Circulating microRNAs are new and sensitive biomarkers of myocardial infarction

Abstract

Aims: Circulating microRNAs (miRNAs) may represent a novel class of biomarkers; therefore, we examined whether acute myocardial infarction (MI) modulates miRNAs plasma levels in humans and mice.

Methods and results: Healthy donors (n = 17) and patients (n = 33) with acute ST-segment elevation MI (STEMI) were evaluated. In one cohort (n = 25), the first plasma sample was obtained 517 ± 309 min after the onset of MI symptoms and after coronary reperfusion with percutaneous coronary intervention (PCI); miR-1, -133a, -133b, and -499-5p were ~15- to 140-fold control, whereas miR-122 and -375 were ~87-90% lower than control; 5 days later, miR-1, -133a, -133b, -499-5p, and -375 were back to baseline, whereas miR-122 remained lower than control through Day 30. In additional patients (n = 8; four treated with thrombolysis and four with PCI), miRNAs and troponin I (TnI) were quantified simultaneously starting 156 ± 72 min after the onset of symptoms and at different times thereafter. Peak miR-1, -133a, and -133b expression and TnI level occurred at a similar time, whereas miR-499-5p exhibited a slower time course. In mice, miRNAs plasma levels and TnI were measured 15 min after coronary ligation and at different times thereafter. The behaviour of miR-1, -133a, -133b, and -499-5p was similar to STEMI patients; further, reciprocal changes in the expression levels of these miRNAs were found in cardiac tissue 3-6 h after coronary ligation. In contrast, miR-122 and -375 exhibited minor changes and no significant modulation. In mice with acute hind-limb ischaemia, there was no increase in the plasma level of the above miRNAs.

Conclusion: Acute MI up-regulated miR-1, -133a, -133b, and -499-5p plasma levels, both in humans and mice, whereas miR-122 and -375 were lower than control only in STEMI patients. These miRNAs represent novel biomarkers of cardiac damage.

Figure 1

MicroRNAs plasma levels in patients with ST-segment elevation myocardial infarction (Group 1). (A–D) miR-1, -133a, -133b, and -499-5p exhibited a 15- to 140-fold increase in plasma samples collected 517 ± 309 min after the onset of myocardial infarction symptoms, i.e. Day 0. At Day 5 after myocardial infarction, these microRNAs were back to levels comparable to those in healthy subjects. (E and F) At Day 0, miR-122 (E) and miR-375(F) were lower than in control subjects and miR-122 level remained below control both at Day 5 and at Day 30. (Control healthy subjects = 17; ST-segment elevation myocardial infarction patients = 25 at Day 0; 7 at Day 5 and 7 at Day 30. Values indicate fold changes of each microRNA vs. its level in control healthy subjects, arbitrarily set at 1 as indicated by the red bar; yellow bars indicate microRNA values after myocardial infarction; results are reported as mean ± SEM; *P < 0.01; ^§P < 0.05 vs. control; NS, not significant.)

Figure 2

Time course of microRNAs plasma levels and TnI in patients with ST-segment elevation myocardial infarction (Group 2). In these patients, the first plasma sample was obtained 156 ± 72 min after the onset of symptoms (T0); other samples were obtained at different times thereafter, as indicated. miR-1, -133a, and -133b achieved their peak before troponin I, whereas miR-499-5p exhibited a slower time course. The data have been normalized to the peak level that each microRNA and troponin I achieved in each patient and the time of the peak-fold increase vs. healthy control was not identical among patients. On the average, miR-1 achieved a 48.3 ± 17.4-fold peak at T0; miR-133a achieved a 5426 ± 3047-fold peak at T0; miR-133b achieved a 312.2 ± 182.6-fold peak at T0; miR 499-5p achieved a 299.1 ± 106.4-fold peak at 9 h. miR-122 and -375 were never above control; their lowest level was 0.26 ± 0.12 and 0.53 ± 0.10 control and occurred at the 45 and 33 h time points, respectively. Troponin I peak increase of 1066 ± 200-fold was achieved at the 3 h time point. (Control healthy subjects = 17; ST-segment elevation myocardial infarction patients = 5–8 at each time point; results for each microRNA and troponin I are reported as mean ± SEM; *P < 0.01; ^§P < 0.05 vs. control.)

Figure 3

Time course of microRNAs plasma levels and troponin I in mice with acute myocardial infarction. miR-1, -133a, and -133b achieved their peak either at the 6 or the 18 h time point. In contrast, miR-499-5p achieved its peak 24 h after coronary occlusion. Changes in miR-122 and -375 were minor. It is noteworthy that the magnitude of the increase was highest for miR-499-5p and that this microRNA, 15 min to 6 h after coronary occlusion, closely paralleled the increase in troponin I which was monitored in a separate group of animals. Fold changes were calculated against the mean value of the sham at each time point (for microRNAs, at each time point, n = 4–5 both for myocardial infarction and sham-operated mice; for troponin I, n = 5 at each time point from 15 to 180 min and n = 2 at each time point from 6 to 24 h; results for each microRNA and troponin I are reported as mean ± SEM; NS, not significant; *P < 0.01, ^§P < 0.05 vs. sham-operated control mice.) Red bars indicate sham controls at each time point, arbitrarily set at 1; yellow bars indicate microRNA values at each time point after myocardial infarction; and blue bars indicate troponin I values after myocardial infarction.

Figure 4

MicroRNAs cardiac levels in mice with acute myocardial infarction. The cardiac expression level of miR-1, -133a, -133b, -499-5p, -122, and -375 was evaluated in the border zone and infarct area of mice, 3 and 6 hr following coronary artery ligation, as well as in the left ventricle of sham-operated mice at the same time points. miR-499-5p exhibited a decrease at both time points, both in the infarct and border zones. miR-1, -133a, and -133b decreased, either in the border area, the infarct, or both, and this response was apparent either 3 or 6 h after coronary occlusion. Interestingly, miR-122 exhibited an increase in the infarct both 3 and 6 h after coronary occlusion. miR-375 exhibited a minor increase in the infarct area which was apparent only at the 3 h time point (at each time point, n = 4–5 for myocardial infarction and n = 4–5 for sham operated; results for each microRNA are reported as mean ± SEM; NS, not significant; *P < 0.01, ^§P < 0.05 vs. sham-operated control mice.) Control values were arbitrarily set at 1 as indicated by the red bar.

Figure 5

MicroRNAs plasma levels in mice with acute hind-limb ischaemia. miR-1, -133a, -133b, -499-5p, -122, and -375 plasma levels were evaluated in mice with acute hind-limb ischaemia, 6 and 24 h after femoral artery dissection. At 6 h, miR-1, -133a, and -133b exhibited a marked decrease, whereas miR-375 increased; all microRNAs were back to control value at 24 h. In contrast, miR-499-5p and miR-122 exhibited no significant change. (n = 5 for hind-limb ischaemia and n = 5 for sham operated; values indicate fold changes of each microRNA vs. sham-operated mice; results for each microRNA are reported as mean ± SEM; NS, not significant; ^§P < 0.05). Control values were arbitrarily set to 1 as indicated by the red bar.