Velocity vector imaging echocardiography and N-terminal pro- brain natriuretic peptide study of people with preclinical hypertrophic cardiomyopathy

Background: To investigate whether familial hypertrophic cardiomyopathy (HCM) gene mutation carriers without overt left ventricular hypertrophy have subclinical changes in left ventricular function. Methods: We studied Eighteen HCM families with pathogenic mutations, 45 patients with overt HCM (gene positive/phenotype positive (G+/P+)), 40 patients without myocardial hypertrophy (gene positive/phenotype negative G+/P-)), and 48 genotype-negative related healthy controls. Conventional echocardiography and velocity vector imaging (VVI) were performed, and blood levels of N- terminal pro- brain natriuretic peptide (NT- pro- BNP) were analyzed. Results: Although the global longitudinal, circumferential and radial strain was similar between the G+/P-group and the control group, the longitudinal strain of basal inferoseptum and basal anteroseptum was lower in G+/P- patients than in controls, while the basal and middle inferolateral longitudinal strains were signicantly higher. Compared with the controls, G+/P+ patients had signicantly lower global and segmental longitudinal and radial strains. There were no signicant differences between the normal control and G+/P+ groups for global and segmental circumferential strains. The middle of the left ventricle (LV) was clockwise in G+/P+ patients (opposite to normal).The rotation angle of the mid LV rotation in the G+/P+ group were signicantly higher than those in the G+/P- subjects and controls. The NT-proBNP levels were higher in G+/P+ patients than in G+/P- people and controls. Conclusions: Sarcomere gene mutation carriers without overt left ventricular hypertrophy have subclinical segmental systolic dysfunction. Velocity vector imaging is feasible for differentiating HCM, G+/P-patients from controls. in diastolic; LVMWT, left ventricular wall maximum thickness; BSA, body surface area; DBP, diastolic blood pressure; SBP,systolic blood pressure; GLS, global longitudinal strain; GCS, global circumferential strain; GRS, global radial strain; PBr, Peak basilar rotation angle; PMr, Peak mid rotation angle; PAr, peak apex rotation angle; Ptw, peak left ventricular twist; NT-pro-BNP, N-terminal pro-brain natriuretic peptide; VVI, velocity vector imaging.


Background
Hypertrophic cardiomyopathy (HCM) is one of the most common autosomal dominant cardiovascular diseases, and it is the primary cause of sudden death in young people and athletes. In most patients, gene mutation is the primary cause of HCM. Most mutations are sarcomere protein gene mutations encoding myocardium that exhibit autosomal dominant inheritance [1]. HCM demonstrates obvious family clustering, and the genetic probability is 50%. According to statistics, the proportion of patients with familial HCM who eventually develop HCM is 40%-100%. Early recognition of and intervention for cardiac function changes are particularly important.
Familial HCM gene mutation carriers without overt left ventricular hypertrophy (gene positive/phenotype negative G+/P-) may experience syncope and have other clinical symptoms, including abnormal electrocardiogram (ECG) repolarization, and they may develop subclinical changes in cardiac function before developing myocardial hypertrophy. Therefore, it is urgent to identify these patients via imaging methods.
Velocity vector imaging (VVI) is based on two-dimensional grayscale images that track the spatial motion of cardiovascular tissue to show echo spots. The tracking of multiple regional myocardial segments is performed simultaneously. The velocity and displacement of the regional myocardium are displayed quantitatively as a curve. VVI can be used to analyze the movement and deformation of the myocardium, and it is possible to detect ne space and time distinctions in cardiac deformation in different myocardial segments during systole and diastole [1][2][3][4][5]. Therefore, VVI is valuable for evaluating regional and global cardiac function.
Many studies have shown that VVI is potentially viable for assessing myocardial function [6]. N-terminal pro-brain natriuretic peptide (NT-proBNP) level may be related to cardiovascular damage, re ecting ventricular function [7]. However, there are no VVI parameters for the left ventricle (LV), globally or segmentally, in preclinical HCM. We aimed to evaluate changes in the long and short-axis function of the LV using VVI combined with NT-pro-BNP levels.

