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Abstract

Aims

MicroRNAs (miRNAs) have been implicated in cardiac diseases. This study aimed to characterize the circulating miRNAs in patients with arrhythmogenic right ventricular cardiomyopathy (ARVC) and correlate the miRNAs with the clinical outcomes of ARVC.

Methods and results

This study included 62 patients with ventricular arrhythmia (VA): 28 patients (45%) had definite ARVC, 11 (18%) had borderline or possible ARVC, and 23 (37%) had idiopathic ventricular tachycardia (VT). In addition, 33 age- and sex-matched healthy subjects were enrolled as normal control subjects. The expression of selected miRNAs was analysed in all study subjects. The clinical outcomes of patients with definite ARVC after catheter ablation were further investigated. On the basis of the miRNA polymerase chain reaction array, we selected 11 miRNAs for analysis of their expression in the plasma of all subjects. Definite ARVC patients had significantly higher expression of circulating miR-144-3p, 145-5p, 185-5p, and 494 than the three other groups. Out of 25 definite ARVC patients who underwent radiofrequency catheter ablation, recurrent VA occurred in 8 patients (32%) during the follow-up period (45 ± 20 months). Definite ARVC patients with recurrent VA had a higher level of circulating miR-494 than did those without recurrence. Receiver operating characteristic analysis showed miR-494 to be a predictive factor of recurrent VA (area under the curve: 0.832).

Conclusion

Plasma levels of miR-144-3p, 145-5p, 185-5p, and 494 were significantly elevated in definite ARVC patients with VA. An increased plasma level of miR-494 was associated with the recurrence of VA after ablation in definite ARVC patients.

Arrhythmogenic right ventricular cardiomyopathy, MicroRNAs, Ventricular arrhythmia, miR-494, Recurrence

What’s new?

  • We have demonstrated for the first time that serum levels of miR-144-3p, 145-5p, 185-5p, and 494 are significantly elevated in definite arrhythmogenic right ventricular cardiomyopathy (ARVC) patients with ventricular arrhythmia (VA).

  • Increased serum level of miR-494 was associated with recurrence of VA after catheter ablation in patients with definite ARVC. In H9c2 cells, we demonstrated that miR-494 was associated with apoptosis process. These results suggest that the overexpression of miR-494 may play an important key role in the myocardial apoptosis in ARVC.

Introduction

Arrhythmogenic right ventricular cardiomyopathy (ARVC), an inherited cardiomyopathy, is a leading cause of sudden cardiac death (SCD) and ventricular arrhythmia (VA).1,2 Arrhythmogenic right ventricular cardiomyopathy is characterized by fibro-fatty infiltration of the right ventricular (RV) myocardium according to histopathologic findings, which predisposes to VA and SCD.1,3 The clinical manifestations of ARVC vary widely, ranging from concealed presentation [as idiopathic ventricular tachycardia (VT)], to overt arrhythmias, to profound cardiac dysfunction. These variations cause difficulties in the differential diagnosis, especially for patients with ARVC in an early stage.3 The genetic penetrance is often low, making the diagnosis of ARVC more challenging.4,5 Modified Task Force Criteria have been proposed for the diagnosis of ARVC, but these criteria are not highly sensitive.6

MicroRNAs (miRNAs) are small, non-coding RNAs approximately 22 nucleotides in length that regulate the expression of target genes through sequence-specific hybridization to the 3′ untranslated region of messenger RNAs, resulting in either blocking of translation or direct degradation of their target messenger RNAs.7 miRNAs may play important roles in the genesis of cardiovascular diseases. Recently, Zhang et al.8 reported that miRNAs might be correlated with both the myocardium adipose and fibrosis in the end-stage ARVC patients who underwent heart transplantation. However, the role of miRNAs in the early phases of ARVC patients remains unclear. VAs are often a first manifestation of ARVC, and differentiation between early-phase ARVC and idiopathic VA can be challenging.1 The aim of this study was to characterize the circulating miRNAs in ARVC patients with VA and correlate them with the clinical outcomes of ARVC.

Methods

Study population

This study enrolled 62 patients with VA from 2012 to 2013. Twenty-eight patients (45%) had definite ARVC, 11 (18%) patients had borderline (n = 8) or possible (n = 3) ARVC, and 23 (37%) patients had idiopathic VT. All patients underwent electrophysiological study, and 58 (94%) patients underwent a radiofrequency ablation procedure for VA. One patient with borderline ARVC and three patients with definite ARVC did not have the radiofrequency ablation procedure. There were 33 age- and sex-matched healthy subjects (14 men, mean age 42 ± 7 years) enrolled as normal control subjects, who had no hypertension, diabetes, heart failure, coronary heart disease, arrhythmia, and history of hospitalization for cardiovascular or other chronic diseases. There were no significant differences in age (P = 0.07) and sex (P = 0.37) among the four groups. Ethics approval was granted by the Institutional Review Board of the Veterans General Hospital, Taipei, Taiwan. All subjects gave written informed consent.

