Background/Purpose: Children with juvenile dermatomyositis (JDM) have decreased autophagy, as also confirmed by our RNA seq data in JDM muscle, which may contribute to accumulation of harmful agents. Consistent with that interpretation, our prior work demonstrated accumulation of calcified mitochondria in degenerated muscle fibers of JDM patients. In further mechanistic studies, we demonstrated mitochondrial reactive oxygen species (mtROS) as the main driver of mitochondrial calcification in human skeletal muscle cells. However, what remains to be explored is the effect of mitochondrial calcification on inflammation in calcifying skeletal muscle cells and in phagocytosing immune cells that may participate in clearance of calcified extruded mitochondria.

Methods: Mitochondrial calcification was induced in human skeletal muscle cells (RH30 cells) by addition of moderately high calcium-phosphate medium (Ca-P), and detected by fluorescent stain osteoimage. MtROS was measured using MitoSOX. Targeted metabolomics was performed on RH30 cells to identify potential pathways enriched upon mitochondrial calcification. Inflammation in RH30 cells was measured by RT-qPCR and ELISA. Mitochondrial DNA was measured using qPCR. Cardiolipin exposure was measured by flow cytometry. THP-1 monocytes were used to investigate the inflammatory potential of calcified mitochondria.

Results: Consistent with the role of calcium in mitochondrial metabolism, we found a significant increase in mtROS of calcified RH30 cells compared to untreated cells (p< 0.01). Pathway enrichment analysis on metabolites significantly downregulated in Ca-P treated cells revealed several mitochondrial-related pathways including mitochondrial respiration. In RH30 cells, upon mitochondrial calcification, there is a significant induction of interferon-stimulated gene expression and release of inflammatory cytokines including IL-1β (135 pg/mL vs. 75 pg/mL, p=0.0002). Further, cellular supernatants from calcified RH30 cells enriched in mtDNA could induce interferon responses in an interferon reporter cell line suggesting the extrusion of inflammatory mitochondria-derived components upon calcification of skeletal muscle cells. Consistent with these findings, investigation on isolated calcified mitochondria demonstrated a propensity of calcified mitochondria to release significant amounts mtDNA compared to non-calcified mitochondria (2,233,494 copies/mL vs. 581,655 copies /mL, p< 0.0001) and a trend for increased cardiolipin exposure (aCL MFI 18,500 vs. 10,271, p=0.07), suggesting destabilization of calcified mitochondria. Consistent with the proinflammatory nature of calcified mitochondria, THP-1 monocytes stimulated with calcified mitochondria but not non-calcified mitochondria secreted significant levels of IL-1β.

Conclusion: Mitochondrial calcification, as occurs in JDM, contributes to inflammation in skeletal muscle cells as well as in phagocytosing immune cells.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/mitochondrial-calcification-induced-inflammation-in-human-skeletal-muscle-and-immune-cells/