Study Population
A total of 96 unrelated HCM patients who were diagnosed in our hospital from March 2016 to April 2019 were selected for gene detection, and 45 HCM patients who were carrying sarcomere gene mutations were selected as the gene positive/phenotype positive (G+/P+) group. Gene detection and conventional echocardiography were performed on the rst-degree relatives of 45 unrelated patients (i.e., parents, children, siblings of the same parent). According to the examination results, 40 patients with HCM sarcomere mutation genes but no ventricular wall hypertrophy were selected as the gene positive phenotype negative (G+/P-) group. At the same time, 48 healthy volunteers without gene mutations were selected as normal controls.
The diagnostic criterion of HCM is that the thickness of the left ventricular wall in one or more myocardial segments is greater than or equal to 15 mm. It was necessary to exclude myocardial hypertrophy due to athletics, metabolic diseases, congenital heart diseases and other systemic diseases. In patients with a clear family history, an unexplained left ventricular wall thickness of one or more myocardial segments ≥ 13 mm was observed [1].
All G+/P+ individuals had interventricular septum thickening, with or without other left ventricular wall thickening. Before examination, β-blockers, calcium antagonists and angiotensin-converting enzyme inhibitors were stopped for at least 24 hours. The exclusion criteria were as follows: 1) patients with ventricular wall hypertrophy caused by hypertension, coronary heart disease, diabetes mellitus, valvular disease, congenital heart disease, pulmonary heart disease, metabolic disease or other factors, as well as athletes with cardiac hypertrophy, were excluded after obtaining a medical history and performing a physical examination, ECG and echocardiography; 2) patients with HCM whose left ventricular ejection fraction was less than 50%; 3) accepted patients with HCM who underwent percutaneous septal myocardial ablation, surgical septal myomectomy or permanent pacemaker implantation or experienced atrial brillation.
The inclusion criteria for the G+/P-group were as follows: 1) carrier of a sarcomere mutation gene veri ed by gene generation; 2) maximum left ventricular wall thickness (LVMWT) less than 13 mm detected by echocardiography. The exclusion criteria were as follows: 1) diabetes mellitus and hypertension; 2) cardiac muscle noncompaction and amyloidosis; 3) metabolic diseases and other systemic diseases; 4) signi cant pulmonary lesions; 5) treadmill test, coronary angiography or coronary artery computed tomography (CT) results indicating coronary heart disease, which was de nitively diagnosed by imaging; 6) congenital heart disease; 7) moderate and severe valve stenosis and regurgitation detected by echocardiography.
Thirty rst-degree relatives (parents, children, siblings of the same parent) of 45 unrelated patients were examined by gene testing and routine echocardiography. According to the results of the examination, 40 patients with HCM sarcomere mutation genes but no ventricular wall hypertrophy were selected as the positive gene group, and 48 healthy volunteers without gene mutations were selected as the normal control (G-/P-) group.
This cross-sectional study was conducted with the permission of the Institutional Ethics Committe. All subjects provided written informed consent.

Conventional echocardiography
For the ECG recordings, all subjects laid on their left side. Three short-axis views (mitral valve level, papillary muscle level and apical level) and three long-axis views (apical three-chamber view, apical twochamber view and apical four-chamber view) of the LV were obtained on a Siemens S2000 ultrasound system (Axius, Siemens Medical Solutions, Malvern, PA, USA) with a 4Px probe (2.75-4.25 MHz). All images and clips were stored on the echocardiographic machine for analysis.
The interventricular septal thickness in diastole (IVSD) and left atrial diameter (LAD) were detected in the parasternal long-axis view. The LVMWT was measured in diastole in the basal, mid and apical short-axis views and in the apical long-axis view.
The left atrial volume (LAV), left ventricular end-diastolic volume (LVEDV) and left ventricular end-systolic volume (LVESV) were measured by the Simpson biplane method in the apical two-chamber and fourchamber views. The left atrial volume index (LAVI) was calculated as LAVI = LAV/body surface area (BSA).
The left ventricular out ow tract pressure gradient (LVOT-PG) was measured by continuous-wave Doppler (CW), and the sampling line was placed at the stenosis of the left ventricular out ow tract. In the apical four-chamber view, E/A was measured by pulsed-wave Doppler (PW). The ejection fraction (EF) was estimated using the Simpson biplane method.
All recordings were performed by professional sonographers. All conventional echocardiography parameters were read o ine.

Velocity vector imaging echocardiography
Movie clips were recorded in 3 cardiac cycles and stored, and three apical views of the LV were analyzed o ine using VVI software (Axius, Siemens Medical Solutions). A line was tted along the internal surface of the LV endocardium at end-diastole. We used a frame-by-frame image tracking mode to estimate the movement of the myocardium. The acoustic marker of the myocardium was accurately identi ed and automatically tracked during several consecutive frames.
The longitudinal strain, circumferential strain, and radial strain curves were measured for each LV segment using long-axis and short-axis views according to the 16-segment model of the American Society of Echocardiography [8,9]. In this model, we placed a sampling point on each segment to record the strain experienced during 3 cardiac cycles. The mean value of each measurement was calculated for further analysis.

NT-pro-BNP Test
Fasting blood samples were collected from each patient within 24 hours after enrollment. Blood sampling was standardized without tourniquet and immediately centrifuged twice. NT-pro-BNP was analyzed on a Modular E 170 (Roche Diagnostics, Mannheim, Germany).

