Bone marrow-derived mesenchymal stem cells rescue injured H9c2 cells via transferring intact mitochondria through tunneling nanotubes in an in vitro simulated ischemia/reperfusion model

Abstract

The transplantation of mesenchymal stem cells (MSCs) is considered to be a promising treatment for ischemic heart disease; however, the therapeutic effects and underlying mechanisms of action require further evaluation. Mitochondrial dysfunction is a key event in simulated ischemia/reperfusion (SI/R) injury. The purpose of the present study was to investigate the mechanism of mitochondrial transfer, which may be involved the antiapoptotic action of co-culture with MSCs. An in vitro model of simulated ischemia/reperfusion (SI/R) was used in the present study. The apoptotic indexes were significantly increased when H9c2 cardiomyocytes were induced in the SI/R group. Following co-culture with bone marrow-derived (BM)-MSCs, H9c2 cells exhibited marked resistance against the SI/R-induced apoptotic process. Besides, mitochondrial transfer via a tunneling nanotube (TNT) like structure was detected by confocal fluorescent microscopy. In addition, following pretreated with latrunculin-A (LatA), an inhibitor of TNT formation, the BM-MSCs were not able to rescue injured H9c2 cells from apoptosis, as previously observed. In conclusion, the anti-apoptotic ability of BM-MSCs may be partially attributed to the recovery of mitochondrial dysfunction in SI/R, and the formation of TNTs appears to be involved in this action of mitochondrial transfer between adjacent cells.

Figure 1

Identification of isolated BM-MSCs. Flow cytometric analysis of (A) CD90 (B) CD45, (C) CD29 and (D) CD31. The results showed >90% of the BM-MSCs were positive for CD90 or CD29, whereas <3% of the BM-MSCs were positive for CD45 or CD31. BM-MSCs, bone marrow-derived mesenchymal stem cells; CD, cluster of differentiation.

Figure 2

Effects of co-culture with BM-MSCs on apoptosis in the control (Con), SI/R and SI/R co-cultured with BM-MSCs (Co-culture) groups. (A) Changes in apoptotic incidence in the Con, SI/R and Co-culture groups. Data are presented as the mean ± standard deviation. ^*P<0.05, vs. Con; ^#P<0.05, vs. SI/R. BM-MSCs, bone marrow-derived mesenchymal stem cells; SI/R, simulated ischemia/reperfusion.

Figure 3

Effects of co-culture with BM-MSCs on apoptosis and mitochondrial membrane potential in the control (Con), SI/R and SI/R co-cultured with BM-MSCs (Co-culture) groups. (A) Representative western blots showing the changes in the expression Bax and Bcl-2 in H9c2 cells from the Con, SI/R and Co-culture groups. (B) Changes in caspase-3 activity in H9c2 cells from the Con, SI/R and Co-culture groups. (C) Changes in mitochondrial membrane potential in H9c2 cells from the Con, SI/R and Co-culture groups. Data are presented as the mean ± standard deviation (^*P<0.05, vs. Con; ^#P<0.05, vs. SI/R). BM-MSCs, bone marrow-derived mesenchymal stem cells; SI/R, simulated ischemia/reperfusion; Bcl-2, B cell lymphoma-2; Bax, Bcl-2-associated X protein; JC-1, 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazole-carbocyanide iodine.

Figure 4

Mitochondria transfer following direct co-culture of ischemia/reperfusion-stimulated H9c2 cells with BM-MSCs. Arrows indicate the TNT-like structures. (A) Blue nucleus stained by DAPI; (B) Green fluorescence from GFP BM-MSCs; (C) Red mitochondria stained by MitoTracker Deep Red; (D) Merge of A–C. The arrows indicate the TNT-like structures. SI/R, simulated ischemia/reperfusion; BM-MSCs, bone marrow-derived mesenchymal stem cells; GFP, green fluorescence protein. GFP, green fluorescence protein; BM-MSCs, bone marrow-derived mesenchymal stem cells.

Figure 5

Evaluation of apoptotic incidence in the SI/R, Co-culture and Pre-LatA groups. Data are presented as the mean ± standard deviation (^*P<0.05, vs. SI/R. ^#P<0.05, vs. Co-culture). BM-MSCs, bone marrow-derived mesenchymal stem cells; SI/R, simulated ischemia/reperfusion; Co-culture, SI/R co-cultured with BM-MSCs; LatA, latrunculin-A; Pre-LatA, pretreated with LatA.

Figure 6

Effects of co-culture with BM-MSCs on apoptosis and mitochondrial membrane potential in the SI/R, Co culture and Pre LatA groups. (A) Representative western blotting of the changes in the expression levels of Bax and Bcl-2 in H9c2 cells. (B) Changes in caspase-3 activity in H9c2 cells from the different groups. (C) Changes in mitochondrial membrane potential in H9c2 cells from the different groups. Data are presented as the mean ± standard deviation (^*P<0.05, vs. SI/R; ^#P<0.05, vs. Co-culture). BM-MSCs, bone marrow-derived mesenchymal stem cells; SI/R, simulated ischemia/reperfusion; Co-culture, SI/R co-cultured with BM-MSCs; LatA, latrunculin-A; Pre-LatA, pretreated with LatA; Bcl-2, B cell lymphoma-2; Bax, Bcl-2-associated X protein; JC-1, 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazole-carbocyanide iodine.