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Updated: Apr 14, 2026
Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue
Published on: March 23, 2015
Mitochondrial Membrane Potential and Nuclear and Gene Expression Changes During Human Disc Cell Apoptosis: In Vitro
Helen E Gruber1, Gretchen L Hoelscher, Synthia Bethea
1From the Department of Orthopaedic Surgery, Carolinas HealthCare System, Charlotte, NC.
Study Design:
A study using cultured human annulus cells and human annular tissue.
Objective:
To further explore and define mitochondrial mechanisms related to disc cell apoptosis in vitro and in vivo.
Summary Of Background Data:
Mitochondrial-dependent intrinsic signaling pathways are a well-recognized component of apoptosis (programmed cell death). Disc cell apoptosis is important because it is a major mechanism by which cell numbers decrease during disc degeneration. Our objective was to further explore and define mitochondrial mechanisms related to disc cell apoptosis.
Methods:
High-content screening techniques were used to study nuclear morphology and mitochondrial membrane potentials in cultured annulus cells. Gene expression in annulus tissue was studied with microarray analysis.
Results:
Cultured cells showed significantly increased nuclear size (an indicator of apoptosis) with increasing Thompson grade (P < 0.00001 by analysis of variance). A significant negative correlation for mitochondrial potential (which results from the difference in electrical potential generated by the electrochemical gradient across the inner membrane of the mitochondrion) versus Thompson grade was identified in cultured human annulus cells in control conditions (r = 0.356, P < 0.0001). When exposed to the K ionophore valinomycin at sublethal levels to induce apoptosis, a significant reduction in mitochondrial potential was identified versus nontreated cells. Gene expression patterns in more degenerated Thompson grade III, IV, and V discs versus healthier grade I and II discs showed significant upregulation of a number of genes with well-recognized apoptosis roles in mitochondrial potential decline (ITM2B, beta-2-microglobulin, and cathepsin B, DAP, GAS1, and PDCD5) and TNF-α associations (cathepsin B, RAC1, and PPT1).
Conclusion:
Data presented here show the in vivo expression of apoptosis-related genes associated with the loss of mitochondrial membrane integrity and decreased mitochondrial membrane potential with increasing Thompson scores. These data, which mimic our novel, direct cell-based in vitro findings, stress the importance of mitochondrial changes related to apoptosis and TNF-α during human disc degeneration.
Level Of Evidence:
N/A.