Interobserver and Intraobserver Variability
To assess the interobserver variability, which was expressed as the coe cient of variation (CV), 2 independent investigators who were blinded to each other's results analyzed 30 randomly selected VVI movie clips. For intraobserver variability, 30 VVI movie clips were analyzed 3 times within an interval of 2 weeks by one investigator who was blinded to the previous results.

Data and Statistical analysis
All measurement data are expressed as the mean ± standard deviation (SD) and were analyzed using the SPSS 17.0 statistical software package (IBM Corp., Chicago, IL, USA). Signi cant differences between the two groups were analyzed by one-way ANOVA, and comparisons between the two groups were conducted using independent sample t tests. Pearson's correlation analysis was used if the independent variables and dependent variables were normally distributed. The plasma concentration of NT-pro-BNP was logarithmically converted to log NT-pro-BNP, and the normal distribution was analyzed by analysis of variance. P < 0.05 and P < 0.01 indicated signi cant differences. Correlations between VVI parameters and NT-pro-BNP levels were analyzed by linear regression.

Clinical characteristics
There were no signi cant differences in age, sex, BSA, heart rate, or blood pressure among the 3 groups (Table 1).

Conventional echo parameters
The IVSD, LVMWT, LAD, LAVI, and LVOT-PG of the HCM patients were signi cantly higher than those of the patients in the G+/P-group and the control subjects. Meanwhile, G+/P+ patients had a signi cantly lower E/A. However, none of the conventional echo parameters were signi cantly different between the G+/P-group and the control group. In addition, there were no signi cant differences in LVEDV, LVESV or EF among the three groups (Table 1).

Regional longitudinal peak systolic strain
The longitudinal peak systolic strain of the basal inferoseptum and basal anteroseptum in the G+/Pgroup was signi cantly lower than that in the control group (P < 0.05). The longitudinal peak systolic strain of the basal and middle segments of the inferolateral in the G+/P-group was signi cantly higher than that in the control group (P < 0.05). The peak longitudinal strain of each segment in the G+/P+ group was signi cantly lower than that in the control group, especially in the basal and middle segments of the inferoseptum, anterior wall and anteroseptum (P < 0.01). The longitudinal peak systolic strain of each left ventricular wall segment in the G+/P+ group was signi cantly lower than that in the G+/P-group (P < 0.01) In the G+/P-group, the longitudinal peak systolic strain of the basal inferoseptum and basal anteroseptum was signi cantly lower than that of other ventricular wall segments (P < 0.05). In the G+/P+ group, the longitudinal peak systolic strain of the basal and middle segments of the inferoseptum, anterior wall and anteroseptum was signi cantly lower than that of the corresponding segments of the left ventricular wall (P < 0.05) (Table 2) ( Figure 1) ( Figure 2).

Regional circumferential peak systolic strain
There were no signi cant differences among the normal control, G+/P-and G+/P+ groups for GCS values at all levels. The circumferential systolic strain increased from base to apex in the three groups (P < 0.01) ( Table 3) ( Figure 1) ( Figure 2).

Regional radial peak systolic strain
In the G+/P-group, the peak radial strain at all levels no signi cant differences compared with the control group (P < 0.05). The peak radial strain of each segment in the G+/P+ group was signi cantly lower than those in the control and G+/P-groups (P < 0.05).
In the normal control group, G+/P-group and G+/P+ group, there were signi cant differences in peak systolic strain among different segments of the same ventricular wall; the strain was greater in the papillary muscle level than in the apical and mitral valve level (P < 0.05). In the G+/P+ group, the peak radial strain of the anteroseptum, anterior wall and Inferoseptum was signi cantly lower than that of the other ventricular wall segments (P < 0.05) ( Table 4) ( Figure 1) ( Figure 2).

Global longitudinal, circumferential and radial strain
There was no signi cant difference in systolic longitudinal, circumferential, or radial strain between the G+/P-group and the control group (P > 0.05). The systolic GLS of the G+/P+ group was lower than that of the control group and the G+/P-group, and the difference was very signi cant (P < 0.01). The systolic GRS of the G+/P+ group was lower than that of the control group and the G+/P-group, and the difference was signi cant (P < 0.05). The systolic GCS of the G+/P+ group was not signi cantly different from that of the control group or the G+/P-group (P > 0.05) (Table 5) (Figure 3).