The diagnosis of ARVC was based on the revised Task Force Criteria of 2010.6 Idiopathic VT was defined as VT that was not associated with structural heart disease or coronary artery disease. The arrhythmogenic focus was localized by determining the successful site of radiofrequency ablation (RV outflow tract, n = 18; aortic cusp, n = 4; and RV, n = 1). To investigate the expression of circulating miRNAs in our study group, we collected blood samples before electrophysiological study and determined miRNA plasma levels in each subject (see Supplementary Material and Supplementary material online, Figure S1). Baseline demographic, structural, electrocardiographic, and invasive electrophysiological characteristics and the results of the ablation procedures were collected for patients with VA at the time of enrolment. All patients had a resting surface electrocardiogram, a signal-averaged electrocardiogram (SAECG), echocardiography, and 24-h Holter investigations. Right ventricular function was assessed by cardiac magnetic resonance imaging (MRI) or right ventriculography. Left ventricle (LV) systolic function was assessed by echocardiography (see Supplementary Material). In definite ARVC patients, eight patients had an implantable cardioverter defibrillator (ICD) before enrolment, and two patients received an ICD after enrolment in the study.

Electrophysiological study, mapping, radiofrequency catheter ablation, and clinical follow-up

The electrophysiological study, mapping, and radiofrequency catheter ablation were performed, and patients were followed up as described previously.9 For detailed information, see Supplementary Material.

Determination of microRNAs in plasma of study population

RNA isolation

Total RNA, including miRNAs, was isolated using the miRNeasy Serum/Plasma Kit (Qiagen) according to the manufacturer’s protocol. Five volumes of QIAzol lysis reagent were added to the plasma sample, the sample was incubated for 5 min at room temperature, and 3.5 μl of miRNeasy/Plasma Spike-In Control was added to the sample and mixed thoroughly. An equal volume of chloroform was added to the starting sample, the sample was incubated for 3 min at room temperature, and the sample was centrifuged for 15 min at 12 000× g at 4 °C. The aqueous phase containing the RNA was carefully removed, and RNA was precipitated by addition of 100% ethanol. The mixture was added to an RNeasy MinElute spin column and washed several times, and RNA was eluted by the addition of 14 μl of RNase-free water. To date, no housekeeping miRNA has been established and validated to normalize for the miRNA content. Therefore, we chose to use a fixed volume of plasma per sample and a synthetic Caenorhabditis elegans miR-39 (cel-miR-39, 20 fmol/sample, synthesized by Qiagen) as a spiked-in control to normalize for individual RNA-isolation-related variations (see Supplementary Material).

MicroRNA polymerase chain reaction array

Six healthy volunteers were compared with six patients with definite ARVC admitted to the hospital for VT. The miScript miRNA polymerase chain reaction (PCR) Array kit (Qiagen) for 84 miRNAs that known to exhibit altered expression during cardiovascular disease, and development was used to study the circulating miRNAs expression in the patients. To validate the results of the miRNA PCR array, we finally selected the miRNAs whose blood levels in the patients with definite ARVC were four times higher or four times lower than those in normal subjects for further analysis by real-time PCR.

Validation by real-time polymerase chain reaction

Thirty-three healthy control and 62 patients with VA were studied. All samples were measured in duplicates. For detailed information, see Supplementary Material.

Apoptotic effect of miR-494

Our present study demonstrated that miR-494 was correlated with the recurrence of VA after catheter ablation (see Results). Poor ventricular substrate and fibrosis have been reported to be associated with the recurrence of VA after catheter ablation. Therefore, we investigated the impact of miR-494 on the apoptosis in ARVC. The protein expression of cleaved caspase 3 and caspase 3 was investigated in H9c2 cells transfected by miR-494 lentivirus. For detailed information, see Supplementary Material.

Statistical analysis

Continuous data are reported as mean values ± standard deviation (SD). Data for miRNAs are reported as mean values ± standard error (SE). Categorical data are reported as absolute values and percentages. Numerical variables were compared by the Mann–Whitney U test. Data variables among the groups were compared by the Kruskal–Wallis test, and if P-value was <0.05, follow-up comparisons of the different groups were performed by the Dunn’s test. Categorical data were compared by the χ2 test or the Fisher’s exact test. Receiver operating characteristic (ROC) analysis was used to assess diagnostic accuracy of miRNAs (ARVC 2−ΔCT/Average normal 2−ΔCT). P-value < 0.05 was considered to indicate statistical significance. The analysis was performed by a senior biostatistician using SPSS statistical software (Version 22.0, SPSS Institute, Chicago, IL, USA).

Results

Clinical characteristics of study subjects

There were no statistically significant differences between the three groups in age, sex, or presence of hypertension, diabetes, coronary heart disease, or heart failure (Table 1). In echocardiographic findings, LV end-systolic diameter was significantly higher in patients with definite and borderline or possible ARVC than in those with idiopathic VT. Left ventricular fractional shortening was significantly lower in patients with definite and borderline or possible ARVC than in those with idiopathic VT. Right ventricular ejection fraction (RVEF) measured by angiography was significantly lower in patients with definite ARVC than in those with idiopathic VT. According to SAECG, filtered QRS duration in patients with definite and borderline or possible ARVC and duration of terminal QRS in patients with definite ARVC were significantly longer than in patients with idiopathic VT. There were no significant differences in baseline characteristics between patients with definite and borderline or possible ARVC.

