Background/Purpose: Calcinosis, the accumulation of calcium crystals in soft tissues, is often a locus of infection and a debilitating manifestation of chronic juvenile dermatomyositis (JDM), contributing to long-term disability and sometimes death. Mechanistic understanding of calcinosis in JDM remains poorly understood, thus impeding therapeutic strategies. Our prior findings that the presence of calcified mitochondria and their remnants in degenerated muscle fibers and blood of JDM associated with calcinosis suggested that mitochondrial calcification plays a central role in JDM calcinosis. In this study, we hypothesized that the pathophysiological process in chronic active JDM contributes to mitochondrial activation and impairment, leading to mitochondrial calcification and extrusion.

Methods: To facilitate mitochondrial calcification, human skeletal muscle cells (RH30 cell line) were incubated in calcium phosphate (Ca-P) solution in the absence or presence of CoCl₂ (100 µM) or recombinant interferon-α (IFN-α) to mimic hypoxia and inflammation, respectively. Calcification in cells was assessed using Osteoimage, a stain for hydroxyapatite calcium salt crystals, by flow cytometry and microscopy. The extrusion of calcified mitochondria was determined by fluorescence microscopy. Mitochondrial damage was analyzed by flow cytometric analysis of mitochondrial ROS (mtROS) using fluorescent stain MitoSOX. Inflammation in RH30 cells in response to calcification was quantified by RT-PCR and ELISA.

Results: Hypoxia-mimetic CoCl₂ augmented Ca-P media-induced calcification in RH30 cells (% of osteoimage+ cells, 86.85 vs. 52.25; MFI 66929 vs. 12649, p< 0.05). Hypoxia caused mitochondrial formation of ROS, a process deemed essential for calcification as mitoTEMPO, a mitochondrial ROS scavenger, completely abrogated mitochondrial calcification. Fluorescent microscopy revealed an extrusion of calcified mitochondria in extracellular vesicles, resembling calcinosis. Muscle cells undergoing mitochondrial calcification demonstrated inflammation characterized by increased expression of interferon-induced genes and secretion of IL-6 in cellular supernatants. Finally, we hypothesized that IFN-α, one of the pathogenic factors of JDM found in muscle tissue, would promote mitochondrial calcification. Accordingly, we found a dose-dependent increase in the calcification of RH30 cells cultured in the Ca-P medium supplemented with IFN-α (p< 0.05).

Conclusion: Mitochondrial ROS is a chief regulator of mitochondrial calcification and extrusion in in vitro systems, promoting inflammation in muscle cells. Calcinosis associated with JDM is dystrophic, which, by definition, occurs in injured tissues despite the presence of normal systemic levels of calcium and phosphate. Hence, pathologic calcification in JDM does not regress in response to therapy of calcium depletion suggesting a role of local mechanisms in calcium dyshomeostasis. Our findings suggest that therapeutic agents targeting mtROS may prevent mitochondrial impairment and subsequent mitochondrial calcification/extrusion in skeletal muscle cells reducing calcinosis.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/mitochondrial-ros-as-a-regulator-of-calcinosis-in-juvenile-dermatomyositis/