Cooperative sensing of mitochondrial DNA by ZBP1 and cGAS promotes cardiotoxicity

Measuring mtDNA turnover, synthesis, and supercoiling via selective bromodeoxyuridine incorporation

“…B) Assay to measure turnover showing BrdU-labelled mtDNA 24h, 48h, 72h, and 96h following a 24h BrdU-pulse in U2OS cells. C) Assay to measure mtDNA supercoiling, adapted from Lei et al 3 , illustrates DNA isolated from wild-type and TFAM +/- MEFs, demonstrating changes in the relative amounts of linear, supercoiled, and relaxed mtDNA.…”

“…In addition, our method is robust enough to not only measure total levels of mtDNA for synthesis and turnover, but also separate linear, supercoiled and relaxed forms of mtDNA for subsequent quantification and analysis. As a result, our protocols allow for pinpointing the molecular mechanisms underlying changes in mtDNA copy number, as we have shown in models of mitochondrial dysfunction such as loss of TOP1MT 1,2 , or reduced TFAM expression 3 .…”

“…Here we describe a protocol that selectively incorporates bromodeoxyuridine into mtDNA for the measurement of mtDNA turnover, synthesis or supercoiling via an adapted immunoblot and Southern blot protocol. For complete use and execution of this protocol see Al Khatib et al 1,2 and Lei et al 3…”

B-Cell Immune Checkpoints Come of Age in Cardio-oncology

“…Pharmacological inhibition or genetic ablation of this pathway improved cardiac function in DIC models. 8,9 Inflammatory cytokines, likely derived from macrophages or monocytes, such as IFN-γ (interferon-gamma), TNF (tumor necrosis factor), and IL-1β (interleukin-1β) have also been shown to contribute to doxorubicin mediated apoptosis. 10 In this issue of Circulation Research, the authors explored the role of adaptive immunity, and specifically B-cells, in the cardioprotective effects conferred by exercise against DIC.…”

Generation of inducible mitophagy mice

“…Third, robust induction of mitophagy may also induce lysosomal exocytosis and released mtDNA stimulates innate immunity via TLR9 (Arnoult et al, 2011). Finally, intracellular mtDNA leakage and subsequent activation of inflammation via the DNA sensor, cGAS-STING pathway (Lei et al, 2023;White et al, 2014). When mitophagy occurs in large mitochondria, the autophagosome tears off a small piece from the mitochondrial body (Yamashita et al, 2016).…”