Electron Transport Chain Complex II Sustains High Mitochondrial Membrane Potential in Hematopoietic Stem and Progenitor Cells

The role of mitochondria in the fate determination of hematopoietic stem and progenitor cells (HSPCs) is not solely limited to the switch from glycolysis to oxidative phosphorylation, but also involves alterations in mitochondrial features and properties, including mitochondrial membrane potential (ΔΨ_mt). Several research groups have used mitochondrial dyes and have showed that long term multi-lineage reconstitution is enriched in low ΔΨ_mt fraction. However, hematopoietic stem cells (HSCs) exhibit higher pump activity than mature populations, and this causes the enhanced extrusion of mitochondrial dyes used for measuring ΔΨ_mt, such as tetramethylrhodamine methyl ester (TMRM), which in turn can lead to biased results (Bonora M. et al, 2018). In this study, while considering the activity of xenobiotic efflux pumps in HSCs, we have assessed the equilibrium between electron transport chain (ETC) complexes and ATP production in order to elucidate the mechanism that sustain mitochondrial membrane potential in HSPCs.

We first used flow analysis of HSCs and other bone marrow populations, stained by TMRM in presence of Verapamil, an efflux pump inhibitor, to show a downward trend in ΔΨ_mt along with hematopoietic differentiation. To validate high ΔΨ_mt as a key feature of HSCs, we measured Ki67 positivity to assess whether ΔΨ_mt is associated with cell cycle quiescence in HSPCs. When Lin^-Sca-1⁺c-Kit⁺ (LSK) cells were separated into two fractions, based on their TMRM intensity, we found that the percentage of Ki67⁺ cells in LSK-High was lower than the one in LSK-Low, and were comparable to CD150⁺CD48^- HSC-enriched fraction. Consistently, phenotypic HSCs preferentially reside in the TMRM high population, with less Ki67 positivity.

Since ΔΨ_mt levels in the cells is determined by the balance between proton pumping (by ETC) and proton flow (by ATP synthase/complex V), we next assessed ETC complexes. The expression of ATP5A, a key subunit of complex V, and of NDUFV1, a subunit of complex I, were particularly weak in HSPCs and drastically increased following differentiation process, while no differences were detected in complex II subunit SDHA expression between HSCs and mature populations. Likewise, the activity of complex I increased following differentiation process, while the activity of complex II remained stable among HSC, LSK, and Lin⁻ fractions. Interestingly, when the respective ratios of complex I and II to complex V were calculated, compared to complex I, a significantly higher ratio of complex II: complex V was found in HSPCs. Collectively, these data support the hypothesis that HSPCs have low proton flow comparing mature populations, but similar proton pumping activity, especially due to complex II, which finally results in a higher ΔΨ_mt.

In order to deeply investigate the contribution of complex I, II and III to sustain ΔΨ_mt, the reduction of TMRM intensity after the administration of low dosages of their specific inhibitors (Rotenone, TTFA and Antimycin A, respectively) was analyzed. The reduction of TMRM intensity by Rotenone was observed in the committed cells, and the addition of Antimycin A led to a drop in TMRM intensity in all hematopoietic lineages. Critically, complex II inhibition by TTFA caused a substantial decrease of ΔΨ_mt, particularly in HSPCs. Finally, we investigated the functional importance of each ETC complex in HSCs, founding that TTFA, but not Rotenone, caused a reduction in in vitro colony-replating capacity, and a similar effect was observed after administration of Antimycin A.

Altogether this study highlights complex II as a key regulator of ΔΨ_mt in HSPCs and suggests the distinct roles of complex I and complex II in hematopoiesis. Further characterization of the precise mechanisms regulating mitochondrial controls in HSCs will contribute to a better understanding of an active role of mitochondria in HSC homeostasis.