Table 1
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Patient characteristics

Definite ARVC (n = 28)Borderline or possible ARVC (n = 11)Idiopathic VT (n = 23)P-value
Age (years)48 ± 1348 ± 1940 ± 100.099
Male17 (60%)5 (45%)8 (34%)0.331
Hypertension10 (35%)4 (36%)5 (21%)0.546
Diabetes1 (3%)2 (18%)1 (3%)0.229
Coronary heart disease2 (7%)2 (18%)0 (0%)0.130
Heart failure2 (7%)2 (18%)1 (4%)0.394
Echocardiography
 Left atrial diameter (mm)36.0 ± 4.736.6 ± 9.135.2 ± 4.20.826
 LVDd (mm)51.5 ± 5.952.8 ± 4.749.0 ± 5.30.278
 LVDs (mm)35.1 ± 5.8**35.5 ± 7.5*27.7 ± 4.40.001
 Left ventricular EF (%)54.0 ± 9.852.5 ± 14.557.8 ± 6.70.386
 Left ventricular FS (%)31.3 ± 7.4**32.9 ± 12.2*43.3 ± 7.30.001
Angiography
 Right ventricular EF (%)40.4 ± 13.0**43.8 ± 15.753.7 ± 10.60.006
MRI
 Right ventricular EDV/BSA (mL/m2)73.6 ± 12.774.1 ± 27.370.9 ± 21.20.886
SAECG
 Filtered QRS duration (ms)122.9 ± 26.9*123.8 ± 26.1*106.6 ± 7.10.033
 Duration of terminal QRS (µV)48.9 ± 25.4*45.4 ± 21.934.2 ± 6.90.033
 Root-mean-square of terminal 40 ms (µV)21.7 ± 12.623.8 ± 11.833.5 ± 19.10.075
Genetic mutation screening
 Desmoglein-21 (3%)1 (9%)N/A0.490
 Desmoplakin6 (21%)1 (9%)N/A0.649
 Plakophilin-23 (10%)1 (9%)N/A1.000
Definite ARVC (n = 28)Borderline or possible ARVC (n = 11)Idiopathic VT (n = 23)P-value
Age (years)48 ± 1348 ± 1940 ± 100.099
Male17 (60%)5 (45%)8 (34%)0.331
Hypertension10 (35%)4 (36%)5 (21%)0.546
Diabetes1 (3%)2 (18%)1 (3%)0.229
Coronary heart disease2 (7%)2 (18%)0 (0%)0.130
Heart failure2 (7%)2 (18%)1 (4%)0.394
Echocardiography
 Left atrial diameter (mm)36.0 ± 4.736.6 ± 9.135.2 ± 4.20.826
 LVDd (mm)51.5 ± 5.952.8 ± 4.749.0 ± 5.30.278
 LVDs (mm)35.1 ± 5.8**35.5 ± 7.5*27.7 ± 4.40.001
 Left ventricular EF (%)54.0 ± 9.852.5 ± 14.557.8 ± 6.70.386
 Left ventricular FS (%)31.3 ± 7.4**32.9 ± 12.2*43.3 ± 7.30.001
Angiography
 Right ventricular EF (%)40.4 ± 13.0**43.8 ± 15.753.7 ± 10.60.006
MRI
 Right ventricular EDV/BSA (mL/m2)73.6 ± 12.774.1 ± 27.370.9 ± 21.20.886
SAECG
 Filtered QRS duration (ms)122.9 ± 26.9*123.8 ± 26.1*106.6 ± 7.10.033
 Duration of terminal QRS (µV)48.9 ± 25.4*45.4 ± 21.934.2 ± 6.90.033
 Root-mean-square of terminal 40 ms (µV)21.7 ± 12.623.8 ± 11.833.5 ± 19.10.075
Genetic mutation screening
 Desmoglein-21 (3%)1 (9%)N/A0.490
 Desmoplakin6 (21%)1 (9%)N/A0.649
 Plakophilin-23 (10%)1 (9%)N/A1.000

ARVC, arrhythmogenic right ventricular cardiomyopathy; BSA, body surface area; EDV, end-diastolic volume; EF, ejection fraction; FS, fractional shortening; LVDd, left ventricular end-diastolic diameter; LVDs, left ventricular end-systolic diameter; mm, millimetre; MRI, magnetic resonance imaging; SAECG, signal-averaged electrocardiogram; VT, ventricular tachycardia.

*

P < 0.05 vs. idiopathic VT.

**

P < 0.01 vs. idiopathic VT.

Table 1
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Patient characteristics

