Burden of premature ventricular contractions beyond nonsustained ventricular tachycardia is related to the myocardial extracellular space expansion in patients with hypertrophic‐cardiomyopathy

Abstract Background Although nonsustained ventricular tachycardia (NSVT) is a risk factor for sudden cardiac death in hypertrophic‐cardiomyopathy (HCM), the impact of premature ventricular contraction (PVC) burden, in the absence of NSVT, is not well‐known. Hypothesis PVC burden may be associated with myocardial fibrosis and genetic mutations in patients with HCM. Methods Of the 212 patients prospectively enrolled to the HCM registry of genetics, 84 were evaluated with both cardiac magnetic resonance, 24‐hour Holter monitoring and genetic analysis. Among them, 71 patients have not been diagnosed with NSVT. Results Patients with NSVT (n = 13) had a higher late gadolinium enhancement (LGE) amount, extracellular volume fraction (ECV), and prevalence of sarcomere mutations compared with patients without NSVT. Among patients without NSVT, those with LGE (n = 46) had a higher total PVC (109 ± 332 vs 7 ± 13, P = .003) and PVC burden (0.114 ± 0.225 vs 0.008 ± 0.014%, P = .003) during 24‐hour Holter monitoring compared with others. The %LGE and global ECV were correlated with PVC burden (r = 0.377, P = .001; r = 0.401, P = .001). The optimal cutoff value for PVC number for LGE was 45 (37.0% and 100% sensitivity and specificity, respectively) with 0.733 of the area under the receiver operating characteristic‐curve (P < .001). Thick filament gene mutation was more prevalent in the higher PVC burden group (41.2% vs 16.7%, P = .048). Conclusion Total PVC burden is significantly related to increase in myocardial fibrosis in HCM patients without NSVT.

Thick filament gene mutation was more prevalent in the higher PVC burden group (41.2% vs 16.7%, P = .048).
Conclusion: Total PVC burden is significantly related to increase in myocardial fibrosis in HCM patients without NSVT. as a risk factor for SCD in patients with HCM. 2,3 However, there are some cases that NSVT is not detected in a 24 to 48 hour Holter monitoring, a recommendation of the current guidelines. A fibrotic myocardium could trigger and act as a substrate for both premature ventricular contraction (PVC) and NSVT. 4 High-PVC burden may reflect myocardial fibrosis even in the absence of NSVT. Moreover, sarcomere-associated gene mutations have been associated with ventricular arrhythmia. 5 We investigated (1) the association between NSVT and myocardial fibrotic burden, (2) the association between PVC burden and myocardial fibrotic burden in HCM patients without NSVT, and (3) contribution of sarcomere-associated mutations for NSVT and PVC burden in a 24-hour Holter monitoring.

| Study population
A total of 432 patients were enrolled in the HCM registry from 2006 to 2014 at our hospital. After excluding those patients who discontinued follow-ups, were unable to visit the clinic due to migration or death, or were denied participation in the study, 212 patients were consecutively enrolled and underwent genetic testing. Of the 212 patients prospectively enrolled to the HCM registry of genetics, 6

| 24-hour Holter monitoring analysis
A 24-hour Holter monitoring was performed with SEER light (GE Healthcare) machine and was analyzed by both manual and automatic reviews of all 24-hour echocardiography (ECGs). NSVT was defined as an episode of ventricular tachycardia (VT) with a heart rate of at least 120 beats per minute, lasting for at least three beats and persisting less than 30 seconds. Total PVC number was calculated, and two PVCs in a row were defined as couplet. PVC burden was defined as percent of total PVC number divided by total beats during monitoring.

| Conventional echocardiography
A routine standard echocardiography study was performed to measure the systolic and diastolic parameters according to the current American Society of Echocardiography guidelines. 7 Continuous wave Doppler was used to measure peak velocity across the LV outflow tract (LVOT), and the pressure gradient was calculated using the Bernoulli equation as follows: 4x (peak velocity across the LVOT). . It was measured at resting and during Valsalva maneuver. LVOT obstruction was defined as a systolic pressure gradient ≥30 mmHg.
Details are described in Supplementary Method S1.

| Cardiac magnetic resonance imaging
Cardiac magnetic resonance (CMR) imaging was performed using a 1.5-T scanner (Magnetom Avanto; Siemens Medical Solutions, Erlangen, Germany) with a phased array body coil. We analyzed LGE, T1, and extracellular volume fraction (ECV) for presenting myocardial fibrosis. The presence of LGE involvement in each segment and the total number of LGE-involving segments was measured. Native T1 mapping with the modified Look-Locker technique was performed during the mid-diastolic phase, and post-T1 mapping was performed 15 minutes after contrast media injection using the same slice axis and parameters as the pre-T1 mapping. The percentage of LGE in LV mass was measured using dedicated quantitative analysis software (QmassMR 7.5 or 8.1, Medis, Leiden, Netherland) on LGE images with phase-sensitive inversion recovery (PSIR). To improve the reproducibility, a radiologist and a cardiologist, both with more than 10 years of experience analyzed the LGE sizes. Boundaries of contrast-enhanced areas were automatically traced in each short-axis slice image. On LGE-CMR images, the myocardium with an abnormal enhancement was defined as an area of hyperenhancement of more than five standard deviations from the remote myocardium. Remote myocardium was defined as nonenhanced myocardium, which is the opposite of hyperenhanced myocardium. Maximal signal was determined by computer-assisted window thresholding of the enhanced area. Obvious artifacts, such as those caused by motion, were excluded using a tool from the software package. Total LGE volume was calculated by summing the LGE volumes of all the slices. 8 Using the QMap RE (Medis, Leiden, Netherland), native T1, post-T1, and ECV analyses were performed. The myocardial ECV was automatically calculated with the following equation: where R1 = 1∕T1 and ΔR1 = postcontrast R1−precontrast R1. 9 The LGE, T1, and ECV values were measured according to the American Heart Association (AHA) 16-segment model. Currently LGE, reflecting replacement fibrosis, is the most widely proven prognostic marker of SCD in HCM, therefore we chose LGE as a targeted value to determine the cutoff value of PVC burden. included 82 genes (33 sarcomere-associated protein genes, 5 phenocopy genes, and 44 nuclear genes linked to mitochondrial cardiomyopathy). 6 The HCM genes consisted of 8 validated sarcomere genes and 25 putative HCM genes. 10 We divided the sarcomere genes into two subgroups: thick filament genes (MYH7, MYBPC3, MYH6, and MYL3) and thin filament genes (other sarcomere genes except for thick filament genes). The details are described in Supplementary Method S2.