Left ventricular rotation parameters andNT-pro-BNP level
In the control, G+/P-and G+/P+ groups, the pattern of cardiac rotation and torsion was the same: the apical part rotated counterclockwise, and the basal part rotated clockwise. However, the rotation of the midventricle was clockwise in G+/P+ group which was different from the control and G+/P-group. In the control and G+/P-groups, the rotation of the midventricle followed the apex. whereas, in G+/P+ groups, the midventricle rotated in the same direction as the base.
The rotation angle of the middle of the LV of the G+/P+ group was signi cantly higher than those of the normal group and the G+/P-group (P < 0.05). However, there were no signi cant differences in the rotation angle of the base, middle or apex of the LV and the global torsion angle of the LV between the G+/P-group and the control group (P > 0.05) (Figure 3).
NT-pro-BNP levels were signi cantly higher in HCM patients compared with the control group and G+/Pgroup. There no detectable differences in G+/P− individuals compared with healthy controls (Table 5).

Discussion
In recent years, studies have shown that the primary cause of familial HCM is mutations in the genes encoding sarcomere proteins and other modi cation genes. Most of the mutations are in genes encoding sarcomere proteins, and point mutations of the β-myosin heavy chain gene (MYH7), myosin binding protein C (MYBPC3), troponin T (TNNT2) and troponin I (TNNI3) are relatively common [10][11][12][13][14]. Abnormal genetic regulation can lead to the disordered arrangement of myocardial cells and abnormal thickening of the myocardium [15][16][17] and can change calcium sensitivity and muscle ber tension, thus affecting myocardial contractile and diastolic function.
In this study, subjects were analyzed from longitudinal, radial and circumferential viewpoints. The results showed that there were no signi cant differences in the global longitudinal, circumferential or radial strains of the systolic period of the LV between the mutation gene carriers and the control group, while the longitudinal strain of the basal inferoseptum and basal anteroseptum was signi cant lower, and the longitudinal strain of the basal and mid inferolateral was signi cantly higher than those of the normal control group. This indicates that the regional myocardial segmental systolic function was impaired in the carriers of the HCM sarcomere gene mutation, and the impairment was limited to the inferoseptum and anteroseptum basal segment. The elevation of longitudinal strain of inferolateral remains unclear.
Maybe regional myocardium experiencing higher longitudinal strain occurs as a cause of adjacent myocardial deformity (with lower strain). Germans et al. [18] found that in HCM gene mutation carriers who did not have ventricular wall hypertrophy, even if the results of conventional echocardiography and ECG were normal, cardiac magnetic resonance technology detected that 81% of HCM gene mutation carriers had a recess in the basal and intermediate segments of the interventricular septum, which may indicate early disease in the HCM gene mutation carriers that will eventually develop into HCM. At the same time, Germans et al. also found that the abnormal myocardial structure of carriers of the HCM gene involved local myocardial segments rather than all myocardial segments, and the interventricular septum were the most obviously involved. HCM gene mutation carriers exhibit disordered arrangement and degeneration of cardiac myocytes, mild brosis in the intercellular matrix and increased myocardial stiffness, and the longitudinal myocardial bers under the endocardium of these patients are more prone to interstitial brosis [19]. The regional radial systolic strain (the basal inferoseptum and basal anteroseptum) of the G+/P-group remained similar to that of the control group. The may be because changes in LV radial systolic function occur later than do changes in longitudinal systolic function.
Our study showned that NT-proBNP levers in HCM patients were signi cant higher and correlated with myocardial deformation and interventricular septal thickness. Among genotype-negative individuals, we also found that there were no difference in NT-proBNP concentrations compared with control relatives, but their local segmental deformation parameters were different, which was different from the Doroteia Silva et al [20] who identify mutation carriers of hypertrophic cardiomyopathy by tissue Doppler imaging.

Conclusions
In conclusion, the GLS and GRS were diminished in HCM subjects, whereas a compensatory mechanism existed that tended to maintain the GCS. Although the GLS, GRS and GCS of HCM gene mutation carriers were still within the normal range, the longitudinal strain of local myocardial segments was diminished.
VVI can provide quantitative information for the early diagnosis of HCM sarcomere gene mutation carriers without myocardial hypertrophy to improve early diagnosis and identi cation.

Declarations
Ethics approval and consent to participate All protocols pertaining to human subjects were rst approved by the Institutional Ethics Committee of Second Xiangya Hospital of Central South University. Informed consent was obtained from all of the patients.

Consent for publication
Not applicable

Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests
All authors declares that there no con icts of interest.  & P < 0.05, compared with the G+/P-group.
⊙P < 0.05; the same segment in the same group compared with other ventricular walls. Table 3 Comparison of left ventricular circumferential peak systolic strain among all groups Longitudinal, circumferential and radial strain of 16 segments in the three groups. a. Longitudinal strain; b. circumferential strain; c. radial strain.
Page 20/20  Comparison of the peak mid rotation angle of left ventricular and global systolic strain among the three groups.