RESUMEN
NP cells of the intervertebral disc and articular chondrocytes reside in avascular and hypoxic tissue niches. As a consequence of these environmental constraints the cells are primarily glycolytic in nature and were long thought to have a minimal reliance on mitochondrial function. Recent studies have challenged this long-held view and highlighted the increasingly important role of mitochondria in the physiology of these tissues. However, the foundational understanding of mechanisms governing mitochondrial dynamics and function in these tissues is lacking. We investigated the role of mitochondrial fusion protein OPA1 in maintaining the spine and knee joint health in mice. OPA1 knockdown in NP cells altered mitochondrial size and cristae shape and increased the oxygen consumption rate without affecting ATP synthesis. OPA1 governed the morphology of multiple organelles, including peroxisomes, early endosomes and cis-Golgi and its loss resulted in the dysregulation of NP cell autophagy. Metabolic profiling and 13C-flux analyses revealed TCA cycle anaplerosis and altered metabolism in OPA1-deficient NP cells. Noteworthy, Opa1AcanCreERT2 mice with Opa1 deletion in disc and cartilage showed age-dependent disc degeneration, osteoarthritis, and vertebral osteopenia. Our findings underscore that OPA1 regulation of mitochondrial dynamics and multi-organelle interactions is critical in preserving metabolic homeostasis of disc and cartilage.
RESUMEN
The contribution of mitochondria to the metabolic function of hypoxic NP cells has been overlooked. We have shown that NP cells contain networked mitochondria and that mitochondrial translocation of BNIP3 mediates hypoxia-induced mitophagy. However, whether BNIP3 also plays a role in governing mitochondrial function and metabolism in hypoxic NP cells is not known. BNIP3 knockdown altered mitochondrial morphology, and number, and increased mitophagy. Interestingly, BNIP3 deficiency in NP cells reduced glycolytic capacity reflected by lower production of lactate/H+ and lower ATP production rate. Widely targeted metabolic profiling and flux analysis using 1-2-13C-glucose showed that the BNIP3 loss resulted in redirection of glycolytic flux into pentose phosphate and hexosamine biosynthesis as well as pyruvate resulting in increased TCA flux. An overall reduction in one-carbon metabolism was noted suggesting reduced biosynthesis. U13C-glutamine flux analysis showed preservation of glutamine utilization to maintain TCA intermediates. The transcriptomic analysis of the BNIP3-deficient cells showed dysregulation of cellular functions including membrane and cytoskeletal integrity, ECM-growth factor signaling, and protein quality control with an overall increase in themes related to angiogenesis and innate immune response. Importantly, we observed strong thematic similarities with the transcriptome of a subset of human degenerative samples. Last, we noted increased autophagic flux, decreased disc height index and aberrant COL10A1/collagen X expression, signs of early disc degeneration in young adult bnip3 knockout mice. These results suggested that in addition to mitophagy regulation, BNIP3 plays a role in maintaining mitochondrial function and metabolism, and dysregulation of mitochondrial homeostasis could promote disc degeneration.Abbreviations: ECAR extracellular acidification rate; HIF hypoxia inducible factor; MFA metabolic flux analysis; NP nucleus pulposus; OCR oxygen consumption rate; ShBnip3 short-hairpin Bnip3.