Definite ARVC (n = 28)Borderline or possible ARVC (n = 11)Idiopathic VT (n = 23)P-value
Age (years)48 ± 1348 ± 1940 ± 100.099
Male17 (60%)5 (45%)8 (34%)0.331
Hypertension10 (35%)4 (36%)5 (21%)0.546
Diabetes1 (3%)2 (18%)1 (3%)0.229
Coronary heart disease2 (7%)2 (18%)0 (0%)0.130
Heart failure2 (7%)2 (18%)1 (4%)0.394
Echocardiography
 Left atrial diameter (mm)36.0 ± 4.736.6 ± 9.135.2 ± 4.20.826
 LVDd (mm)51.5 ± 5.952.8 ± 4.749.0 ± 5.30.278
 LVDs (mm)35.1 ± 5.8**35.5 ± 7.5*27.7 ± 4.40.001
 Left ventricular EF (%)54.0 ± 9.852.5 ± 14.557.8 ± 6.70.386
 Left ventricular FS (%)31.3 ± 7.4**32.9 ± 12.2*43.3 ± 7.30.001
Angiography
 Right ventricular EF (%)40.4 ± 13.0**43.8 ± 15.753.7 ± 10.60.006
MRI
 Right ventricular EDV/BSA (mL/m2)73.6 ± 12.774.1 ± 27.370.9 ± 21.20.886
SAECG
 Filtered QRS duration (ms)122.9 ± 26.9*123.8 ± 26.1*106.6 ± 7.10.033
 Duration of terminal QRS (µV)48.9 ± 25.4*45.4 ± 21.934.2 ± 6.90.033
 Root-mean-square of terminal 40 ms (µV)21.7 ± 12.623.8 ± 11.833.5 ± 19.10.075
Genetic mutation screening
 Desmoglein-21 (3%)1 (9%)N/A0.490
 Desmoplakin6 (21%)1 (9%)N/A0.649
 Plakophilin-23 (10%)1 (9%)N/A1.000
Definite ARVC (n = 28)Borderline or possible ARVC (n = 11)Idiopathic VT (n = 23)P-value
Age (years)48 ± 1348 ± 1940 ± 100.099
Male17 (60%)5 (45%)8 (34%)0.331
Hypertension10 (35%)4 (36%)5 (21%)0.546
Diabetes1 (3%)2 (18%)1 (3%)0.229
Coronary heart disease2 (7%)2 (18%)0 (0%)0.130
Heart failure2 (7%)2 (18%)1 (4%)0.394
Echocardiography
 Left atrial diameter (mm)36.0 ± 4.736.6 ± 9.135.2 ± 4.20.826
 LVDd (mm)51.5 ± 5.952.8 ± 4.749.0 ± 5.30.278
 LVDs (mm)35.1 ± 5.8**35.5 ± 7.5*27.7 ± 4.40.001
 Left ventricular EF (%)54.0 ± 9.852.5 ± 14.557.8 ± 6.70.386
 Left ventricular FS (%)31.3 ± 7.4**32.9 ± 12.2*43.3 ± 7.30.001
Angiography
 Right ventricular EF (%)40.4 ± 13.0**43.8 ± 15.753.7 ± 10.60.006
MRI
 Right ventricular EDV/BSA (mL/m2)73.6 ± 12.774.1 ± 27.370.9 ± 21.20.886
SAECG
 Filtered QRS duration (ms)122.9 ± 26.9*123.8 ± 26.1*106.6 ± 7.10.033
 Duration of terminal QRS (µV)48.9 ± 25.4*45.4 ± 21.934.2 ± 6.90.033
 Root-mean-square of terminal 40 ms (µV)21.7 ± 12.623.8 ± 11.833.5 ± 19.10.075
Genetic mutation screening
 Desmoglein-21 (3%)1 (9%)N/A0.490
 Desmoplakin6 (21%)1 (9%)N/A0.649
 Plakophilin-23 (10%)1 (9%)N/A1.000

ARVC, arrhythmogenic right ventricular cardiomyopathy; BSA, body surface area; EDV, end-diastolic volume; EF, ejection fraction; FS, fractional shortening; LVDd, left ventricular end-diastolic diameter; LVDs, left ventricular end-systolic diameter; mm, millimetre; MRI, magnetic resonance imaging; SAECG, signal-averaged electrocardiogram; VT, ventricular tachycardia.

*

P < 0.05 vs. idiopathic VT.

**

P < 0.01 vs. idiopathic VT.

Screening of plasma microRNAs

Figure 1 demonstrates screening process to determine the specific miRNAs in patients with definite ARVC. In the first stage, 84 cardiac-related miRNAs were evaluated in six patients with definite ARVC and six normal subjects by the miScript miRNA PCR Array kit. The blood levels of 11 miRNAs (hsa-let-7e, miR-122-5p, 133a, 144-3p, 145-5p, 185-5p, 195-5p, 206, 208a, 208b, and 494) were found to be up-regulated in patients with definite ARVC. The blood levels of six miRNAs (miR-107, 125a-5p, 133b, 142-3p, 150-5p, and 378a-3p) were found to be down-regulated in patients with definite ARVC (Figure 2). To validate the results of the miRNA array, we selected the miRNAs whose blood levels in the patients with definite ARVC were four times higher (hsa-let-7e, miR-122-5p, 144-3p, 145-5p, 185-5p, 195-5p, and 494) or four times lower (miR-107, 142-3p, 150-5p, and 378a-3p) than those in normal subjects for further analysis by real-time PCR. The circulating miRNA levels from real-time PCR were compared with the levels from the PCR array (see Supplementary material online, Figure S2).

Flowchart demonstrating screening process to determine the specific miRNAs in definite ARVC patients. ARVC, arrhythmogenic right ventricular cardiomyopathy; miRNA, microRNA; PCR, polymerase chain reaction; VT, ventricular tachycardia.
Figure 1

Flowchart demonstrating screening process to determine the specific miRNAs in definite ARVC patients. ARVC, arrhythmogenic right ventricular cardiomyopathy; miRNA, microRNA; PCR, polymerase chain reaction; VT, ventricular tachycardia.

