Exploring circulating mitochondrial DNA signature across the cardiovascular spectrum

Background Mitochondrial dysfunction is a hallmark of cardiometabolic diseases, but current methods do not account for different fractions of circulating mitochondrial DNA (mtDNA). We investigated whether patients across the heart failure (HF) spectrum have a specific signature of the total circulating mtDNA profile. Methods We performed a complete clinical assessment, including blood tests, ultrasound (US) and cardiopulmonary exercise. US congestion was defined at rest as inferior vena cava of ≥21 mm, lung B-lines ≥4, or discontinuous renal venous flow. In fasting whole blood and plasma samples collected at rest, we simultaneously measured the copy number of mtDNA's cellular and cell-free components by real-time quantitative polymerase chain reaction. We calculated the ratio of cell mtDNA to cell-free mtDNA as an index of mitochondrial efficiency. Results We enrolled 120 consecutive patients: 50 with HF and preserved ejection fraction (HFpEF), 40 with HF and reduced ejection fraction (HFrEF) and 30 at risk of developing HF (Stages A-B). Cell-free mtDNA was increased in patients with HF (with higher levels in HFrEF than HFpEF) than in Stages A-B (Figure 1). Cell-free mtDNA was also higher in patients with systemic inflammation (expressed by high-sensitivity C-reactive protein [hs-CRP] ≥0.2 mg/dL with neutrophil-lymphocyte ratio [NLR] >3) and more US signs of congestion (Figure 2). The cell/cell-free mtDNA ratio showed opposite trends (all p<0.01), but there were no significant differences in cell mtDNA. Cell-free mtDNA had a mild correlation with cell mtDNA levels (r 0.269), NT-proBNP (r 0.314), hs-Troponin T (r 0.248) and aldosterone (r 0.222) while showing a moderate correlation with peak oxygen consumption (VO2, r -0.441) and ventilation/carbon dioxide production slope (VE/VCO2, r 0.455). The opposite relationships were observed when considering mtDNA ratio (NT-proBNP, r -0.242; hs-Troponin T, r -0.303; aldosterone, r -0.215; peak VO2, r 0.409; VE/VCO2, r -0.422), while cell mtDNA had only a mild correlation with hs-Troponin T (r -0.212). Cell-free mtDNA and mtDNA ratio independently predicted the presence of ≥2 US signs of congestion and peak VO2 <16 mL/kg/min at ROC analysis (Figure 1). Using multivariable regressions adjusted for age, sex, hs-CRP, NLR, high-sensitivity Troponin T, NT-proBNP and left ventricle ejection fraction, cell-free mtDNA and mtDNA ratio independently predicted the presence of ≥2 US signs of congestion (odds ratio [OR] 3.329, 95% confidence interval [CI] 1.017 – 10.902 and OR 0.302, 95% CI 0.101 to 0.903) and peak VO2 (rpartial -0.249, p=0.025 and rpartial 0.292, p=0.021) while cell mtDNA did not. Conclusions Patients across the HF spectrum have an altered circulating mtDNA signature. Cell-free mtDNA and mtDNA ratio are associated with impaired response to exercise, higher systemic inflammation and increased congestion, regardless of ejection fraction, whereas cellular mtDNA appears less relevant.