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 HSCs and MPPs.

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

Hematopoietic stem cells (HSCs) exhibit a distinctive antioxidant profile during steady-state and stress hematopoiesis. HSCs and multipotential progenitors (MPPs) are metabolically coupled to bone marrow mesenchymal stromal cells through mitochondrial transfer, a process dependent on hematopoietic connexin 43 (Cx43) and low adenosine monophosphate–activated protein kinase (AMPK) activity. However, the mechanism by which Cx43 preserves mitochondrial functionality in HSCs remains elusive. Here, through integrated transcriptomic, proteomic, metabolomic, phenotypic, and functional analyses of HSCs and their isolated mitochondria, we identified that Cx43 is present on the inner and outer mitochondrial membranes of HSCs/MPPs, in which it primarily regulates mitochondrial metabolism and adenosine triphosphate synthesis by preserving the mitochondrial cristae, activation of mitochondrial AMPK, and 2-oxoglutarate dehydrogenase, a rate-liming enzyme in tricarboxylic acid cycle and electron transfer chain. During replicative stress, Cx43-deficient HSCs/MPPs fail to adapt metabolically and accumulate mitochondrial Ca²⁺, leading to increased mitochondrial AMPK activity, mitochondrial fission, mitophagy, and production of reactive oxygen species, thereby limiting HSC/MPP regeneration potential. Disruption of hyperfragmentation of mitochondria and mitophagy by Drp1 dominant-negative mutant (Drp1^K38A) or restoration of mitochondrial function through ex vivo heteroplasmy prevents 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 indicates 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.