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Blood levels of miRNAs in six definite ARVC patients and six normal subjects in miRNA array. (A) Evaluation of up-regulated or down-regulated miRNA. A total of 84 miRNAs were evaluated in definite ARVC patients compared with normal subjects. (B) The levels of 11 miRNAs were found to be up-regulated in definite ARVC patients. (C) The levels of six miRNAs were found to be down-regulated in definite ARVC patients. Graphs B and C depict fold change of miRNAs in the definite ARVC patients compared with normal subjects. ARVC, arrhythmogenic right ventricular cardiomyopathy; miRNA, microRNA.
Figure 2

Blood levels of miRNAs in six definite ARVC patients and six normal subjects in miRNA array. (A) Evaluation of up-regulated or down-regulated miRNA. A total of 84 miRNAs were evaluated in definite ARVC patients compared with normal subjects. (B) The levels of 11 miRNAs were found to be up-regulated in definite ARVC patients. (C) The levels of six miRNAs were found to be down-regulated in definite ARVC patients. Graphs B and C depict fold change of miRNAs in the definite ARVC patients compared with normal subjects. ARVC, arrhythmogenic right ventricular cardiomyopathy; miRNA, microRNA.

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Expression of selected microRNAs in different groups

In subgroup analyses, patients with definite ARVC had significantly higher expression of circulating miR-144-3p, 145-5p, 185-5p, and 494 than patients in the three other groups (Figure 3 and Supplementary material online, Table S1). On the ROC analysis for the identification of definite ARVC in the patients with VA, the best cut-off value for miR-144-3p, 145-5p, 185-5p, and 494 were 1.88 [fold change, area under the curve (AUC): 0.728], 0.93 (fold change, AUC: 0.619), 13.1 (fold change, AUC 0.740), and 1.72 (fold change, AUC 0.891), respectively (Figure 4).

Comparison of circulating miRNA among control subjects, definite ARVC patients, borderline or possible ARVC patients, and idiopathic VT patients. Graph depicts fold change of miRNAs in each group as compared to control. *P < 0.05, **P < 0.01, ***P < 0.001. ARVC, arrhythmogenic right ventricular cardiomyopathy; miRNA, microRNA; VT, ventricular tachycardia.
Figure 3

Comparison of circulating miRNA among control subjects, definite ARVC patients, borderline or possible ARVC patients, and idiopathic VT patients. Graph depicts fold change of miRNAs in each group as compared to control. *P < 0.05, **P < 0.01, ***P < 0.001. ARVC, arrhythmogenic right ventricular cardiomyopathy; miRNA, microRNA; VT, ventricular tachycardia.

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Receiver operating characteristic (ROC) analysis for the identification of definite arrhythmogenic right ventricular cardiomyopathy in the patients with ventricular arrhythmia. (A) ROC analysis of miR-144-3p. (B) ROC analysis of miR-145-5p. (C) ROC analysis of miR-185-5p. (D) ROC analysis of miRNA-494. AUC, area under the curve; miRNA, microRNA.
Figure 4

Receiver operating characteristic (ROC) analysis for the identification of definite arrhythmogenic right ventricular cardiomyopathy in the patients with ventricular arrhythmia. (A) ROC analysis of miR-144-3p. (B) ROC analysis of miR-145-5p. (C) ROC analysis of miR-185-5p. (D) ROC analysis of miRNA-494. AUC, area under the curve; miRNA, microRNA.

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The blood levels of miR-144-3p, 145-5p, and 494 were down-regulated in patients with borderline or possible ARVC or idiopathic VT but were up-regulated in patients with definite ARVC (Figure 3). Although the blood levels of miR-185-5p were slightly up-regulated in patients with borderline or possible ARVC or idiopathic VT, these levels were much lower than those in patients with definite ARVC. The blood levels of hsa-let-7e, miR-122-5p, and 195-5 p were not significantly higher in patients with definite ARVC than in those with either borderline or possible ARVC or idiopathic VT. The blood levels of 107, 142-3p, 150-5p, and 378a-3p were not significantly lower in patients with definite ARVC than in those with either borderline or possible ARVC or idiopathic VT.

Comparison of clinical data in patients with definite arrhythmogenic right ventricular cardiomyopathy with or without recurrent ventricular arrhythmia

Three definite ARVC patients did not receive catheter ablation because they were in stable condition due to additional medical therapy. Out of 25 definite ARVC patients who underwent catheter ablation, successful ablation was achieved in all patients (100%). During the follow-up period (45 ± 20 months), recurrent VA after ablation occurred in eight definite ARVC patients (32%). Table 2 lists the baseline clinical characteristics of definite ARVC patients with or without recurrent VA. LV fractional shortening was significantly lower in definite ARVC with recurrent VA compared with those without recurrent VA. However, other variables were similar in definite ARVC patients with or without recurrent VA. In addition, there were no significant differences in characteristic of VAs, multiple sites, or epicardial approaches in catheter ablation procedure and genetic mutation screenings between the two groups.

