Mitochondrial Respiration and The Biology of Growth Plate Chondrocytes

Cell differentiation is a complex process in which bioenergetic metabolism, specifically glycolysis and oxidative phosphorylation (OxPhos), plays a pivotal role. This role extends beyond ATP production and encompasses factors like the accumulation of intermediate metabolites such as 2-hydroxyglutarate (2HG). In fetal growth plates, hypertrophic chondrocytes are central to bone development. Despite their importance, our understanding of their regulation by bioenergetic metabolism is still in its infancy. The fetal growth plate is a hypoxic and high glycolytic structure. Hypoxia-inducible factor 1α (HIF1) is crucial for the survival of growth plate chondrocytes. HIF1 promotes glycolysis and lactate fermentation while reducing OxPhos activity. The survival role of HIF1 relies on its partial inhibition of OxPhos. Recently, we generated a mutant mouse lacking Mitochondrial Transcription Factor A (TFAM) in limb bud mesenchyme. TFAM, encoded by the nuclear genome, primarily regulates the transcription of mitochondrial-encoded electron transport chain subunits. Loss of TFAM did not impact the differentiation of mesenchymal progenitor cells into chondrocytes, chondrocyte proliferation, survival, or physiological death. However, it significantly delayed chondrocyte hypertrophy, leading to shorter bones. Thus, despite the high glycolytic activity of the fetal growth plate, its proper development still relies on OxPhos. While TFAM is not a known nuclear transcription factor, single-cell RNA sequencing (scRNA seq) of mutant and control growth plates at birth revealed significant changes in the expression of numerous genes involved in chondrocyte biology. These changes included reduced expression of HIF1 target genes and increased expression of genes involved in chondrocyte biology. Intriguingly, enhancing HIF1 activity appeared to slow down endochondral bone development, suggesting that decreased HIF1 signaling does not mediate the delayed hypertrophy observed in TFAM mutant growth plates. Untargeted metabolomics analysis revealed that levels of intermediate metabolites like 2HG were significantly elevated in mutant cells. Additionally, exposing control cells to hypoxia, thus activating the HIF1 signaling pathway, resulted in a similar increase in 2HG and other intermediate metabolites. 2HG plays a role in controlling cell differentiation by modulating epigenetic events and chromatin accessibility. Based on our preliminary data, we will test the following hypothesis: 1. loss of TFAM in growth plate chondrocytes delays hypertrophy by suppressing OxPhos, resulting in elevated levels of intermediate metabolites, such as 2HG, which modulate chromatin remodeling, ultimately affecting the nuclear transcriptome; 2. Augmented HIF1 activity replicates the effects of TFAM loss by delaying chondrocyte hypertrophy, reducing OxPhos activity, and increasing 2HG levels or other intermediate metabolites.