Circulating mitochondrial DNA signature in cardiometabolic patients

Abstract

Background: Mitochondrial dysfunction is a hallmark of cardiometabolic diseases. Circulating mitochondrial DNA (mtDNA) profiles could refine risk stratification, but current methods do not account for different fractions of circulating mtDNA. We investigated whether patients with type 2 diabetes and/or heart failure (HF) have a specific signature of the total circulating mtDNA profile, including intracellular and cell-free fractions.

Methods: We performed a complete clinical assessment, including blood tests, 12-lead ECG and ultrasound at rest and during cardiopulmonary exercise. Ultrasound 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 the cellular and cell-free components of mtDNA by real-time quantitative polymerase chain reaction (qPCR) using custom standards. We calculated the ratio of cell mtDNA to cell-free mtDNA as an index of mitochondrial efficiency.

Results: We enrolled 120 consecutive patients: 50 (42%) with HF and preserved ejection fraction (HFpEF), 40 (33%) with HF and reduced ejection fraction (HFrEF) and 30 (25%) at risk of developing HF; 42/120 (35%) had diabetes. Cell-free mtDNA was increased in patients with HF (with higher levels in HFrEF than HFpEF) and those with diabetes. 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 ultrasound signs of congestion. The cell/cell-free mtDNA ratio showed opposite trends (all p < 0.05), but there were no significant differences in cell mtDNA. Cell-free mtDNA and mtDNA ratio independently predicted the presence of ≥ 2 ultrasound signs of congestion and effort intolerance (peak oxygen consumption < 16 mL/kg/min) at ROC analysis and using multivariable regressions after adjustment for age, sex, hs-CRP, NLR, high-sensitivity Troponin T and NT-proBNP.

Conclusions: Patients with HF and diabetes have an altered circulating mtDNA signature characterised by higher cell-free mtDNA and lower mtDNA ratio, whereas cellular mtDNA remains unaffected. Cell-free mtDNA and mtDNA ratio are associated with impaired response to exercise, higher systemic inflammation and increased congestion. Circulating mitochondrial profile could be a new biomarker of mitochondrial status in cardiometabolic diseases.

Keywords: Circulating mitochondrial DNA; Congestion; Effort intolerance; Heart failure; Type 2 diabetes.

Fig. 1

Distribution of the cell-free mtDNA and mtDNA ratio presented as violin plots showing median and interquartile range in patients with and without diabetes (A, B), with and without inflammation (C, D) and according to the absence or presence of increasing ultrasound (US) signs of congestion (E, F). Inflammation was defined as the co-presence of C-Reactive Protein ≥ 0.2 mg/dL and Neutrophil-Lymphocyte Ratio > 3

Fig. 2

Univariate correlation matrix using Spearman’s rank correlation. *P < 0.01, **P < 0.001, ***P < 0.0001. Red shading indicates positive correlations, and blue shading indicates inverse correlations. White boxes are non-significant (P > 0.05). eGFR: estimated glomerular filtration rate; VE: ventilation; VOC2: carbon dioxide production; VO₂: oxygen consumption

Fig. 3

Receiver-operator characteristic (ROC) curves illustrating the accuracy of cell mtDNA, cell-free mtDNA and mtDNA ratio in predicting ≥ 2 ultrasound (US) signs of congestion and peak oxygen consumption (VO₂) < 16 mL/kg/min. AUC: area under the curve