Table 2
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Comparisons of patient characteristics in patients with definite ARVC with or without recurrent VA

Recurrent VA (n = 8)No recurrent VA (n = 17)P-value
Echocardiography
 Left atrial diameter (mm)34.2 ± 3.736.1 ± 4.30.287
 LVDd (mm)50.0 ± 6.351.0 ± 5.90.636
 LVDs (mm)37.4 ± 6.432.7 ± 5.80.197
 Left ventricular EF (%)51.5 ± 9.656.7 ± 8.10.255
 Left ventricular FS (%)25.3 ± 4.835.1 ± 7.70.003
Angiography
 RVEF (%)44.8 ± 12.941.1 ± 10.60.406
MRI
 Right ventricular EDV/BSA (mL/m2)69.8 ± 3.574.5 ± 14.00.582
SAECG
 Filtered QRS duration (ms)123.2 ± 24.5120.7 ± 28.50.549
 Duration of terminal QRS (µV)47.5 ± 25.346.5 ± 24.80.754
 Root-mean-square of terminal 40 ms (µV)26.3 ± 17.221.2 ± 9.30.932
Characteristic of VAs
 Ventricular premature complexes2 (25.0%)7 (41.2%)0.723
 Non-sustained VT1 (12.5%)2 (11.7%)
 Sustained VT5 (62.5%)8 (47.1%)
Radiofrequency catheter ablation
 Multiple sites ablation5 (62.5%)6 (35.2%)0.389
 Epicardial ablation2 (25.0%)1 (5.8%)0.231
Genetic mutation screening
 Desmoglein-21 (12.5%)0 (0.0%)0.320
 Desmoplakin2 (25.0%)3 (17.6%)1.000
 Plakophilin-21 (12.5%)2 (11.7%)1.000
Recurrent VA (n = 8)No recurrent VA (n = 17)P-value
Echocardiography
 Left atrial diameter (mm)34.2 ± 3.736.1 ± 4.30.287
 LVDd (mm)50.0 ± 6.351.0 ± 5.90.636
 LVDs (mm)37.4 ± 6.432.7 ± 5.80.197
 Left ventricular EF (%)51.5 ± 9.656.7 ± 8.10.255
 Left ventricular FS (%)25.3 ± 4.835.1 ± 7.70.003
Angiography
 RVEF (%)44.8 ± 12.941.1 ± 10.60.406
MRI
 Right ventricular EDV/BSA (mL/m2)69.8 ± 3.574.5 ± 14.00.582
SAECG
 Filtered QRS duration (ms)123.2 ± 24.5120.7 ± 28.50.549
 Duration of terminal QRS (µV)47.5 ± 25.346.5 ± 24.80.754
 Root-mean-square of terminal 40 ms (µV)26.3 ± 17.221.2 ± 9.30.932
Characteristic of VAs
 Ventricular premature complexes2 (25.0%)7 (41.2%)0.723
 Non-sustained VT1 (12.5%)2 (11.7%)
 Sustained VT5 (62.5%)8 (47.1%)
Radiofrequency catheter ablation
 Multiple sites ablation5 (62.5%)6 (35.2%)0.389
 Epicardial ablation2 (25.0%)1 (5.8%)0.231
Genetic mutation screening
 Desmoglein-21 (12.5%)0 (0.0%)0.320
 Desmoplakin2 (25.0%)3 (17.6%)1.000
 Plakophilin-21 (12.5%)2 (11.7%)1.000

ARVC, arrhythmogenic right ventricular cardiomyopathy; BSA, body surface area; ECG, electrocardiogram; EDV, end-diastolic volume; EF, ejection fraction; FS, fractional shortening; LVDd, left ventricular end-diastolic diameter; LVDs, left ventricular end-systolic diameter; mm, millimetre; MRI, magnetic resonance imaging; RVEF, right ventricular ejection fraction; SAECG, signal-averaged electrocardiogram; VA, ventricular arrhythmia, VT, ventricular tachycardia.

Table 2
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Comparisons of patient characteristics in patients with definite ARVC with or without recurrent VA

