Decreased Peripheral Mitochondrial DNA Copy Number is Associated with the Risk of Heart Failure and Long-term Outcomes

Abstract

Mitochondrial DNA (mtDNA) copy number variation (CNV), which reflects the oxidant-induced cell damage, has been observed in a wide range of human diseases. However, whether it correlates with heart failure, which is closely related to oxidative stress, has never been elucidated before. We aimed to systematically investigate the associations between leukocyte mtDNA CNV and heart failure risk and prognosis. A total of 1700 hospitalized patients with heart failure and 1700 age- and sex-matched community population were consecutively enrolled in this observational study, as well as 1638 (96.4%) patients were followed prospectively for a median of 17 months (12-24 months). The relative mtDNA copy number of leukocyte of peripheral blood or cardiac tissue was measured in triplicate by quantitative real-time PCR method. Patients with heart failure possessed much lower relative mtDNA copy number compared with control subjects (median 0.83, interquartile range [IQR] 0.60-1.16 vs median 1.00, IQR 0.47-2.20; P < 0.001), especially for the patients with ischemic etiology (median, 0.77 for ischemic and 0.91 for non-ischemic, P < 0.001). Patients with lower mtDNA copy number exhibited 1.7 times higher risk of heart failure (odds ratio 1.71, 95% confidence interval [CI] 1.48-1.97, P < 0.001). Long-term follow-up (median of 17 months) showed that decreased mtDNA copy number was significant associated with both increased cardiovascular deaths (hazard ratio [HR] 1.58, 95% CI 1.16-2.16, P = 0.004) and cardiovascular rehospitalization (HR 1.48, 95% CI 1.21-1.82, P < 0.001). After adjusting for the conventional risk factors and medications, lower mtDNA copy numbers were still significantly associated with 50% higher cardiovascular mortality (P = 0.035). In conclusion, mtDNA copy number depletion is an independent risk factor for heart failure and predicts higher cardiovascular mortality in patients with heart failure.

FIGURE 1

Relative mtDNA copy number as a function of age in control subjects and heart failure patients. Relative mtDNA copy number, expressed as the natural log (Ln) of the mtDNA copy number to the nuclear one copy gene ratio, is plotted as a function of age. The open circles represent 1700 controls (A) or 1700 patients (B), and linear regression lines are drawn to elucidate the correlations. Correlation coefficients (r) between Ln (relative mtDNA copy number) and age were 0.10 (P < 0.001) for controls (A) and −0.04 (P = 0.067) for heart failure patients (B). Yearly increase of relative mtDNA copy number was 0.012 in controls and 0.002 in heart patients.

FIGURE 2

The mtDNA copy number distributions in control subjects and heart failure patients. Y axis represents the relative mtDNA copy number. (A) The mtDNA copy number distributions of controls, patients and subgroups additionally stratified by etiologies. (B) The mtDNA copy number distributions stratified by New York Heart Association functional class. Bold black horizontal lines represent the median of relative mtDNA copy number with the top and button lines of the boxes representing the interquartile ranges.

FIGURE 3

Clinical outcomes in the follow-up heart failure population and prespecified subgroups. (A) Kaplan-Meier curves for the primary end point (deaths from cardiovascular causes) and second endpoint (cardiovascular rehospitalization). Red lines represent the patients whose mtDNA copy number levels are below the median level of control population and black lines represent those patients whose copy number are above the median. X axis represents the follow-up time and the Y axis represents cumulative risk. (B) Subgroup analyses of the primary and secondary endpoint according to various baseline characteristics. The position of blue square represents the hazard ratio and the size of the square corresponds to the number of patients in each subgroup. Black lines represent the 95% confidence interval.

FIGURE 4

The correlation between peripheral blood cells and cardiomyocytes. (A) The correlation of mtDNA contents between peripheral leukocytes and heart tissues from end-stage heart failure patients in a subgroup of patients (n = 10) quantified by real-time quantitative PCR analysis using the nuclear gene HBB as an endogenous control. (B) Representative fluorescence imagines of reactive oxygen species (ROS) productions (red fluorescent) in cardiomyocytes incubated with dihydroethidium (DHE) (left panel, scale bars: 50 mm). The average levels of fluorescence intensity of DHE form control group (n = 3) and patients (n = 5) were summarized in right panel. (C) ROS generation in peripheral lymphocytes from heart failure patients or control subjects (left panel). In the right panel, Y axis represents average fluorescence intensity of DCFH-DA counted per 10,000 cells by flow cytometry (n = 5 for each group).