A translational study of circulating cell-free microRNA-1 in acute myocardial infarction

Abstract

miRNAs (microRNAs) participate in many diseases including cardiovascular disease. In contrast with our original hypothesis, miRNAs exist in circulating blood and are relatively stable due to binding with other materials. The aim of the present translational study is to establish a method of determining the absolute amount of an miRNA in blood and to determine the potential applications of circulating cell-free miR-1 (microRNA-1) in AMI (acute myocardial infarction). The results revealed that miR-1 is the most abundant miRNA in the heart and is also a heart- and muscle-specific miRNA. In a cardiac cell necrosis model induced by Triton X-100 in vitro, we found that cardiac miR-1 can be released into the culture medium and is stable at least for 24 h. In a rat model of AMI induced by coronary ligation, we found that serum miR-1 is quickly increased after AMI with a peak at 6 h, in which an increase in miR-1 of over 200-fold was demonstrated. The miR-1 level returned to basal levels at 3 days after AMI. Moreover, the serum miR-1 level in rats with AMI had a strong positive correlation with myocardial infarct size. To verify further the relationship between myocardial size and miR-1 level, an IP (ischaemic preconditioning) model was used. The results showed that IP significantly reduced circulating miR-1 levels and myocardial infract size induced by I/R (ischaemia/reperfusion) injury. Finally, the levels of circulating cell-free miR-1 were significantly increased in patients with AMI and had a positive correlation with serum CK-MB (creatine kinase-MB) levels. In conclusion, the results suggest that serum miR-1 could be a novel sensitive diagnostic biomarker for AMI.

Figure 1. miR-1 is able to be released by necrotic cardiac myocytes in vitro

(A) Representative images from vehicle (PBS) or Triton X-100-treated cardiac cells. (B) The relative levels of miR-1 in culture medium of rat cardiac cells treated with vehicle (PBS) or different concentrations of Triton X-100 for 20 min determined by qRT-PCR. Note: The miR-1 level in vehicle-treated group is expressed as 1. n=6; *P<0.05 compared with PBS control. (C) The stability assay of miR-1 in cultured medium. The cardiac cells were treated 2% Triton X-100 for 20 min. Then, the cultured medium was collected and was kept at 37°C up to 24 h. The miR-1 levels were determined by qRT-PCR at 0, 6, 12 and 24 h after medium collection. Note: n=6.

Figure 2. Establish of the quantitative method for serum miR-1 assay

(A) Amplification chart of different concentrations of miR-1. (B) Standard curve of the miR-1 concentration in serial dilutions vs. cycle number (CT). Each dot represents the result of triplicate PCR amplification for each dilution.

Figure 3. Circulating cell-free miR-1 in rats with or without acute myocardial infarction (AMI)

(A) Myocardial infarction in rat heart was induced by left anterior descending coronary artery ligation and the infarct size was determined by pathological staining in heart slices. Note: Color blue is Evans blue staining. The region without Evans blue staining is myocardial ischemic area at risk (IAR). Color red is the triphenyltetrazolium chloride (TTC) staining. TTC unstained area within IAR was the infarcted area. Infarct size is expressed as a percentage of the IAR (% IAR). (B) The relative serum miR-1 levels in 8 sham-opened control rats, and in 8 rats before (0 h) and at different time points after AMI. Note: the mean value of serum miR-1 before AMI was expressed as 1. n=8, *P<0.05 compared with the group before AMI (0 h). (C) The absolute amount of serum miR-1 sham-opened control rats, and in rats before (0 h) and at different time points after AMI. Note: n=8, *P<0.05 compared with the group before AMI.

Figure 4. The relationship between serum miR-1 levels and myocardial infarct sizes in rats with AMI

AMI was induced by I/R in 12 rats, the infarct sizes and serum miR-1 levels were determined in rats at 3 h after reperfusion. Note: n=12; a strong positive correlation was demonstrated between the two variables (r=0.88; p<0.05).

Figure 5. Ischemic preconditioning (IP) reduces myocardial infarct size and serum miR-1 level

(A) Myocardial infarct size induced by ischemia-reperfusion injury (I/R) was reduced by IP. Note: n=6; *P<0.05 compared with I/R group. (B) Representative heart slice images from rats in sham-opened group, IP and I/R group, and I/R group. Note: Color blue is Evans blue staining. The region without Evans blue staining is myocardial ischemic area at risk (IAR). Red color was the triphenyltetrazolium chloride (TTC) staining. TTC unstained area within IAR is the infarcted area. (C) The increased serum miR-1 induced by I/R isinhibited by IP. Note: n=6; *P<0.05 compared with I/R group.

Figure 6. Serum miR-1 is increased in patients with AMI

(A)The serum miR-1 levels were determined from patients within 24h of AMI. The serum from age matched normal controls was used as the control group. Note: n=31 in AMI group and n=20 in control group; P< 0.05 compared with the control group. (B) The relationship between serum miR-1 levels and CK-MB levels in patients. Note: n=31; a positive correlation was demonstrated between the two variables (r=0.68; p<0.05).