Recurrent VA (n = 8)No recurrent VA (n = 17)P-value
Echocardiography
 Left atrial diameter (mm)34.2 ± 3.736.1 ± 4.30.287
 LVDd (mm)50.0 ± 6.351.0 ± 5.90.636
 LVDs (mm)37.4 ± 6.432.7 ± 5.80.197
 Left ventricular EF (%)51.5 ± 9.656.7 ± 8.10.255
 Left ventricular FS (%)25.3 ± 4.835.1 ± 7.70.003
Angiography
 RVEF (%)44.8 ± 12.941.1 ± 10.60.406
MRI
 Right ventricular EDV/BSA (mL/m2)69.8 ± 3.574.5 ± 14.00.582
SAECG
 Filtered QRS duration (ms)123.2 ± 24.5120.7 ± 28.50.549
 Duration of terminal QRS (µV)47.5 ± 25.346.5 ± 24.80.754
 Root-mean-square of terminal 40 ms (µV)26.3 ± 17.221.2 ± 9.30.932
Characteristic of VAs
 Ventricular premature complexes2 (25.0%)7 (41.2%)0.723
 Non-sustained VT1 (12.5%)2 (11.7%)
 Sustained VT5 (62.5%)8 (47.1%)
Radiofrequency catheter ablation
 Multiple sites ablation5 (62.5%)6 (35.2%)0.389
 Epicardial ablation2 (25.0%)1 (5.8%)0.231
Genetic mutation screening
 Desmoglein-21 (12.5%)0 (0.0%)0.320
 Desmoplakin2 (25.0%)3 (17.6%)1.000
 Plakophilin-21 (12.5%)2 (11.7%)1.000
Recurrent VA (n = 8)No recurrent VA (n = 17)P-value
Echocardiography
 Left atrial diameter (mm)34.2 ± 3.736.1 ± 4.30.287
 LVDd (mm)50.0 ± 6.351.0 ± 5.90.636
 LVDs (mm)37.4 ± 6.432.7 ± 5.80.197
 Left ventricular EF (%)51.5 ± 9.656.7 ± 8.10.255
 Left ventricular FS (%)25.3 ± 4.835.1 ± 7.70.003
Angiography
 RVEF (%)44.8 ± 12.941.1 ± 10.60.406
MRI
 Right ventricular EDV/BSA (mL/m2)69.8 ± 3.574.5 ± 14.00.582
SAECG
 Filtered QRS duration (ms)123.2 ± 24.5120.7 ± 28.50.549
 Duration of terminal QRS (µV)47.5 ± 25.346.5 ± 24.80.754
 Root-mean-square of terminal 40 ms (µV)26.3 ± 17.221.2 ± 9.30.932
Characteristic of VAs
 Ventricular premature complexes2 (25.0%)7 (41.2%)0.723
 Non-sustained VT1 (12.5%)2 (11.7%)
 Sustained VT5 (62.5%)8 (47.1%)
Radiofrequency catheter ablation
 Multiple sites ablation5 (62.5%)6 (35.2%)0.389
 Epicardial ablation2 (25.0%)1 (5.8%)0.231
Genetic mutation screening
 Desmoglein-21 (12.5%)0 (0.0%)0.320
 Desmoplakin2 (25.0%)3 (17.6%)1.000
 Plakophilin-21 (12.5%)2 (11.7%)1.000

ARVC, arrhythmogenic right ventricular cardiomyopathy; BSA, body surface area; ECG, electrocardiogram; EDV, end-diastolic volume; EF, ejection fraction; FS, fractional shortening; LVDd, left ventricular end-diastolic diameter; LVDs, left ventricular end-systolic diameter; mm, millimetre; MRI, magnetic resonance imaging; RVEF, right ventricular ejection fraction; SAECG, signal-averaged electrocardiogram; VA, ventricular arrhythmia, VT, ventricular tachycardia.

Although the blood levels of miR-144-3p, 145-5p, 185-5p, and 494, which were significantly up-regulated in patients with definite ARVC, were compared in patients with definite ARVC with or without recurrent VA, definite ARVC patients with recurrent VA had a higher circulating miR-494 than those without recurrence (Figure 5A). On the ROC analysis for the identification of the recurrent VA in definite ARVC patients, the best cut-off value for miR-494 was 26.6 (fold change, AUC: 0.832) as shown in Figure 5B.

MicroRNAs in definite ARVC patients with and without recurrent VA after catheter ablation. (A) Levels of miRNA-144-3p, 145-5p, 185-5p, and 494 in definite ARVC patients with (n = 8) or without (n = 17) recurrent VA. (B) Receiver operating characteristic analysis for the identification of recurrent VA after catheter ablation in definite ARVC patients. *P < 0.05. ARVC, arrhythmogenic right ventricular cardiomyopathy; AUC, area under the curve; miRNA, microRNA; VA, ventricular arrhythmia.
Figure 5

MicroRNAs in definite ARVC patients with and without recurrent VA after catheter ablation. (A) Levels of miRNA-144-3p, 145-5p, 185-5p, and 494 in definite ARVC patients with (n = 8) or without (n = 17) recurrent VA. (B) Receiver operating characteristic analysis for the identification of recurrent VA after catheter ablation in definite ARVC patients. *P < 0.05. ARVC, arrhythmogenic right ventricular cardiomyopathy; AUC, area under the curve; miRNA, microRNA; VA, ventricular arrhythmia.

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Protein expression of cleaved caspase 3 in H9c2 cells transfected by miR-494 lentivirus

Figure 6A illustrates examples of the fluorescence in H9c2 cells with or without miR-494 transfection. The fluorescence levels were evaluated in the miR-494-transfected group and the mock group (Figure 6B). The ratio of cleaved caspase 3 to caspase 3 in the miR-494-transfected group was significantly higher than that in the mock group (Figure 6C and D).

Apoptotic effect of miR-494. (A) Fluorescence in H9c2 cells after transfection of lentiviral vectors (pLenti-GIII-CMV-GFP-2A-Puro) expressing miR-494. Right panel shows an example of fluorescence in the miR-494 group. Left panel shows an example of fluorescence in the mock group. Fluorescence was increased in the miR-494 group compared with the mock group. (B) The fluorescence levels in the miR-494-transfected group (right panel) and the mock group (left panel). (C) Protein expression of cleaved caspase 3 and caspase 3 in each group by western blot analysis. (D) The ratio of cleaved caspase 3 to caspase 3 was evaluated in each group. ***P < 0.001.
Figure 6

Apoptotic effect of miR-494. (A) Fluorescence in H9c2 cells after transfection of lentiviral vectors (pLenti-GIII-CMV-GFP-2A-Puro) expressing miR-494. Right panel shows an example of fluorescence in the miR-494 group. Left panel shows an example of fluorescence in the mock group. Fluorescence was increased in the miR-494 group compared with the mock group. (B) The fluorescence levels in the miR-494-transfected group (right panel) and the mock group (left panel). (C) Protein expression of cleaved caspase 3 and caspase 3 in each group by western blot analysis. (D) The ratio of cleaved caspase 3 to caspase 3 was evaluated in each group. ***P < 0.001.

