# Mitochondrial calcification in juvenile dermatomyositis

> **NIH NIH R21** · UNIVERSITY OF WASHINGTON · 2020 · $245,328

## Abstract

Project Summary
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 contribu...

## Key facts

- **NIH application ID:** 10027222
- **Project number:** 1R21AR077565-01
- **Recipient organization:** UNIVERSITY OF WASHINGTON
- **Principal Investigator:** Jan Christian Lood
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $245,328
- **Award type:** 1
- **Project period:** 2020-09-01 → 2022-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10027222

## Citation

> US National Institutes of Health, RePORTER application 10027222, Mitochondrial calcification in juvenile dermatomyositis (1R21AR077565-01). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10027222. Licensed CC0.

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