| Statistical analysis
Continuous variables with normal distributions are reported as the mean ± SD or 95% confidence interval. The Student t tests were used to compare the means of the continuous variables that were approximately normally distributed between the two groups. Normality was determined using the Shapiro-Wilk test. Categorical variables were reported as counts (or percentages) and were compared using the χ 2 tests. For the reproducibility test, paired sample t-tests were performed and Pearson's correlation coefficients were calculated.
Receiver operating characteristic (ROC) analysis was performed to identify the optimal cutoff values of burden of PVC for the presence of LGE. All clinical statistical analyses were performed using the SPSS version 25.0 statistical package (IBM Corp., Armonk, NewYork). A two-sided P-value <.05 was considered to be statistically significant.
PVC couplet was more prevalent in the NSVT group compared with the no NSVT group (61.5% vs 21.4%, P = .006). In the echocardiographic analysis, the NSVT group had higher maximal wall thickness

| PVC burden and genetics according to the presence of LGE in patients without NSVT
We analyzed the characteristics of the patients without NSVT (n = 71).
Moreover, ECV correlated with total PVC beats (r = 0.387, P = .001) and PVC burden (r = 0.401, P = .001). Post-T1 tended to be correlated with PVC burden and total PVC beats (all P < .10). The optimal cutoff value for PVC number for the presence of LGE was 45 (37.0% and 100% sensitivity and specificity, respectively) with 0.733 of the area under the ROC curve (P = .001) (Figure 1).

| DISCUSSION
This study reports on major findings with regard to ventricular arrhythmia in HCM. First, patients with NSVT showed higher myocardial fibrosis burden compared with patients without NSVT. Second, after excluding patients who have been diagnosed with NSVT, higher PVC burden was associated with higher %LGE and ECV, which represent myocardial extracellular space expansion. Third, a pathogenic or probably pathogenic sarcomere-associated mutation was associated with NSVT and higher PVC burden.  12 Previous studies reported that frequent PVC was a predictor of future VT in postinfarction patients. 13,14 Therefore, although the dominant mechanism is unclear, we can suggest that frequent PVC may be associated with NSVT in HCM by triggered automaticity or re-entry formation. However, a 24-hour Holter monitoring cannot always catch the presence of NSVT; therefore, prolonged ECG monitoring is recommended despite the inconvenience in its performance. 15 In addition, previous studies revealed that PVC burden was related with SCD in the general population. 16

| Sarcomere mutations, PVC burden, and NSVT in HCM
Mutations in sarcomere genes could activate proliferative and profibrotic signals to produce pathogenic cardiac remodeling as a primary phenotype in HCM. 19 Sarcomere-associated gene mutation may be associated with ventricular arrhythmia due to its contribution to myocardial fibrosis, although the results of previous studies were controversial. 5,20 Our study results also show that patients with pathogenic sarcomere mutation had higher LGE amount and ECV and the patients with NSVT had more prevalent pathogenic or likely pathogenic mutation and VUS, and a tendency of more prevalent thick filament mutation in sarcomere genes compared with others. After exclusion of patients with NSVT, higher PVC burden group had a tendency of more prevalent pathogenic or likely pathogenic sarcomere-associated mutation and significantly higher prevalence of thick filament mutation compared with the lower PVC burden group. It has not been clarified whether thick filament mutation is associated with higher rate of ventricular arrhythmia in HCM. 21 F I G U R E 2 Comparisons of late gadolinium enhancement (LGE) amount and global extracellular volume fraction (ECV) between patients with higher premature ventricular complex (PVC) burden and lower PVC burden but without nonsustained ventricular tachycardia outcomes could not be established. A follow-up study is needed to investigate the predictive value of current PVC burden to future events.

| CONCLUSIONS
Total beats and PVC burden are significantly related to the increase in myocardial fibrosis in patients with HCM but without NSVT. In the absence of NSVT, high-PVC burden may provide information for individualized therapy and follow-up plan to prevent fatal ventricular arrhythmia in HCM. Moreover, the presence of sarcomereassociated gene mutation may be also considered as one of the risk factors for ventricular arrhythmia. Further study is needed to investigate the direct relation between PVC burden and SCD in HCM.