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Discussion

The main findings of this study are the following: (i) the expression of circulating miR-144-3p, 145-5p, 185-5p, and 494 was significantly up-regulated in patients with definite ARVC. (ii) Patients with definite ARVC with recurrent VA after ablation had higher circulating levels of miR-494 than those without recurrence. (iii) miR-494 was associated with apoptosis in H9c2 cells. These findings bring insight on understanding the characteristics of the definite ARVC patients with VA.

Relationship between arrhythmogenic right ventricular cardiomyopathy and selected microRNAs

Although the importance of tissue miRNAs during disease progression has been demonstrated, miRNAs in blood plasma or serum are clinically regarded as ideal risk markers for cardiovascular diseases because they are stable and easily detectable as a result of cellular damage or secretion.10,11 However, the correlation between circulating and tissue miRNAs in ARVC is still unclear, and the expression of miRNAs may be different between circulating and tissue miRNAs. In this study, the levels of miRNAs were not linear among idiopathic VT, borderline or possible ARVC, and definite ARVC. These results imply that borderline or possible ARVC might include other non-ischaemic cardiomyopathy. Nevertheless, we demonstrated that the expression of circulating miR-144-3p, 145-5p, 185-5p, and 494 was significantly up-regulated in patients with definite ARVC with VA. In addition, on the ROC analysis for the identification of definite ARVC, the AUCs of miR-144-3p, 145-5p, 185-5p, and 494 were 0.728, 0.619, 0.740, and 0.891, respectively. Although differentiation between early-phase ARVC and idiopathic VA can be challenging, these miRNAs may be useful for the diagnosis of ARVC.

miR-494 is associated with recurrence of ventricular arrhythmia after catheter ablation

In our study, definite ARVC patients with recurrent VA had higher circulating levels of miR-494. Additionally, ROC analysis showed miR-494 to be a predictor of recurrent VA (AUC: 0.832). One possible reason for the recurrent VA after ablation is progression of the disease in ARVC.12,13 It is considered that apoptosis contributes to progressive myocardial atrophy and cardiac electrical instability.2 It was previously reported that myocardial cells in patients with ARVC showed high levels of expression of caspase 3, which is essential for the apoptotic process.14,15 Therefore, we demonstrated that H9c2 cells transfected by miR-494 lentivirus had a higher activation of caspase 3 than H9c2 cells without transfection. This embryonic rat ventricular myocardial H9c2 cell line has been widely used to investigate apoptotic mechanisms.16 Wang et al.17 showed that miR-494 exerted cardioprotective effects against acute ischaemia/reperfusion-induced injury via activation of the Akt signalling pathway, whereas they verified that miR-494 was able to target both antiapoptotic and proapoptotic proteins. They speculated that various targets of a miRNA might work unequally to balance a common signalling pathway. Therefore, it is possible that miRNA demonstrate contrasting functions in different types of diseases and diverse conditions. Indeed, it has been reported that miR-494 can play opposing roles (cell proliferation or cell apoptosis) in various types of cancer.18–20 In contrast to acute ischaemia/reperfusion-induced injury, we speculate that myocardial apoptosis induced by chronic overexpression of miR-494 has a key role in the disease progression of ARVC. In this study, LV fractional shortening in definite ARVC with recurrent VA was significantly lower compared with those without recurrent VA. It is considered that LV systolic dysfunction is one of the predictors of adverse outcomes.21 Therefore, lower LV fractional shortening supports that definite ARVC with higher miR-494 may be associated with future adverse events.

Recognition and validation of miRNA targets is essential for the understanding of miRNA function and possible therapy in ARVC. Future studies should focus on the potential of specific plasma miRNAs as biomarkers for screening patients with VA for ARVC.

Conclusions

We have demonstrated for the first time that plasma levels of miR-144-3p, 145-5p, 185-5p, and 494 are significantly elevated in definite ARVC patients with VA. An increased plasma level of miR-494 was associated with recurrent VA after ablation in definite ARVC patients.

Supplementary material

Supplementary material is available at Europace online.

Conflict of interest: none declared.

Funding

This study was supported by NSC101-2314-B-075-056-MY3, MOST 105-2314-B-075-036, Taipei Veterans General Hospital grant V104E7-002, V105C-116, V105C-121, VGHUST104-G7-3-1 and -2, and VGHUST105-G7-9-1 and -2.

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Author notes

Shinya Yamada and Ya-Wen Hsiao authors contributed equally to this work.

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2017. For permissions, please email: [email protected].
This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://dbpia.nl.go.kr/journals/pages/about_us/legal/notices)
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Clinical Research > Arrhythmogenic right ventricular cardiomyopathy
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