METHODS
Ethical approval for this study was sought but deemed unnecessary by the Leicestershire Research Ethics Committee, and the study protocol was approved by the Research and Development Office of the University Hospitals of Leicester National Health Service Trust. The study group was identified by screening the University Hospitals of Leicester NHS Trust Cardiology Department’s audit databases for patients who had undergone an electrophysiology study (EPS) with programmed electrical stimulation as part of clinical risk stratification for ICD implantation between 1st January 2004 and 31stJuly 2009. Patients were excluded if they had a history of ischemic heart disease or Brugada syndrome (these patients are being investigated in a separate study). This identified 53 suitable patients, 3 of whom were later excluded because of insufficient data due to breakthrough / non-captured beats, providing a final cohort of 50 patients: 33 with dilated cardiomyopathy (DCM) and 17 with ‘other conditions’ – 5 with myotonic dystrophy, 5 with non-compaction cardiomyopathy, 3 with arrhythmogenic right ventricular dysplasia, 1 cardiac sarcoidosis, 1 hypertrophic cardiomyopathy, 1 cardiac amyloidosis and 1 apparently normal heart ventricular tachycardia.
A 30 patient control group was selected from patients undergoing an EPS for clinical management of supraventricular tachycardia in 2010. Controls were excluded prior to analysis if they had: insufficient surface ECG data recorded, an abnormal echocardiogram, family history of SCD or diabetes mellitus (because of potential for associated autonomic dysfunction and silent ischemic heart disease). During data analysis one control patient was excluded because the quadripolar catheter became displaced from the right ventricular apex during the study. All control subjects had normal left ventricular function as judged by visual assessment of 2D transthoracic echocardiograms. Endpoints were assigned by an independent three member committee with access to clinical records.
The EP studies for both cohorts of patients were conducted according to standard departmental protocol. Subjects were fasted and antiarrhythmic drugs withheld 4-5 half-lives prior to the procedure. The procedure was performed under minimal sedation. Catheters were positioned as was appropriate for the clinical procedure, and the study pacing protocol delivered through a 6F quadripolar catheter advanced via femoral transvenous access to the right ventricular apex. Standard 12-lead ECGs were recorded using Labsystem Pro (BARD, Lowell) at a 1kHz sampling rate with a high pass filter set at 0.01 Hz and a low pass filter set to 50 Hz. The EPS protocol consisted of a single extrastimulus programmed ventricular stimulation with 8 or 10 beat trains at drive cycle lengths (DCL)s of 600ms/400ms; reduced as necessary due to breakthrough beats. The extrastimulus was started at 500ms or 360ms followed by decrements of 20ms to the effective refractory period. The S1S2 coupling interval describes the interval between the last beat of the drive train and the first extrastimulus. Measurements taken from the final S1 and the S2 beats for each drive train are used to calculate the R2I2/PERS.
Calculation of R2I2 and PERS has been described in full previously.11 Surface ECG recordings taken during the study were exported from Labsystem Pro (BARD, Lowell) at 16-bit resolution. Recordings were then analysed using custom software written by WBN using MATLAB (Mathworks, Natick, MA, USA) with further work to refine the software by Madeiro et al. 13 All points were manually verified and corrected where appropriate by MIS and WBN. The T wave peak (Tp) was preselected as the body surface surrogate for the end of the APD as has been previously used for R2I2 and PERS.12 The diastolic interval was represented by the period from the Tp of the last S1 beat to the QRS onset (QRSo) of the S2 beat (T wave peak to QRS onset - TpQ), while the APD was taken as the interval between the QRSo and Tp (QRS onset to T wave peak - QTp) of the S2 beat. Individual APD restitution slopes for all 12 ECG leads were calculated using the technique of Taggart et al. 14 R2I2 was taken as the mean of the standard deviations of the difference from the mean slope for all of the leads. PERS was calculated by taking the mean restitution curve slope at each S1–S2 coupling interval across the 12 ECG leads with the maximum value defined as PERS.
It was necessary to censor out some data points according to predetermined rules: 1. Breakthrough beat occurring within the last 3 beats of the drive train (122/929 censored), 2. Point measurement being indeterminate because of artefact, unclear morphology or baseline wander (298/10081). Points that have near or identical TpQ measurements across multiple QTp intervals produce extremely steep, non-physiological gradients. To avoid skewing of the data, gradients exceeding ± 10 were censored from analysis (1.6% of gradients).
Example QTp/TpQ plots for study and control patients with low and high values are shown in Figure 1. Figure 1A is a control patient with low R2I2 and PERS, all 12 ECG leads are seen to follow relatively homogenous, shallow restitution slopes. Figure 1B is a second control patient, PERS is high and R2I2 is moderately low in this patient reflecting the steep but relatively uniform course of the patient’s ECG restitution slopes. Figure 1C is from a patient with non-ischemic cardiomyopathy and low R2I2 and PERS who did not reach the endpoint of VA/death; this patient’s ECG restitution slopes are less homogenous than Figure 1A but contrast with Figure 1D a non-ischemic cardiomyopathy patient with high R2I2 and PERS who reached the endpoint of VA/death and whose ECG restitution slopes follow markedly contrasting paths.
The study group sample size was informed by a two-sample t-test power calculation using the Satterthwaite approximation for unequal variances and assuming that study patients would have R2I2 values in keeping with our previous retrospective study of patients with ischemic cardiomyopathy (R2I2 in VA/death group compared with No VA/death group (mean±SD: 1.30±0.25 vs 1.03±0.27)).12 To achieve 80% power at a 5% significance level, to show that R2I2 was significantly higher in study patients reaching the endpoint of VA/death versus those not, required 10 patients reaching endpoint. Screening of departmental electrophysiology audit databases captured all available, appropriate patients but because event rates were lower than anticipated, 9 patients reached the endpoint of VA/death over median follow up of 5.6 years [IQR 1.9 years]. Hence this study is underpowered for the primary endpoint.
The predefined cut-off for a positive R2I2 test was set at ≥1.03 (no units) and the cut-off for a positive PERS test was ≥1.21 (no units) as has been used in previous studies.11,12 Previously it has been found that the best discrimination of patients reaching an endpoint of VA/SCD was achieved using a combined R2I2+PERS marker: patients positive for both R2I2 and PERS, patients positive for either R2I2/PERS, patients negative for both R2I2 and PERS.
Parametric data are expressed as mean± standard error of the mean (SEM) and were analysed with the Student t-test; nonparametric data are expressed as median [interquartile range (IQR)] and were analysed with the Mann-Whitney U test. Proportions were analysed using a two-sided Fisher’s exact test or chi-squared test as appropriate. A receiver operator characteristic curve using R2I2 and PERS singly and combined was constructed in the study cohort and the area under the curve calculated. Kaplan–Meier survival curves were constructed for patient subgroups partitioned by R2I2≥1.03, PERS≥1.21 and combined R2I2+PERS; comparison of cumulative endpoints was based on logarithmic transformations. Survival was recorded as time to first VA / death or the end of follow-up, which was capped at 6 years in view of the potential for arrhythmia substrate to change over time. In the study group Cox proportional hazards models were used to estimate hazard ratios for R2I2≥1.03 and PERS≥1.21 and to look for independence of combined R2I2+PERS from aetiology; independence from aetiology was chosen for multivariate analysis because of the preponderance of events in the ‘other conditions’ group. Spearman’s rank correlation was used to look for correlation between parametric and non-parametric data. P<0.05 was considered statistically significant. All analyses were performed using STATA (StataCorp LP, College Station, TX, USA).