Sixty-nine consecutive HOCM patients with symptomatic left ventricular (LV) outflow tract obstruction who underwent surgical myectomy at Fuwai hospital,Beijing between 2015 and 2020 were retrospectively studied. All patients underwent a detailed cardiovascular evaluation, including medical history, clinical examination, 12-lead ECG, and CMR.

HCM was diagnosed by the criteria mentioned previously [12]..LV outflow obstruction was defined as an instantaneous peak doppler LV outflow tract gradient (LVOTG) ≥30 mmHg at rest, or an exercised LVOTG ≥50 mmHg [13]. Patients with obstructive coronary artery disease, history of alcohol septal ablation, history of surgery or trauma within the previous 6 months before the surgery, severe valvular disease,stages 3 to 5 chronic kidney disease (CKD),connective tissue disease and osteoarthropathy were excluded from the study.

Control myocardium from the LV septal wall was collected at autopsy of 9 individuals (6 males,3 females, mean age 45.4 ± 14.3 years) who died from accidents, showing no signs of macroscopic or microscopic cardiac lesions.

A standard 12-lead ECG (0.5–150 Hz, 25 mm/s, 10 mm/mV) was recorded in the supine position during quiet respiration before the surgeries. The fQRS was defined as previously described [14],as follows: in patients with QRS duration < 120 milliseconds, (1) an additional R wave (R prime), (2) notching in nadir of the S wave, (3) notching of R wave, or (4) the presence of more than 1 R prime in 2 contiguous leads corresponding to the left ventricular (LV) segment; in patients with right or left bundle branch block (QRS duration≥120 milliseconds), (1) various RsR’ pattern with > 2 R’, (2) > 2 notches in the R wave, or (3) > 2 notches in the downstroke or upstroke of the S wave, in 2 contiguous leads corresponding to the LV segment. The presence of fQRS in 2 contiguous anterior leads (V1–V5), lateral leads (I, aVL, and V6), or inferior leads (II, III, and aVF) was assigned to detect myocardial fibrosis in the anterior, lateral, or inferior segments, respectively. The Q-fQRS method quantifies the total amount of fragmentation in all 12 routine ECG leads together. The summation of absolute numbers of positive and negative deflection points in each first QRS complex of each ECG lead were labeled as a fQRS count value. The first deviation from the iso-electric line and the last transition from the last deflection to the iso-electric line were excluded [11].

Transthoracic echocardiography was performed by an experienced cardio-sonographer using the Phillips iE33 Color Doppler Ultrasound System (Philips Healthcare, Andover, MA, USA). M-mode, twodimensional, and pulsed and continuous-wave Doppler studies were utilized in the standard evaluation according to the guidelines of the American Society of Echocardiography [15]. All patients underwent resting LVOT gradient measurements with continuous-wave Doppler echocardiography, while LVOT gradient after provocation was only determined in those with a resting LVOT gradient < 50 mmHg. The severity of mitral regurgitation was measured with flow Doppler imaging as a qualitative index (mild =1, moderate = 2, moderate to severe = 3, and severe = 4) [16].

CMR imaging was performed using a 1.5-T speed clinical scanner (Siemens Medical Solutions, Erlangen, Germany) before ventricular septal myectomy. All images were acquired with an electrocardiographically gated breath-hold technique.

To evaluate functional parameters, electrocardiographic gating cine images were then acquired using a segmented, balanced, steady-state-free precession sequence. The images were acquired during multiple short breath holds (8–15 s). After scout images, cine imaging was performed in four chamber, three-chamber and two-chamber long- and short axis views with the following protocol: 6-mm-thick sections with a 2-mm gap between sections, repetition time, 2.7 ms; eho time, 1.2 ms; 70° flip angle; temporal resolution, 40 ms; field of view, 360 × 315 mm2; matrix, 192 × 162 pixels; pixel size, 1.9 × 1.3;slice thickness, 6 mm.

