  1 , Youngjun Lee  2 , Yong-Soo Choi  3
Affiliations

Delivery of exogenous mitochondria via centrifugation enhances cellular metabolic function

Mi Jin Kim et al. Sci Rep. .

Abstract

Mitochondria are essential organelles involved in the maintenance of cell growth and function, and have been investigated as therapeutic targets in various diseases. Recent studies have demonstrated that direct mitochondrial transfer can restore cellular functions of cells with inherited or acquired mitochondrial dysfunction. However, previous mitochondrial transfer methods are inefficient and time-consuming. Here, we developed a simple and easy mitochondrial transfer protocol using centrifugation, which can be applied to any cell type. By our simple centrifugation method, we found that the isolated mitochondria could be successfully transferred into target cells, including mitochondrial DNA-deleted Rho0 cells and dexamethasone-treated atrophic muscle cells. We found that mitochondrial transfer normalised ATP production, mitochondrial membrane potential, mitochondrial reactive oxygen species level, and the oxygen consumption rate of the target cells. Furthermore, delivery of intact mitochondria blocked the AMPK/FoxO3/Atrogene pathway underlying muscle atrophy in atrophic muscle cells. Taken together, this simple and rapid mitochondrial transfer method can be used to treat mitochondrial dysfunction-related diseases.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Confocal microscopic analysis of target cells following mitochondrial transfer. (A) Experimental scheme for mitochondrial transfer and further application. The picture was drawn by us. (B) Representative images of UC-MSCs co-stained with fluorescent mitochondrial dyes (MitoTracker Green and MitoTracker Red CMXRos) at 24 h after mitochondrial transfer in the before mitochondrial transfer (upper panels) and after mitochondrial transfer (lower panels). Green: endogenous mitochondria of UC-MSCs (recipient cells), red: transferred mitochondria isolated from UC-MSCs, yellow: merged mitochondria. (C–E) Three confocal sections are shown in Z-stack overlay mode. Transferred mitochondria (red) within UC-MSCs were detected in the orthogonal view (upper panels; Z) and the corresponding signal profile (lower panels; S) together with endogenous mitochondria (green). Results are from the centre of the mitochondrial network of UC-MSCs (D) and 2 μm below (C) and 2 μm above (E) it. Z: Z stack image-ortho analysis, S: signal profile of each section. Scale bar, 50 μm.
Figure 2
Figure 2
Effect of mitochondrial dose on mitochondrial delivery efficiency. (A) Flow cytometric analysis of MitoTracker Green fluorescence in UC-MSCs at 24 h after transfer of various amounts of mitochondria (expressed as μg of protein). (B) qPCR analysis of human mtDNA (h-mtDNA), rat-mtDNA and rat-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in rat L6 muscle cells after transfer of mitochondria isolated from UC-MSCs. (C) Real-time PCR analysis of the mean h-mtDNA copy number of mitochondria isolated from UC-MSCs for transfer (prepared mitochondria in bar definition), rat L6 cells after mitochondrial transfer via centrifugal force (centrifugation) and rat L6 cells after 1 day of co-culture (co-culture). Relative mtDNA copy numbers normalised to the GAPDH level are shown. All values are mean ± SEM. N = 3, *P < 0.05 vs. normal UC-MSCs (0 μg). (D) Comparison of the transfer efficiency, as determined by the intensity of green fluorescence, by various cell types upon centrifugation for 5 min at 1,500 × g. The information of all cell lines were represented in Supplementary information section.
Figure 3
Figure 3
Changes in UC-Rho0 cell metabolism after delivery of intact mitochondria isolated from UC-MSCs. All analyses were performed 48 h after transfer of various amounts of mitochondria (expressed as μg of protein). Changes in cell proliferation (A), intracellular ATP content (B), MMP (C) and mROS level (D) were obtained and compared to the effects of mitochondrial transfer under uridine-free (−) conditions. Scale bar, 100 μm. (E,F) Immunoblot analysis of AMPK (E) and PGC-1α (F). Representative western blots and quantified expression levels (normalised to β-actin expression) are shown. (G) OXPHOS activity. The OCR (pmol/min) was measured in triplicate in three experiments. The OCRs of basal respiration (H), maximal respiration (I), ATP production (J) and spare respiratory capacity (K) were determined in UC-MSCs or UC-Rho0 cells. Cells cultured with uridine were used as control since it is needed to sustain viability of UC-Rho0 cells. All data represent the mean ± SEM. N = 3, *P < 0.05 vs. UC-Rho0 cells, #P < 0.05 vs. UC-MSCs. The grouping of blots were obtained from different parts of the same gel. Full length image of results by Western blot were represented in Fig. S7.
Figure 4
Figure 4
Metabolic changes in Dexa-treated atrophic L6 muscle cells after transfer of intact or damaged mitochondria. All analyses were performed 48 h after transfer of various amounts of mitochondria (expressed as μg of protein). Mitochondria were prepared from UC-MSCs grown under normal conditions (intact MT) or treated with oligomycin (damaged MT). Changes in cell proliferation (A), intracellular ATP content (B), MMP (C) and mROS level (D) were obtained and compared among the groups. (E–H) Immunoblot analysis of AMPK (E), PGC-1α (F), FoxO3α (G) and MuRF-1 (H). Representative western blots and quantified expression levels (normalised to β-actin expression) are shown. All data represent the mean ± SEM. N = 3, *P < 0.05 vs. Dexa (+) group, #P < 0.05 vs. UC-MSCs. The grouping of blots were obtained from different parts of the same gel. Full length image of results by Western blot were represented in Fig. S7.
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