Metabolic Adaptation of Regenerative Hematopoiesis Depends on Docking-Independent Mitochondrial Connexin-43 Available to Purchase

• Antioxidant activity of Cx43 depends on its autonomous activity in mitochondria of hematopoietic stem cells and multipotential progenitors.

• Cx43 activity depends on hemichannels that control mitochondrial Ca2+, a-ketoglutarate dehydrogenase and electron transfer chain activity.

Hematopoietic stem cells (HSC) exhibit a distinctive antioxidant profile during steady-state and stress hematopoiesis. HSC and multipotential progenitors (HSC/MPP) are metabolically coupled to bone marrow (BM) mesenchymal stromal cells through mitochondrial transfer, a process dependent on hematopoietic connexin 43 (Cx43) and low AMP-activated protein kinase (AMPK) activity. However, the mechanism by which Cx43 preserves mitochondrial functionality in HSC remains elusive. Here, through integrated transcriptomic, proteomic, metabolomic, phenotypic, and functional analyses of HSC and their isolated mitochondria, we identified that Cx43 is present on inner and outer mitochondrial membranes of HSC/MPP, where it primarily regulates mitochondrial metabolism and ATP synthesis by preserving the mitochondrial cristae, activation of mitochondrial AMPK and 2-oxoglutarate dehydrogenase (OGDH)-a rate liming enzyme in TCA cycle and electron transfer chain. During replicative stress, Cx43 deficient HSC/MPP fail to adapt metabolically, accumulate mitochondrial Ca2+, increase mitochondrial AMPK activity, mitochondrial fission, mitophagy, and production of reactive oxygen species, thereby limiting HSC/MPP regeneration potential. Disruption of hyper mitochondrial fragmentation and mitophagy by Drp1 dominant negative mutant (Drp1K38A) or restoration of mitochondrial function through ex vivo heteroplasmy prevent the harmful effects of Cx43 deficiency on mitochondrial metabolism and restore HSC activity in serial transplantation experiments. Re-expression analysis of Cx43 structure function mutants indicate that Cx43 hemichannels are sufficient to reset HSC mitochondrial metabolism, dynamics, Ca2+ levels, and regeneration capacity. This report defines the cell-autonomous mechanism of action behind the role of Cx43 in HSC activity and opens a venue to translational applications in transplantation.