All CMR images were analyzed using standard ventricular analysis software (Argus,VE36A; Siemens Medical Solutions). For all patients, wall thickness at the septal, posterior and LV end-diastolic dimensions were all determined in the short-axis view (at the midpapillary level). Epicardial and endocardial borders of the LV myocardium were manually traced during the whole cardiac phase on each cine short-axis image to obtain LV and RV end-diastolic and end-systolic volumes, ejection fractions, and myocardial mass. Myocardial mass was calculated by multiplying the volume of the myocardium calculated at end-diastole by the specific gravity of the myocardium (1.05 g/ml). The end-diastolic volume index, end-systolic volume index, and mass index were indexed to body surface area. Late Gadolinium Enhanced (LGE) images were obtained 10–15 min after injection of 0.2 mmol/Kg gadolinium-DPTA.The extent of scarred myocardium was determined automatically by computer counting of all hyperenhanced pixels in the myocardium on each of the shortaxis images. Hyperenhanced pixels resembling LGE were defined as those with image intensities of 2SD, 4SD, 6SD, and 8SD above the mean of image intensities in a remote myocardial region in the same image. A percentage of LGE was then generated by the ratio of total LGE mass to total LV mass.

We applied extended septal myectomy evolving from the classic Morrow procedure. The hypertrophic ventricular septal leading to systolic anterior motion of the anterior mitral valve and LVOT obstruction was resected. The resection range in the long-axis direction started from approximately 4 mm below the aortic ring to the apex of the left ventricle beyond the bases of the papillary muscles. In the shortaxis direction, the myectomy started rightward to the nadir of the right aortic cusp and to the left and terminated near the mitral anterior commissure. Part of the LV anterior free wall detached to the ventricular septal causing LVOT narrowing may also need to be resected. Furthermore, the anomalous chordal attachments between the mitral valve leaflflets or papillary muscle and the ventricular septal were also excised. Additional surgery was performed based on expert consensus among the experienced cardiac surgeons. If intraoperative transoesophageal echocardiography detected a postoperative LVOT gradient > 30 mmHg or more-than-moderate mitral valve regurgitation after weaning from cardiopulmonary bypass, reoperation was required.

The septal myocardium samples were immediately fixed in 10% buffered formalin and embedded in paraffin. The samples were sectioned and stained with Masson’s trichrome staining for evaluating MF. Four images of every section were acquired with a projection microscope (× 200;Fig. 3). Subsequent image analysis was performed using Image-Pro Plus 6.0 image analysis software (Media Cybernetics Inc., Buckinghamshire, UK). To determine extent of myocardial fibrosis, which was expressed as collagen volume fraction (CVF,%). CVF was calculated as the ratio of collagen-specific staining to the total area of the myocardium in each septal myocardium sample. The endocardium was excluded from analysis. This histological evaluation was performed by two well-trained cardio-pathologists without knowledge of which patient provided the tissue sections.

Continuous variables were expressed as a mean ± standard deviation (SD) or median and interquartile range according to their distribution. Categorical variables were presented as percentage frequency. Differences between groups were compared with the use of the Student t test (for parametric variables) and Mann–Whitney test (for nonparametric variables) for continuous variables. Categorical variables were compared with chi-square tests or Fisher’s exact chi-square tests. The Spearman’s correlation coefficient method was utilized to test for association between continuous variables. Patients were categorized in 2 subgroups according to values of CVF within (subgroup with normal CVF) and above (subgroup with high CVF) the upper limit of normality (established as mean ± 1.96SD obtained in control subjects and equal to 6.09%). Receiver operating characteristic (ROC) analysis was plotted and the accuracy of the ROC curve was determined by measuring the area underneath it (AUC). Sensitivity, specifificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy for the presence of fQRS were defined as previously reported [17]. Multivariate regression analysis was performed using stepwise of the following variables: age, sex, body surface area, Q-fQRS and LGE at 6SD to evaluate if these variables were independently associated with CVF, provided to have a p < 0.10 in univariate analysis. This study assessed interobserver reproducibility for CVF by correlation and Bland-Altman analysis. All statistical analyses were conducted by using statistical software package SPSS Statistics V22.0. A value of P < 0.05 was considered significant.