Introduction:

Inflammation, oxidative stress and cell death by necrosis or apoptosis contribute to coronary artery disease (CAD) and myocardial infarction.   Recently, blood levels of circulating cell-free DNA have emerged as novel biomarkers in a variety of diseases. 

Circulating cell-free mitochondrial DNA (cf-mtDNA) can also be found in circulation and was suggested to possess both prognostic and biological relevance as activator of pro-inflammatory innate immune signalling. However, the significance of cf-mtDNA in coronary artery disease remains unresolved. 

Aim of this study is to analyse secretion mechanisms and prognostic relevance of cf-mtDNA in CAD. 

Methods:

Currently, we are analysing serum samples from our coronary artery disease biobank consisting of 789 patients undergoing coronary angiography. Patients were stratified in three groups (No CAD, chronic coronary syndrome: CCS and acute coronary syndrome: ACS). Total cell-free DNA was isolated by silica membrane technology. Afterwards, expression of the mitochondrial DNA genes ND1 and ND6 (NADH-ubiquinone oxidoreductase chain) and of the nuclear gene GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) were assessed by quantitative PCR. ND1 and ND6 levels as markers of mtDNA were normalized to GAPDH as housekeeping gene as marker for nuclear DNA.

Additionally, in vitro experiments using human coronary artery endothelial cells (HCAEC) were performed. HCAEC were treated with stressors of mitochondrial homeostasis (CDDO, MitoBloCK-6) and as a comparison with stressors of endoplasmic reticulum homeostasis (Tunicamycin, Thapsigargin) for 6 and 24 hours. Total cell-free DNA was isolated from the supernatant and processed accordingly.

Results:

With our isolation method, total cf-DNA was detectable in all patients in sufficient amounts for usage in quantitative PCR. Patients with invasively confirmed CAD had a higher cf-mtDNA / cf-nuclearDNA ratio than patients with no CAD. Interestingly, cf-mtDNA / cf-nuclearDNA was more pronounced in patients with CCS than in patients with ACS. Patient data is currently analysed for the primary endpoint of one-year mortality.

In vitro, incubation with stressors of endoplasmic reticulum homeostasis (tunicamycin, thapsigargin) did not shift the cf-mtDNA / cf-nuclearDNA ratio in the supernatant of stressed HCAEC. Accordingly, the mitochondrial stressors CDDO did not influence the cf-mtDNA / cf-nuclearDNA ratio. However, MitoBlock-6, an inhibitor of mitochondrial protein import and potent stressor of mitochondrial homeostasis, unexpectedly lowered the cf-mtDNA / cf-nuclearDNA ratio.

Conclusion:

Cell-free DNA of both mitochondrial and nuclear origin can be detected in patients with CAD. Patients with CCS have higher levels of cf-mtDNA than patients with no CAD or patients with ACS. Future studies are required to elucidate the mechanisms of mtDNA release in the context of ACS and ischemia. Additionally, the relevance of mitochondrial stressors and mtDNA / nuclear DNA release in the cardiovascular system must be further analysed to provide insights into the mechanisms and significance of mtDNA release.