Circulating E3 ligases are novel and sensitive biomarkers for diagnosis of acute myocardial infarction

Abstract

Ubiquitin ligase (E3) is a decisive element of the ubiquitin-proteasome system (UPS), which is the main pathway for intracellular protein turnover. Recently, circulating E3 ligases have been increasingly considered as cancer biomarkers. In the present study, we aimed to determine if cardiac-specific E3 ligases in circulation can serve as novel predictors for early diagnosis of acute myocardial infarction (AMI). By screening and verifying their tissue expression patterns with microarray and real-time PCR analysis, six of 261 E3 ligases, including cardiac-specific Rnf207 and cardiac- and muscle-enriched Fbxo32/atrogin-1, Trim54/MuRF3, Trim63/MuRF1, Kbtbd10/KLHL41, Asb11 and Asb2 in mouse heart, were selected for the present study. In the AMI rats, the levels of five E3 ligases including Rnf207, Fbxo32, Trim54, Trim63 and Kbtbd10 in the plasma were significantly increased compared with control animals. Especially, the plasma levels of Rnf207 was markedly increased at 1 h, peaked at 3 h and decreased at 6-24 h after ligation. Further evaluation of E3 ligases in AMI patients confirmed that plasma Rnf207 level increased significantly compared with that in healthy people and patients without AMI, and showed a similar time course to that in AMI rats. Simultaneously, plasma level of cardiac troponin I (cTnI) was measured by ELISA assays. Finally, receiver operating characteristic (ROC) curve analysis indicated that Rnf207 showed a similar sensitivity and specificity to the classic biomarker troponin I for diagnosis of AMI. Increased cardiac-specific E3 ligase Rnf207 in plasma may be a novel and sensitive biomarkers for AMI in humans.

Figure 1. Detection of E3 ligases specifically expressed in the mouse heart

(A) Microarrays analysis of ubiquitin E3 ligases expression in heart, aorta, kidney and brains from mouse. (B) Real-time PCR analysis of ubiquitin E3 ligases expression in eight organs from mouse (n=9 per group). Sk.M, skeletal muscle.

Figure 2. Measurement of plasma E3 ligases in rats with acute myocardial infarction

(A) The plasma was collected from the rats at different times (0, 0.5, 1, 3, 6, 12 and 24 h) after coronary artery occlusion (n=8–10 per group), and the levels of circulating E3 ligases and cTnI were measured by using ELISA kit. (B) The plasma was collected from rats of non-operation (non-op), sham-operation (sham-op) and ligation groups at 3 h after coronary artery occlusion (n=5 per group). Data are expressed as mean±S.D. *, P<0.05.

Figure 3. Measurement of plasma E3 ligases in patients with acute myocardial infarction

(A) The plasma was collected from patients at various time points (0.5, 1, 3, 6, 12 and 24 h) and the levels of circulating E3 ligases and cTnI were measured by using ELISA kit (n=12–15 per group). (B) The plasma was collected from acute myocardial infarction (n=65), patients with coronary heart disease but without acute myocardial infarction (coronary heart disease, n=31), and healthy volunteers (healthy, n=28). Data are expressed as mean±S.D. *, P<0.05.

Figure 4. Evaluation of plasma E3 ligases for the diagnosis of acute myocardial infarction

(A) Receiver operating characteristic curves (ROC) were drawn of 65 AMI patients and 28 healthy volunteers with the data of plasma ubiquitin E3 ligases. AUC, the area under curve. (B) Sensitivity and specificity of plasma ubiquitin E3 ligases levels in the diagnosis of AMI. The dashed line indicates a 100% specificity threshold. Filled circle, AMI patients; inverted triangle, healthy volunteers.