Mitochondria are intracellular organelles involved in metabolism, inflammation and cell death. Mitochondria also play an important role in regulating intracellular calcium levels, scavenging cytosolic calcium and inorganic phosphate, trapping it in its crystalline form (hydroxyapatite) within the mitochondria to rescue the cell from cytosolic Ca2+ overload. Intramitochondrial calcification has been observed in case reports of adult dermatomyositis (DM) as well as in experimental models of cutaneous calcinosis, a debilitating manifestation of juvenile DM (JDM). However, the underlying mechanisms and consequences of intramitochondrial calcification have not been investigated. Though mainly found intracellularly, mitochondria can be extruded upon ROS-mediated damage, partaking in induction of inflammation. The premise of this application is that JDM patients have mitochondrial calcification promoting mitochondrial extrusion, calcium crystal accumulation (calcinosis), and inflammation. To investigate this hypothesis we have two specific aims.
The first aim will look into mechanisms contributing to mitochondrial calcification and extrusion in vitro. We hypothesize that mitochondrial ROS generation will support mitochondrial calcification and extrusion, contributing to inflammation and calcinosis. Experimentally, primary human skeletal muscle cells will be incubated in calcium rich medium in presence of stress agents, including hypoxia, TLR agonists, or JDM sera, and mitochondrial function assessed using flow cytometry, qPCR, immunofluorescence microscopy and metabolomics. Mass spectrometry-based phosphoproteomics will be used to establish which pathways are involved in mitochondrial calcification. Potential targets, including mitochondrial ROS, will be blocked by using small molecules, e.g. mitoTEMPO.
The second aim will investigate mechanisms by which extruded mitochondria are cleared. We hypothesize that calcified mitochondria are phagocytosed, but not degraded, remaining in the cytosolic compartment triggering TLR9 and inflammasome activation. In brief, mitochondria will be incubated with primary human monocytes and neutrophils and assessed for uptake, intracellular localization, signaling pathways (phosphoproteomics) and cytokine induction. Key pathways involved in induction of mitochondrial- mediated inflammation, e.g. DNA sensors TLR9 and cGAS, as well as inflammasome activation, will be targeted using chemical inhibitors. The capacity of cells from JDM children and healthy individuals to support clearance of mitochondria will be compared in vitro. Finally, JDM immune cells will be analyzed for presence of hydroxyapatite-containing mitochondria in vivo using flow cytometry and confocal microscopy. We believe that our proposal is highly innovative and significant as i) we will define underlying mechanisms involved in mitochondrial calcification; and ii) we will for the first time explore if, and how, extruded mitochondria contribute to inflammation and calcinosis in JDM. These findings may also be applicable to other conditions in which the calcification process is prominent, including atherosclerosis.
Dying cells release several toxic components, including their bacterial-resembling power plants, mitochondria, a process known to promote inflammation and autoimmunity. However, we still do not fully understand how the released mitochondria contribute to inflammation and disease. In the current proposal, we aim to study how mitochondria are calcified and removed by our immune cells to avoid unwarranted inflammation and autoimmunity, with the ambition to identify novel pathways that could be targeted in mitochondrial-mediated diseases, including juvenile dermatomyositis.
